Re: Ancient Etruscans, and their cattle, were immigrants from Anatolia,

On Jun 18, 11:07 am, "Uwe Müller" <uwemuel...@xxxxxxxxxx> wrote:
"Jack Linthicum" <jacklinthi...@xxxxxxxxxxxxx> schrieb im Newsbeitragnews:1182123532.988436.75870@xxxxxxxxxxxxxxxxxxxxxxxxxxxxxx



Genetic differences in current population of the Etruscan region were
found in populations in Turkey.

"We found that the DNA samples from individuals from Murlo and
Volterra were more closely related those from near Eastern people than
those of the other Italian samples", Professor Alberto Piazza, from
the University of Turin, Italy, says at the European Society of Human
Genetics

"In Murlo particularly, one genetic variant is shared only by people
from Turkey, and, of the samples we obtained, the Tuscan ones also
show the closest affinity with those from Lemnos."

Scientists had previously shown this same relationship for
mitochondrial DNA (mtDNA) in order to analyse female lineages. And in
a further study, analysis of mtDNA of ancient breeds of cattle still
living in the former Etruria found that they too were related to
breeds currently living in the near East.

Public release date: 16-Jun-2007

Contact: Mary Rice
m...@xxxxxxxxxxxxxxxxxxx
European Society of Human Genetics
Ancient Etruscans were immigrants from Anatolia, or what is now Turkey
Geneticists find the final piece in the puzzle
snip >
"We think that our research provides convincing proof that Herodotus
was right", says Professor Piazza, "and that the Etruscans did indeed
arrive from ancient Lydia. However, to be 100% certain we intend to
sample other villages in Tuscany, and also to test whether there is a
genetic continuity between the ancient Etruscans and modern-day
Tuscans. This will have to be done by extracting DNA from fossils;
this has been tried before but the technique for doing so has proved
to be very difficult."
snip

http://www.eurekalert.org/pub_releases/2007-06/esoh-aew061307.php

Lets say they provided evidence for the anatolian origin theory. What they
lack is a precise dating of the movement of the people, which can't be done
with modern DNA. So they prooved an immigration, but they can't know if it
happened in the Iron Age, during the Greek colonisation, in Roman times or
during the times of direct Byzantine envolvement in the area (or even
later).

And if extracting of prehistoric or historic DNA works with bones from
Neanderthals or chickens, for bronce age burials or medieval graves it
should work with Etruscan bones too.

So, to be 100% certain they'd need to look at Etruscan bones not at modern
day Tuscans.

have fun

Uwe Mueller

If you were this group, working on a problem of considerable
dimensions, would you unload all of your findings in one release?
Of course not, they get a grant, they fulfill the grant or part of it
and move on. Piazza is giving a paper at a meeting not writing a book.
It would also seem that the study of Etruscan bones from the 7th to
the 3rd centuries BC has been done by some of the same team.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1181945


Am J Hum Genet. 2004 April; 74(4): 694-704.
Published online 2004 March 10.
Copyright © 2004 by The American Society of Human Genetics. All rights
reserved.
The Etruscans: A Population-Genetic Study
Cristiano Vernesi,1 David Caramelli,2 Isabelle Dupanloup,1,* Giorgio
Bertorelle,1 Martina Lari,2 Enrico Cappellini,2 Jacopo Moggi-Cecchi,2
Brunetto Chiarelli,2 Loredana Castrì,3 Antonella Casoli,4 Francesco
Mallegni,5 Carles Lalueza-Fox,6 and Guido Barbujani1
1Dipartimento di Biologia, Università di Ferrara, Ferrara, Italy;
2Dipartimento di Biologia Animale e Genetica, Laboratori di
Antropologia, Università di Firenze, Firenze, Italy; 3Dipartimento di
Biologia Evoluzionistica e Sperimentale, Università di Bologna,
Bologna, Italy; 4Dipartimento di Chimica Generale e Inorganica,
Chimica Analitica, Chimica Fisica, Università di Parma, Parma, Italy;
5Dipartimento di Scienze Archeologiche, Università di Pisa, Pisa,
Italy; and 6Unitat de Biologia Evolutiva, Departament de Ciències
Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona,
Spain
Address for correspondence and reprints: Dr. Guido Barbujani,
Dipartimento di Biologia, via Borsari 46, I-44100 Ferrara, Italy. E-
mail: g.barbujani@xxxxxxxx
*Present affiliation: Centre integratif de genomique (CIG), Lausanne,
Switzerland.
Received December 5, 2003; Accepted January 28, 2004.

