Hum Genet
DOI 10.1007/s00439-007-0403-6ORIGINAL INVESTIGATION
Genetic variation in prehistoric Sardinia
David Caramelli · Cristiano Vernesi · Simona Sanna · Lourdes Sampietro · Martina Lari · Loredana Castrì ·
Giuseppe Vona · Rosalba Floris · Paolo Francalacci · Robert Tykot · Antonella Casoli · Jaume Bertranpetit ·
Carles Lalueza-Fox · Giorgio Bertorelle · Guido Barbujani
Received: 4 June 2007 / Accepted: 29 June 2007
© Springer-Verlag 2007
Abstract We sampled teeth from 53 ancient Sardinian
(Nuragic) individuals who lived in the Late Bronze Age
and Iron Age, between 3,430 and 2,700 years ago. After
eliminating the samples that, in preliminary biochemical
tests, did not show a high probability to yield reproducible
results, we obtained 23 sequences of the mitochondrial
DNA control region, which were associated to haplogroups
by comparison with a dataset of modern sequences. The
Nuragic samples show a remarkably low genetic diversity,
comparable to that observed in ancient Iberians, but much
lower than among the Etruscans. Most of these sequences
have exact matches in two modern Sardinian populations,
supporting a clear genealogical continuity from the Late
Bronze Age up to current times. The Nuragic populations
appear to be part of a large and geographically unstructured
cluster of modern European populations, thus making it
diYcult to infer their evolutionary relationships. However,
the low levels of genetic diversity, both within and among
ancient samples, as opposed to the sharp diVerences among
modern Sardinian samples, support the hypothesis of the
expansion of a small group of maternally related individu-
als, and of comparatively recent diVerentiation of the
Sardinian gene pools.
Electronic supplementary material The online version of this
article (doi:10.1007/s00439-007-0403-6) contains supplementary
material, which is available to authorized users.
D. Caramelli · S. Sanna · M. Lari
Dipartimento di Biologia Animale e Genetica,
Laboratorio di Antropologia,
Università di Firenze, Firenze, Italy
C. Vernesi · G. Bertorelle · G. Barbujani (&)
Dipartimento di Biologia ed Evoluzione,
via Borsari 46, 44100 Ferrara, Italy
e-mail: g.barbujani@unife.it
L. Sampietro · J. Bertranpetit
Dept. Ciències de la Salut i de la Vida,
Unitat de Biologia Evolutiva,
Universitat Pompeu Fabra, Barcelona, Spain
L. Castrì
Dipartimento di Biologia Evoluzionistica e Sperimentale,
Università di Bologna, Bologna, Italy
G. Vona · R. Floris
Dipartimento di Biologia Sperimentale,
Università di Cagliari, Cagliari, Italy
P. Francalacci
Dipartimento di Zoologia e Antropologia Biologica,
Università di Sassari, Sassari, Italy
R. Tykot
Department of Anthroopology,
University of South Florida, Tampa, FL, USA
A. Casoli
Dipartimento di Chimica generale ed Inorganica,
Chimica Analitica e Chimica Fisica,
Università di Parma, Parma, Italy
C. Lalueza-Fox
Unitat d’Antropologia, Dept. Biologia Animal,
Universitat de Barcelona,
Barcelona, Spain123

Hum GenetIntroduction
The population of Sardinia is one of the main European
genetic outliers (Cavalli-Sforza and Piazza 1993). When
compared with populations from all over the world, Sardi-
nians are clearly part of a European genetic cluster (Rosen-
berg et al. 2002). However, they diVer sharply from their
European (Barbujani and Sokal 1990) and Italian (Barbu-
jani and Sokal 1991; Barbujani et al. 1995) neighbours, so
much so that they are often excluded from multivariate
analyses, lest all other samples appear identical in compari-
son (Piazza et al. 1988; Semino et al. 2000). Mitochondrial
(Morelli et al. 2000) and Y-chromosome (Semino et al.
2000; Quintana-Murci et al. 2003) haplotypes that are rare
elsewhere in Europe occur at higher frequencies in Sardi-
nia, and an extensive linkage disequilibrium has been
described for autosomal markers (Tenesa et al. 2004). In
addition, unusually strong genetic diVerences are observed
among Sardinian communities, both for allele-frequency
(Barbujani and Sokal 1991) and DNA (Fraumene et al.
2003) polymorphisms.
These peculiar features are the likely evolutionary prod-
uct of the interaction between reproductive isolation and
small population sizes, the former historically documented
and the latter a consequence of the fragmented habitat.
Starting perhaps 10,000 years ago, people of diVerent prov-
enance reached the island (Webster 1996), but strong isolat-
ing factors, such as the Mediterranean Sea and mountain
ranges, as well as cultural barriers (Barbujani and Sokal
1991), have probably enhanced the evolutionary role of
genetic drift, both within the island and between it and the
rest of Europe. However the details of these processes are
not well understood, and it is well known that ages of par-
ticular genealogical lineages present in a geographical
region do not contain information on the arrival of the pop-
ulation in that region (Barbujani and Goldstein 2004).
Therefore, to gain insight into the biological features of
past populations, and into the relationships between modern
populations and their ancient counterparts, the direct study
of the DNA of ancient individuals is a crucial research
priority.
Here we present the Wrst analysis of the hypervariable
region I (HVR-I) of mitochondrial DNA (mtDNA) from
samples of Bronze and Iron Age inhabitants of Sardinia,
who are called Nuragic people from the towers (nuraghi)
that they built. Using a strict set of methodological criteria
that give a high probability of reproducible results (which
forced us to exclude more than half of the initial samples
from the analysis) we obtained sequences that were com-
pared with those of modern European populations, and with
the sequences of the two pre-classical European popula-
tions whose DNA has been described so far, the Etruscans
(Vernesi et al. 2004) and the Iberians (Sampietro et al.
2005). In particular, prior to sequencing, the samples were
treated with Uracil-N-glicosidase, so as to avoid, as far as
possible, artefacts due to post-mortem damage of DNA
(Hofreiter et al. 2001; Gilbert et al. 2003).
Materials and methods
DNA molecules are often scarce and damaged in ancient
samples. As a consequence, experimental artefacts are pos-
sible or even likely, depending on the state of preservation
of the material, during the analysis of DNA from fossil
remains. The risk to mistake modern contaminating DNA
for endogenous genetic material is higher when dealing
with relatively recent human samples, whose DNA
sequence may be similar to that of the archaeologists,
museum personnel and geneticists who manipulated them.
For all these reasons, in order to obtain reproducible results,
the most stringent available standards were followed in this
study, based on criteria listed by Cooper and Poinar (2000)
and Hofreiter et al. (2001).
We collected 106 teeth, 2 from each of 53 diVerent indi-
viduals, coming from excavations at six archaeological
sites of Sardinia (Fig. 1) and dated between 4,300 and
3,000 years before the present (Table 1). The selected teeth
had not been washed, which meant they had not been
touched by anthropologists for morphological analysis. By
analysing dental pulp from inside the tooth we reduced the
risk of contamination from the archaeologists’ DNA, and
care was taken to choose integer teeth without fractures. As
a rule, to avoid the risk of sampling twice the same individ-
Fig. 1 A map of Sardinia showing the six Nuragic sampling sites. The
two-letter codes are used to label the sequences in the text and in Fig. 2
Carbonia (CA)
Fluminimaggiore (FL)
Perdasdefogu (PE)
Seulo (SE)
Alghero (AL)
Santa Teresa di
Gallura (ST)123