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Lerna, 2000 1500 BC:

The integration of bio-molecular, osteological and archaeological data

Abstract
This paper presents the pilot analysis of a new 5-year research project, which focuses on social
change in the Argolid, Greece during the Middle Bronze Age. The pilot analysis involves the
study of the skeletal material from the Middle Bronze Age intramural cemetery at Lerna. Angels
(1971) publication of the human bones from Lerna remains one of the few large-scale studies of
human skeletal material from the Aegean. However, this material needs to be re-examined in the
light of new analytical techniques.
The project will combine traditional archaeological analyses with innovative techniques. First, the
human skeletal material will be systematically re-examined: an osteological analysis will enable
us to reconstruct the biological history of Lernas skeletal population and to examine variation
between different sub-groups. The examination of dental lesions, the analysis of stable isotopes as
well as dental microwear analysis will help us establish dietary patterns of specific age and/or sex
groups.
Second, DNA analysis will target genomic and mitochondrial DNA in order to identify gender
and to reconstruct kinship relations. A pilot analysis of 12 samples (teeth) will give us
preliminary indications about the preservation of the ancient DNA and the success rate of DNA
isolation and amplification. If the first results are encouraging, an application will be made to
analyze a larger number of samples. The selection of skeletons to be sampled will be informed by
the archaeological analysis of burial (kin?) groupings; an effort will be made to include different
age, sex and wealth categories and modes of disposal from each phase
Finally, a contextual statistical analysis of the funerary data will be undertaken in order to
establish variation among burial groupings. This information will be combined with stratigraphic
observations (associations between tombs and houses) in order to establish possible kin groups on
purely archaeological grounds.

Keywords
Lerna, Middle Bronze Age, osteological analysis, aDNA analysis, kinship, stable isotopes
analysis.

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Authors
Dr. Sofia Voutsaki (main author)
Groningen Institute of Archaeology
Groningen, the Netherlands
s.voutsaki@let.rug.nl

Prof. Sofia Kouidou-Andreou

Department of Biological Chemistry
Medical School, Aristotle University
Thessaloniki, Greece
Kouidou@auth.gr

Dr. Leda Kovatsi

Department of Forensic Medicine and Toxicology,
Medical School, Aristotle University
Thessaloniki, Greece
kovatsi@hotmail.com

Dr. Sevi Triantaphyllou

Archaeological Institute of Northern Greece
Thessaloniki, Greece
sevitr@otenet.gr

Ms Eleni Milka
Groningen Institute of Archaeology
Groningen, the Netherlands
e.milka@let.rug.nl

The wider project

This paper presents part of a new 5-year research project financed by the NWO (Netherlands
Organisation for Scientific Research) and the University of Groningen, the Netherlands. The
project is directed by Dr. Sofia Voutsaki, Groningen Institute of Archaeology.
The aim of the wider project is to understand processes of social change and patterns of cultural
interaction in the southern Greek mainland during the Middle Bronze Age (or Middle Helladic
period, approx. 2000 - 1600 BC) and the beginning of the Late Bronze Age (or Late Helladic I
period, approx. 1600 - 1500 BC). The task is undertaken by means of an integrated analysis of
settlement, funerary and skeletal data from the Argolid, northeastern Peloponnese.
In this paper we would like to present the pilot analysis of the funerary data: the study of the
Middle Helladic cemetery at Lerna.

The Middle Helladic cemetery at Lerna

Lerna is located in the southwestern corner of the Argive plain, in a fertile area surrounded by
marshes (Fig. 1). The site is close to the sea and occupies a strategic location, at the cross-roads
of important axes of communication. The site was the focus of habitation from the Neolithic
period to the end of the Late Bronze Age.
Lerna is a very well excavated and documented settlement. The excavation of the site by the
American School of Classical Studies in the 1950s, under the direction of John L. Caskey,
University of Cincinatti established the sequence within the Middle Helladic period and
transformed our knowledge of the Middle Bronze Age of the Greek mainland. Lerna has
therefore become the type-site of the Middle Helladic period.
The settlement consists of small, rectangular or apsidal, self-standing houses (Fig. 2) which are
sometimes re-built in the same location. The houses differ little in terms of size, construction or
contents. 228 graves (Fig. 3) have been found, dug under the floor, or between the houses. They
often form small groupings, which might represent different kin groups. All the graves contain
inhumations; the norm is single burial, but double or even multiple burials occur as well.
Different types of graves (pits, cists, jar burials) are used, and some variation in wealth can be
observed.
The excavation of the site is described in detailed preliminary reports (Caskey 1954; 1955; 1956;
1957; 1958). The tombs were published in preliminary form by E.T. Blackburn (1970), and the
small finds by E.C. Banks (1967). The skeletal material was published by J.L. Angel (1971). The

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final publication of the Middle Helladic tombs and houses is now under preparation by C. Zerner,
University of North Carolina (see also Zerner 1978).