Abstract
Introduction
Material and Methods
Results
Discussion
Electronic-Database Information
References

Abstract
The origins of the Etruscans, a non-Indo-European population of
preclassical Italy, are unclear. There is broad agreement that their
culture developed locally, but the Etruscans' evolutionary and
migrational relationships are largely unknown. In this study, we
determined mitochondrial DNA sequences in multiple clones derived from
bone samples of 80 Etruscans who lived between the 7th and the 3rd
centuries b.c. In the first phase of the study, we eliminated all
specimens for which any of nine tests for validation of ancient DNA
data raised the suspicion that either degradation or contamination by
modern DNA might have occurred. On the basis of data from the
remaining 30 individuals, the Etruscans appeared as genetically
variable as modern populations. No significant heterogeneity emerged
among archaeological sites or time periods, suggesting that different
Etruscan communities shared not only a culture but also a
mitochondrial gene pool. Genetic distances and sequence comparisons
show closer evolutionary relationships with the eastern Mediterranean
shores for the Etruscans than for modern Italian populations. All
mitochondrial lineages observed among the Etruscans appear typically
European or West Asian, but only a few haplotypes were found to have
an exact match in a modern mitochondrial database, raising new
questions about the Etruscans' fate after their assimilation into the
Roman state.

Introduction

Analysis of genetic data in modern populations has proved to be a
powerful tool for reconstructing crucial aspects of human evolutionary
history (Cavalli-Sforza et al. 1994; Ingman et al. 2000; Barbujani and
Bertorelle 2001; Tishkoff and Verrelli 2003). It is now technically
feasible to validate these analyses by directly studying the DNAs of
ancient people, but so far only a handful of European sequences have
been published (Handt et al. 1994; Di Benedetto et al. 2000). In this
study, we present the first extensive genetic data on a European
population of the preclassical period, the Etruscans.

The Etruscan culture developed in central Italy (Etruria) in the first
millennium b.c. The oldest-known inscriptions in Etruscan, a non-Indo-
European language isolate, date back to the end of the 8th century,
right after the shift from the rural Villanovian culture, documented
in the same area in the 9th century b.c., to an urban society
(Bartoloni 1989). The Etruscan cities were independent states that
shared a language and a religion but never formed a political unit.
However, between the 7th and the 5th centuries, leagues of Etruscan
cities established their political and cultural leadership over an
area spanning from the Po Valley to Magna Graecia (fig. 1), including,
during part of the 6th century, Rome. Military defeats, the Roman
expansion, and progressive assimilation caused a decline of the
Etruscan cities, which lost their autonomy when Roman citizenship was
granted to the Roman allies (90-89 b.c.). Immediately afterward, the
language disappeared (Pallottino 1975; Barker and Rasmussen 1998).
Figure 1 Figure 1
Map of Italy showing the area of Etruscan influence (gray) in the 7th
and 6th centuries b.c., from Barker and Rasmussen (1998). A solid line
identifies the boundaries of Etruria proper. Solid circles are
sampling locations: A, Adria (17 samples, 5 DNA (more ...)

Paleoanthropological studies have only proved broad similarities
between the Etruscans and their neighbors of the Iron Age (Barker and
Rasmussen 1998). Archaeological evidence suggests that the Etruscan
culture developed locally, with some features pointing to an Eastern
influence (Pallottino 1975; Barker and Rasmussen 1998). However, it is
not clear if such influence reflects only trading and cultural
exchange or rather some sort of shared biological ancestry. That is a
long-lasting controversy. Dionysius of Halicarnassus (1.30.2) favored
local development, whereas, according to Herodotus (1.94), the
Etruscans were Lydians of Anatolia who were fleeing from famine
(Barker and Rasmussen 1998). No modern archaeologist supports the
latter view, but some affinities between the Lydian and the Etruscan
languages have been recognized (Beekes 2002), and gene flow from the
eastern Mediterranean area is impossible to rule out on archaeological
grounds (Tykot 1994). Unfortunately, no original documents are
available to help solve this question. Indeed, although the Etruscan
alphabet and language are largely understood (Bonfante and Bonfante
2002), the written record is limited to short inscriptions of
religious or funerary content.

By themselves, DNA sequences cannot tell us who the Etruscans were and
where they came from, but they can provide crucial information on two
related questions:

* 1. Were the Etruscans a single population, or were they simply a
set of individuals who shared a language and a culture but not a
common ancestry?
* 2. What are the genetic relationships between the Etruscans and
modern populations, and do these relationships suggest any
genealogical or migrational links between the Etruscans and other
Eurasians?

To address the above questions, we obtained from museums and public
collections fragments of 80 well-preserved skeletons from 10 Etruscan
necropoleis (fig. 1), covering much of Etruria in terms of both
chronology (7th to 2nd centuries b.c.) and geography. All human
remains analyzed come from sites where (1) the material culture has
been identified as Etruscan by archaeologists, and (2) all
inscriptions, if any, are in Etruscan. Two cities, Adria and Capua,
were at the fringes of Etruscan territory, in the Po River valley and
in Campania, respectively. Historical documents and archaeological
evidence, such as inscriptions in Etruscan and decorations on the
pottery, show that these were indeed Etruscan settlements (Barker and
Rasmussen 1998; Bonfante 1999; Haynes 2000), although they were both
locations where hybridization may have occurred-with Veneti (in Adria)
and with Samnium natives or Greek colonizers (in Capua).


Material and Methods

DNA is generally present only in small amounts in ancient samples, and
it is often damaged. Therefore, extreme precautions are needed to
minimize the risk of amplifying and typing modern contaminating DNA
molecules. This is especially important when dealing with relatively
recent human samples, whose genetic material may not be very different
from that of the archaeologists and biologists who manipulated it. To
maximize the probability of extracting and sequencing authentic DNA
from the ancient samples, the strictest available standards (Cooper
and Poinar 2000) were followed throughout this study. All samples that
did not comply with any of Cooper and Poinar's nine criteria were
discarded. In most cases, no information was available on the
archaeologists and museum staff who had previous contact with the bone
material, but all people who manipulated the specimens for this
project had their mtDNA typed and compared with the sequences obtained
from the ancient specimens.

Authentication Methods for Ancient DNA

Physically isolated work area. All work was performed in isolated
areas of the Florence and Barcelona laboratories, where no modern DNA
has ever been introduced (criterion 1 of Cooper and Poinar [2000]).

Amino acid racemization. In living individuals, all amino acids are in
the L-enantiomeric form, but after death a racemization process
begins. Poinar et al. (1996; Poinar and Stankiewicz 1999) described a
correlation between the amount of D-forms of three amino acids (Asp,
Glu, and Ala) and the presence of amplifiable DNA in the sample. To
test for biochemical preservation as indirect evidence for DNA
survival, we thus estimated the degree of amino acid racemization in
each sample, using 5 mg of bone and following the procedures
described in the work of Vernesi et al. (2001) (criterion 7). In
addition, to better estimate the preservation of organic material, 3
mg of bone powder from each individual was also analyzed by a
thermogravimetric assay (Peters et al. 2000).

DNA extraction. After brushing and irradiating each bone surface (1 hr
under ultraviolet light), DNA was extracted from powdered bone by
means of a silica-based protocol (modified from Krings et al. 1997;
see Di Benedetto et al. 2000). For each individual, we performed two
independent extractions from different bones, usually a rib and a
fragment of long bone (criterion 2). For each individual, we also
attempted to amplify longer mtDNA fragments (360 bp and 700 bp), which
have been reported to be unusual in ancient specimens; we sequenced
only the samples in which that attempt failed (criterion 3). A
negative control was included in each extraction (criterion 2).

Quantitation of ancient DNA. Sporadic contamination is considered
unlikely when the number of molecules that PCR will use as template,
or "target DNA," is >1,000. We estimated the amount of target DNA by
competitive PCR, as described in the work of Handt et al. (1996)
(criterion 8). A competitor was used containing a 95-bp deletion
(nucleotide positions 131-225; positions were numbered according to
Anderson et al. [1981], 16,000) in the mitochondrial hypervariable
region I (HVR-I) (Caramelli et al. 2003). PCR components were the same
as described below for the sequencing of HVR-I, and the primers were
the same as those used for the amplification of the second of three
HVR-I fragments. A negative control was included in each amplification
(criterion 2). Thermal cycler conditions consisted of an initial 10
min incubation at 95°C, followed by 45 cycles of 50 s at 94°C, 50 s at
48°C, and 50 s at 72°C, with a final extension step at 72°C for 5 min.