The analysis of the funerary data

The analysis of the funerary data of Lerna includes:
i.

A systematic re-examination of the skeletal material.

ii.

The analysis of ancient DNA of human skeletons.

iii.

A contextual analysis of the archaeological data.

This pilot analysis is unique in Aegean archaeology, because it combines several analytical
techniques and includes an extensive programme of sampling. Furthermore, it sets out to integrate
archaeological, osteological and bio-molecular data, and place them in a wider historical enquiry
about the role of individuals and social groups in processes of social change. Such integration is
essential at a period of increasing specialization and fragmentation in the fields of archaeology
and bio-molecular archaeology.

(i) The re-examination of the skeletal material

Angels study of the people of Lerna (1971), which focused mainly on anthropometrics and
human skeletal variation, was a pioneering analysis and still remains fundamental in physical
anthropology. However, the development in the last three decades of new methodological tools
and theoretical approaches in the field of biological anthropology necessitates a thorough reexamination of the Lerna skeletons.
This task is undertaken by Dr. Sevi Triantaphyllou. The new study gives special emphasis on the
biological quality of life of the Lerna population, and in particular on the effect of biological
parameters to certain population subgroups defined by age and/or sex. Thus the analysis will
explore the dynamics of ancient demography by investigating mortality rates and sex ratios, and
will also examine aspects of health and oral status, dietary patterns as well as broad categories of
physical activities that the Lerna population was engaging with during life.
As regards health status, the study focuses on two broad categories of pathologies:
1) bone lesions associated with mechanical load and occupational activities repeatedly
exercised on the skeleto-muscular system e.g. degenerative joint disease, trauma, vertebral
defects, indicators of muscle attachments and enthesopathies
2) pathological conditions associated with systemic physiological stress and/or acute episodes

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of stress which affected the individual during his/her lifetime e.g. metabolic diseases such as
anemia, enamel hypoplasia and non-specific infections.
The central aim of the re-examination of the material is not merely to identify these features in
individual cases but to recognize patterns of pathological conditions and stress markers at the
level of the entire population. A further aim is to detect variation between age and sex groups, as
well as between social and kin groups (Larsen 1997).
Dietary patterns in general reflect the availability of natural resources, but inform us also about
the management and distribution of resources by means of social practices (such as eating
etiquette, dietary taboos, differential participation in communal eating and feasting, etc.), and
therefore about differentiation along age, sex and status lines. Certain types of dental disease can
be associated with specific food categories- for instance, dental decay can be attributed to a high
consumption of carbohydrates. In order to reconstruct the diet of the prehistoric community at
Lerna, the following methods will be used:
1) The systematic recording of dental disease at a macroscopic level (undertaken by Dr.
Sevi Triantaphyllou).
2) A dental microwear analysis of tooth enamel in order to explore the texture of the food
categories consumed shortly before death (Teaford 1991). This analysis is carried out by
Dr. Sevi Triantaphyllou as part of a separate project financed by the Institute of Aegean
Prehistory, New York.
3) The analysis of stable isotopes of nitrogen and carbon (to be carried out by Dr. Michael
Richards, Palaeodietary Research Group, Department of Archaeological Science,
University of Bradford) will allow us to recognize certain isotopically distinctive
nutrients in the diet (Katzenberg 1992). For instance, it will allow us to establish the
contribution of marine versus terrestrial resources, or the consumption of plant versus
animal proteins, or of C3 versus C4 plants, etc.
The integration of the results obtained by stable isotope and dental microwear analysis will enable
us to reconstruct the life histories of individual members of the Lerna community. The ultimate
aim of the re-examination of the Lerna material will be to treat the skeletal remains as an active
body of information which is seen as reflecting a number of roles such as social status, gender or
age-assigned roles assumed by the Middle Helladic population of Lerna.