Amplification. The following profile was used to amplify 2 l of DNA
extracted from the bone: 94°C for 10 min (Taq polymerase activation),
followed by 50 cycles of PCR (denaturation 94°C for 45 s; annealing
53°C for 1 min; and extension 72°C for 1 min) and a final step at 72°C
for 10 min. The 50- l reaction mix contained 2 U of AmpliTaq Gold
(Applied Biosystems), 200 M of each dNTP, and 1 M of each primer.
The 360-bp-long HVR-I was subdivided in three overlapping fragments by
use of the following primer pairs: L15995/H16132; L16107/H16261; and
L16247/H16402 (Caramelli et al. 2003). Each extract was amplified at
least twice (criterion 4). Since overlapping primers were used
throughout the PCR amplifications, it is highly unlikely that we
amplified a nuclear insertion rather than the organellar mtDNA (Handt
et al. 1996; Greenwood et al. 1999).

Cloning and sequencing. PCR products were cloned (criterion 5) using
the TOPO TA Cloning kit (Invitrogen), according to the manufacturer's
instructions. Screening of white recombinant colonies was accomplished
by PCR. The colonies were transferred into a 30- l reaction mix (67 mM
Tris HCl [pH 8.8], 2 mM MgCl2, 1 M of each primer, 0.125 mM of each
dNTP, and 0.75 U of Taq polymerase) containing M13 forward and reverse
universal primers. After 5 min at 92°C, 30 cycles of PCR (30 s at
90°C, 1 min at 50°C, 1 min at 72°C) were performed and clones with an
insert of the expected size were identified by agarose-gel
electrophoresis. After purification of these PCR products with
Microcon PCR devices (Amicon), a volume of 1.5 l was cycle-sequenced,
according to the BigDye Terminator kit (Applied Biosystems) supplier's
instructions. The sequence was determined using an Applied BioSystems
377 DNA sequencer.

Independent replication. To test for contamination within the
laboratory, three bone fragments were subjected to DNA extraction,
amplification, cloning, and sequencing in Barcelona (criterion 6). In
this lab, the following primer pairs were used: L16055/H16218 and
L16209/H16401.

Associated faunal remains. The presence of human mtDNA sequences in
extracts from nonhuman bones proves that those bones have been
contaminated by human DNA and, hence, that sequences obtained from
human remains in the same burial may also result from contamination.
Cattle (Bos taurus) remains retrieved in Magliano, in the tomb of the
individual with the 5AM sequence, gave us an opportunity to test for
this type of contamination. We tried to amplify the DNA extracted from
the cattle bones by use of both human-specific and bovine-specific
primers. We used primers for a fragment of 152 bp of the Bos taurus
mtDNA hypervariable region and for a fragment of the human HVR-I (the
primers were L16030/H16137 and L16107/H16261, respectively). The PCRs
were performed with 2 l of DNA, 1 M of each primer, 200 M of each
dNTP, 1 × reaction buffer (Applied Biosystems), 1.5 mM MgCl2, and 2 U
of AmpliTaq Gold (Applied Biosystems) in a total volume of 50 l. The
cattle hypervariable region was amplified using the following thermal
cycle: initial denaturation at 94°C for 10 min, followed by 40 cycles
of 94°C for 1 min, 57°C for 1 min, 72°C for 1 min, and final extension
at 72°C for 5 min. The conditions described above for human mtDNA
amplification were used in the amplification of bovine DNA with human-
specific primer. PCR products were obtained using only the primers
specific for cattle. After run on a 1.5% agarose gel, bands of the
appropriate size were excised from the gel and purified with Ultra
Free DNA (Amicon). Cloning and sequencing of the PCR products were
performed as described above (criterion 9).

RFLP: typing of the 14766 site. A short fragment of the coding
mitochondrial region around nucleotide position 14766 was amplified in
a 50- l reaction volume containing 2 l of template DNA, 1.5 mM MgCl2,
200 M of each dNTP, 2 U AmpliTaq Gold (Applied Biosystems), and 1 M
of each primer (L14695/H14792), with the following thermal cycles:
initial denaturation at 94°C for 10 min, followed by 40 cycles of 94°C
for 1 min, 57°C for 1 min, 72°C for 1 min, and final extension at 72°C
for 5 min. Fifteen l of PCR product were digested with 10 U of MseI
(Celbio Italy), using the recommended buffer with overnight incubation
at 37°C. Digestion products were visualized by electrophoresis on 2.5%
agarose gels. To confirm the results of the restriction analysis, the
nucleotide at position 14766 was also determined in each sample by
independent amplification (with the same primer pair and under the
same conditions as those described above), cloning, and sequencing.

Database of Modern Mitochondrial Sequences
The data in the database analyzed by Simoni et al. (2000) were updated
with the following populations: Haviks of India (Mountain et al.
1995), Egyptians (Krings et al. 1999), Syrians and Greeks (Vernesi et
al. 2001), Uighurs, Kazakhs, and Kirghiz of Central Asia (Comas et al.
1998), Armenians and Azerbaijanis (Nasidze and Stoneking 2001), and
Ladin speakers of Italy (Vernesi et al. 2002), which replaced the
previous sample from the same area. Populations and sample sizes are
listed in the legend to figure 4.
Figure 4 Figure 4
Two-dimensional representation of the relationships between
populations (MDS plot) based on FST distances. Population labels and
sample sizes: ETRU: Etruscans, 27; AUST: Austrians, 117; BULG:
Bulgarians, 30; DENM: Danes, 32; ESTO: Estonians, 28; FRAN: (more ...)

Statistical Analysis
Haplotype variation was summarized by a reduced median network
(Bandelt et al. 1999). Sites with lower mutation rates were given
greater weight. Allele sharing was estimated by counting the
occurrences of the haplotypes observed among the Etruscans in the 34
populations of the database.

Pairwise genetic distances (FST) between populations were estimated by
the ARLEQUIN software package (Schneider et al. 2000), considering
both haplotype-frequency differences and numbers of substitutions
between haplotypes. Kimura's two-parameter method was used (assuming a
gamma distribution for rate variation among sites, with =0.26), which
allows for multiple substitutions at a site and for different rates of
transitions and transversions. FST distances calculated in this way
are equivalent to (tm t0)/tm, where tm is the mean coalescence time of
two random haplotypes drawn from the two populations, and t0 is the
mean coalescence time of two random haplotypes drawn from the same
population (Slatkin 1991). From the matrix of FST distances, we
obtained a two-dimensional representation of the data by
multidimensional scaling (MDS) (Kruskal 1964), using the software
STATISTICA.

Patterns of genetic variation within the Etruscan and the Italian
populations were described by analyses of molecular variance (AMOVA)
(Excoffier et al. 1992), as implemented in the ARLEQUIN package. AMOVA
estimated indexes of molecular diversity among members of the same
populations, among populations, and (in the description of Etruscan
diversity across time periods) among groups thereof, and tested for
their statistical significance using a Monte Carlo randomization
approach.

The genetic relationships between the Etruscans and modern Italian
populations were quantified by estimating admixture coefficients for
each of them, representing the relative contribution of potential
parental populations. The purpose of this exercise was not to identify
the historical admixture process giving rise to the Etruscan gene
pool, for which the necessary data will be available only when several
ancient populations have been described at the mitochondrial level.
Rather, we wanted to obtain figures allowing us to quantitatively
compare the composition of modern and ancient gene pools. Thus,
admixture coefficients were inferred from differences in haplotype
frequencies, considering the Etruscans and the modern Italian
populations as hybrids among up to four potential parents (Dupanloup
and Bertorelle 2001). The mitochondrial features of the parental
populations were approximated assuming that the best available
estimates of allele frequencies in past (and unknown) populations is
found in their modern counterparts, as is customary in admixture
studies (e.g., Chakraborty 1986; Alves-Silva et al. 2000; Chikhi et
al. 2002). We chose the Basques as representative of western Europe,
the Turks as representative of the eastern Mediterranean region,
Karelians and Volga Finns as representative of northeastern Europe,
and Egyptians and Algerians as representative of North Africa.
Admixture coefficients can also be estimated from the coalescence
times between haplotypes. However, mitochondrial coalescence times go
back to several tens of thousand years ago in Eurasia (Richards et al.
2000). The specimens in this study are 2,500 years old, and in this
period changes of haplotype frequencies resulting from drift certainly
had a greater effect on genetic diversity than the onset of new
mutations. Therefore, estimates based only on haplotype frequencies
seemed preferable.