(ii) Ancient DNA analysis from Lerna

The pilot analysis of ancient DNA from Lerna has two goals:

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1) The first goal is to establish the sex of the deceased. This method is particularly useful in
cases where the traditional morphological analysis of bones cannot provide sufficient
information, either because the skeletons are poorly preserved or because they are
immature. This is certainly the case in Lerna, an intramural cemetery where graves are
often disturbed and contain a high proportion of infants and children.
2) The second goal is to detect family relationships. This can only be achieved with DNA
analysis. In the case of Lerna, where graves are closely connected with specific
households (the graves are dug under or between houses), DNA analysis will allow us to
establish kinship relations within and between burial groupings and households, as well
as their evolution through time. Reconstructing the network of kinship relations in
Middle Helladic Lerna, a society without overt signs of social differentiation, will allow
us to establish the significance of kinship in social life.
The analysis will be undertaken by Prof. Sofia Kouidou-Andreou and Dr. Leda Kovatsi at the
Laboratory of Biological Chemistry, Medical School, Aristotle University of Thessaloniki.
First, a pilot analysis of 12 samples will be carried out. This will give us indications about the
state of the preservation of the aDNA, and will allow us to assess the chances of extracting and
amplifying the DNA. This careful approach is necessary because of the significance of the Lerna
skeletal assemblage and the need to preserve it for future generations of researchers.
If the results prove encouraging, we will apply for permission to undertake a more extensive
sampling programme. We would like to sample a large number of skeletons which have been
selected on the basis of primarily archaeological criteria. The intention is to include men, women
and children, as well as different wealth categories and modes of disposal from each burial group.
The preservation of the skeletons is, of course, another important consideration. We are planning
to sample teeth as they are a more appropriate source of genetic material, but we may consider
using long bones if teeth are not available. It should be mentioned here that a previous analysis of
aDNA using samples from long bones from Middle Helladic skeletons from Lerna (carried out by
Prof. T. Brown and Dr. K. Brown, both at the Department of Biomolecular Sciences, University
of Manchester) did not yield any DNA (T. Brown and K. Brown, personal communication).
The following method will be used: In order to extract DNA (mitochondrial and genomic), teeth
will be pulverized after their surface has been extensively cleaned and contaminants have been
removed. If long bones are used, a small fragment (up to 5gr) will be removed after extensive
cleaning, and will be pulverized. In either case, DNA will be isolated from the pulverized sample
by means of a technique developed in our laboratory. This protocol involves extensive digestion

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of the sample with proteinase K and extraction of DNA from the digest followed by purification
of the extract (Pfeiffer et al. 1998).
Genomic DNA will be used for the identification of sex. The amelogenin locus will be amplified
with the help of specific commercial kits (Sullivan et al 1993).
In order to establish family relationships, mitochondrial DNA (mtDNA) will be studied.
Hypervariable control regions I and II will be amplified, each in two overlapping fragments, and
the amplicons will be sequenced (Sullivan et al. 1992).
In order to overcome the problem of contamination with contemporary DNA, the samples in our
laboratory are handled and processed under very stringent conditions. Tight segregation is
employed between pre-PCR and post-PCR areas. DNA extractions and PCR setups are carried
out in a dedicated biosafety hood that is located separately from that used for post-PCR analysis.
The use of positive controls is avoided. The extractions are always carried out for each sample
separately, while one negative extraction control (extraction blank) is also being included. The
sample extract and the extraction blank, plus one negative reagent control (PCR blank) are
subjected to PCR amplification. The batches (extraction blank, PCR blank, sample) in which the
extraction blank or PCR blank give a positive result, are rejected.
aDNA analysis is prone to PCR-introduced errors due to both DNA damage and inappropriate
enzyme activity (Krings et al. 1997). In order to ensure that our results are genuine, the target
sequences are amplified and sequenced twice and in some cases the whole procedure is applied to
two different DNA extracts of the same specimen.
The aDNA analysis from MH Lerna will serve a further goal: to develop and promote ancient
DNA analysis in Greece, a country which is very rich in archaeological remains and where the
analysis of human skeletal remains is gradually becoming an important growth area (e.g.
Papathanasiou 2001; Triantaphyllou 2001).