Results

The 80 samples in this study underwent nine tests for authentication
of ancient DNA, and at each step those that did not comply with the
relevant criterion were eliminated. In particular, three samples
yielded a D/L ratio for aspartic-acid enantiomers >0.1, which is
considered incompatible with retrieval of sufficient endogenous DNA
from ancient remains (Poinar et al. 1996) and, hence, were discarded.
In the remaining 77 samples, not only the content of racemized amino
acids was low but also the relative extent of racemization was
Asp>Glu>Ala, both findings generally associated with good preservation
of endogenous macromolecules (Poinar et al. 1996). Low temperatures
are known to facilitate the preservation of DNA in ancient samples
(Kumar et al. 2000; Reed et al. 2003), and, indeed, all samples of
this study come from room burials or caves, where temperatures are low
and constant throughout the year.

Competitive PCR showed that 48 of these 77 samples had sufficient
amounts (initial number >1,000 copies) of template DNA (Handt et al.
1996); 29 samples were discarded at this stage. Thus, 48 samples were
selected for which all tests indicated a good probability to obtain
DNA sequences without artifacts. In all of them, the PCR amplification
strength appeared inversely related to the size of the product, and
PCR products larger than 200 bp were not observed, both findings
consistent with the expected behavior of ancient (as opposed to
contaminating modern) DNA molecules (Hofreiter et al. 2001). Each
extract was subjected to two amplifications of three overlapping
fragments, each PCR product was cloned, and multiple clones were
sequenced, so that each of the three fragments was sequenced at least
eight times (two extracts × two PCRs × two clones or more). Eighteen
individuals yielded incomplete or multiple sequences and were
discarded at this stage. Thirty individuals gave complete PCR products
whose sequences were the same across clones, except for sporadic
misincorporation of nucleotides in single clones. The overall
misincorporation rate was a low 0.27% over the 92,104 nucleotides
sequenced (compared, for example, with 0.39% in the "Iceman" specimen
of Handt et al. 1994), which confirms that there was a fairly large
amount of target DNA in the 30 samples.

The entire procedure was repeated in the Barcelona laboratory, under
double-blind conditions, for three bone specimens, and identical
sequences were obtained. Finally, the DNA from cattle remains found in
the burial site of the individual with the 5AM sequence could be
amplified only using bovine-specific (but not human) primers. The
sequences, determined from multiple clones, match with those of the
Bos taurus sequence deposited in GenBank (accession number NC_001567),
showing no trace of human sequences, and so once again failing to
provide evidence of modern human DNA contamination.

We observed 23 different sequences with 26 variable sites (25
transitions, 1 transversion) (table 1). (Sequences of each clone in
this study can be accessed at the authors' Web site.) All identical
sequences came from different necropoleis, except for three
individuals from the same tomb in Tarquinia. To remove possible
effects of consanguinity on the estimated population statistics, we
excluded two of them from further analyses. A different problem was
represented by sequence 2V, which showed six substitutions-the
069-186-189-223-319-362 motif-that usually occur in distant and
mutually exclusive branches of the mtDNA network (Richards et al.
2000). Independent replicates in two laboratories under double-blind
conditions confirmed the sequence, showing that the motif does not
result from experimental artifacts. At this stage, we can only
speculate that some kind of oxydative damage, possibly resulting from
the specific microclimatic condition of the burial site (Lindahl 1993,
1997), has altered the DNA molecules of that specimen. At any rate,
for the sake of phylogenetic consistency, it seemed prudent not to
consider this sequence in successive statistical analyses.
Table 1 Table 1
Consensus HVR-I Mitochondrial Sequences in 28 Etruscan Individuals
[Note]

Exclusion of the 2V sequence brought the final sample size to 27
individuals with 22 different sequences. Only seven such sequences
(4V, 5AM, 6AM, 8A, 9A, 16S, and 19M) occur in a database of 34 modern
populations comprising 2,481 individuals from western Eurasia and the
southern Mediterranean shores.