(iii) Contextual analysis of the archaeological data and integration

The archaeological analysis of the funerary data from Middle Helladic Lerna, currently carried
out by Eleni Milka, proceeds in two stages. The first stage involves a statistical and contextual
analysis of the archaeological data, and the second the integration of anthropological,
biomolecular and archaeological information.
The aims of the archaeological analysis are:
1) To detect and interpret variation between the graves and burial groupings. The main
question here is whether the variation in the type, construction and size of graves as well

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as in the quantity or quality of funerary offerings accompanying the dead represent
differentiation between age, sex and kin or social groupings (Parker -Pearson 1993; Robb
1994).
2) To reconstruct and explain processes of social change which the Lerna community
underwent through the Middle Helladic period. The Middle Helladic period is
characterized by social regression, poverty and material austerity. The end of the period,
however, sees important changes, especially in the mortuary sphere: the introduction of
more labour intensive tombs, the adoption of more complex burial ritual, and a striking
increase in the wealth deposited with the dead (Voutsaki 1993; Cavanagh and Mee 1998).
The ultimate goal of this analysis is to understand the causes which led to these changes
(Chapman 1991; Voutsaki 1998).
3) To understand the funerary ideology of the Middle Helladic society of Lerna. The first
question to be addressed is whether mortuary ritual was used in order to express or deny
social differentiation. Further, an attempt will be made to reconstruct the attitudes to
death, as well as the cultural values, beliefs and norms underlying the treatment of the
dead in Middle Helladic Lerna (Bloch and Parry 1982; Morris 1991).
These aims will be achieved with a detailed analysis of all aspects of the mortuary data: the
location, size and type of graves and their association with the space of the living; the quality,
quantity and type of funerary offerings; the traces of ritual surrounding the disposal of the
body (Caskey 1954; 1955; 1956; 1957; 1958; Banks 1967; Blackburn 1970; Angel 1971;
Zerner 1978). The emphasis is on the presence / absence of specific features as well as their
correlation or mutual exclusion. Special attention is given to the relation between houses,
habitation floors and clusters of graves in order to detect households and possible kin
groups. The offerings are examined in terms of presence/ absence, quantity, and diversity in
order to understand their use in social strategies. An attempt will also be made to understand
their meaning and significance (both in the mortuary ritual and in everyday life) by studying
their position inside the grave, their preservation and patterns of breakage, as well as the
associations between the various offerings (Zerner 1990; Nordquist 1990; Chapman 2000).
In the second stage of the analysis, the archaeological information will be integrated with the
results of the osteological and biomolecular analysis. The information about pathologies,
occupational activities and nutrition will be correlated with variation in terms of grave
architecture or the accompanying grave goods. The results of the DNA analysis, if positive, will
be used to confirm (or perhaps refute) inferences on kinship connections between individuals,
tomb groupings and houses.

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The integration of archaeological, anthropological and biomolecular information will enable us to
reconstruct the life histories of the inhabitants of Lerna, and the biography of the entire
community of Middle Helladic Lerna.

The next stage: social change in the Middle Helladic Argolid

The pilot analysis of Lerna is the first stage of a wider investigation which will eventually include
all the sites of the Middle Helladic Argolid (Fig. 1). The second stage of the analysis will include
the other two large cemeteries of the Argolid, Asine (Nordquist 1987, Ingvarsson-Sundstrom
2003) and Argos (Deilaki 1980, Pirart & Touchais 1996). The comparison of these three
important sites will allow us to reconstruct their different socio-political trajectories as well as
processes of social and political competition within and between communities. At a further stage,
the data from Mycenae (Karo 1930-33; Mylonas , Alden 2000) and Tiryns, two sites which rise in
importance towards the end of the Middle Helladic period, but are less well preserved and
documented, will be included. During the final stage, the information from lesser sites such as
Prosymna (Blegen 1937 ) will be added.
To conclude, the comparative analysis of all extant sites of the MH Argolid and the integration of
funerary, skeletal and bio-molecular data with the settlement evidence will enable us to
reconstruct the social structure of the communities of the Middle Helladic Argolid, and to
understand the causes of their transformation (Voutsaki 1998; 2001).

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Acknowledgements
We would like to thank the American School of Classical Studies, and in particular Prof. M.
Wiencke, the director of the Lerna excavations, and as Prof. E. Banks for their permission to reexamine the Lerna material. Special thanks go to Dr. C. Zerner for her permission to study the
finds from Lerna and for her continuous help and support. We are also grateful to the Argolid
Ephorate of Classical and Prehistoric Antiquities for permission to study the Lerna material in the
Museum of Argos. We would like to acknowledge the help and advice we received by the
director of the Dutch Institute in Athens, Dr. G.J. van Wijngaarden. Finally, our thanks to Tomek
Hertig, our project assistant, for producing the poster presented at the conference and Dr. J.W.
Veluwenkamp for help with printing it.