The Etruscan sequences show substitutions at sites (069, 126, 223,
270, and 356) known to be prone to recurrent mutation or postmortem
damage (Gilbert et al. 2003). Substitutions at these mutational
hotspots may cause misassignment of sequences to haplogroups (Bandelt
et al. 2002). To achieve a better resolution, we then typed the
restriction site of the coding region 14766 MseI, which is
characteristic of the HV superhaplogroup (Richards et al. 2000).
Restriction cuts on ancient DNA molecules are not always reproducible
(but see Endicott et al. 2003), and so we chose not to use these
markers extensively for assigning each haplotype to a haplogroup.
However, the 14766 MseI restriction cuts were confirmed by sequencing
of multiple clones in the region of interest. The results were
unambiguous across replicates and make phylogenetic sense (with two
exceptions; in these individuals, this position was left undefined)
(table 1). Therefore, typing of the 14766 MseI site both clarified and
further validated the results of HVR-I sequencing.

In the reduced median network based on HVR-I sequences and on the
14766 MseI polymorphism (fig. 2), several clusters are evident. Two
lineages, characterized by substitutions 193-219 and 356,
respectively, have a rather high internal diversity. The former
substitutions are documented in Cornwall, England (but in association
with 186-260-362), and the latter along the eastern and central
Mediterranean shores, including Tuscany, with some derivatives in
northern Europe. The only lineage containing a transversion, 095G-189,
also occurs in Turkey, while the lineage with the 129 substitution is
present in the eastern Mediterranean region and in northern Europe.
The lineages with 126 or 126-362 presumably belong to the pre-HV
haplogroup and have been observed in southeastern Europe and in the
Levant.
Figure 2 Figure 2
Reduced median network of the haplotypes identified among the
Etruscans. Haplotype 5AM is the Cambridge reference sequence (CRS).
Circle sizes are proportional to frequencies. Transitions are numbered
relative to the CRS ( 16,000); 095G is the (more ...)

Six Etruscan sequences show the 126-193 motif. In modern individuals,
these substitutions occur in lineages attributed to the J2, and
occasionally T, haplogroups, accompanied by substitutions at 069 or
294, respectively, which were not observed in these six sequences.
Therefore, on the basis of the HVR-I motifs, these sequences could
belong to either the HV or to the JT haplogroups. However, in all of
them, the MseI site was present at 14766, which rules out the
hypothesis that they could belong to the HV haplogroup. Considering
that postmortem changes lead to false positives (i.e., substitutions
observed in the ancient sample that did not exist in the living
individual) at 069 or 294 but are not known to generate false
negatives there (Gilbert et al. 2003), we see no compelling reason to
imagine that these results are due to postmortem damage. In addition,
these sequences were determined in multiple clones for each
individual, which makes a sequencing error unlikely. Therefore,
despite the absence of substitutions at 069 or 294, the sequences with
the 126-193 motif appear to belong to the JT haplogroup.

Internal genetic diversity within the Etruscans (gene diversity 0.98 ±
0.01; mean number of pairwise sequence differences 3.90 ± 2.02) is
close to the average in the database (0.97 and 4.68, respectively).
AMOVA showed no heterogeneity among Etruscan sites (1.11% of the total
[NS]), whereas differences are significant among contemporary Italian
populations (1.72% of the total, P<.0001); in addition, we could not
demonstrate any significant differences among time periods (7th-6th
centuries vs. 5th-4th vs. 3rd-2nd). None of these tests suggests that
the Etruscans are more of a cultural assemblage than a biological
population.

Only two Etruscan haplotypes (5AM and 6AM, carried by 13.7% of the
individuals) occur in a sample of modern Tuscans who were selected to
represent inhabitants of former Etruria (Francalacci et al. 1996). The
average value in comparisons of pairs of modern European populations
is 27.9% ± 12.0%, showing that the genetic resemblance between the
Etruscans and their modern counterparts is much less than observed
between random European populations with no special evolutionary ties.
Allele sharing is higher not only with the Turks (four haplotypes in
common) but also with other, presumably unrelated, populations, such
as the Cornish or the Germans (five and seven haplotypes in common,
respectively). However, allele sharing may not be the best statistic
summarizing the evolutionary relationships between the Etruscans and
modern populations. Indeed, many haplotypes that so far have been
observed only in the Etruscans differ by just one substitution from
haplotypes that are present, or even common, among modern Europeans.
Therefore, pairwise sequence differences, as well as genetic distances
between populations, are more informative. The shortest genetic
distances between the Etruscan and modern populations are with Tuscans
(FST=0.036; P=.0017) and Turks (FST=0.037; P=.0001); values of
FST<0.050 were also observed for other populations of the
Mediterranean shores and for the Cornish (fig. 3).
Figure 3 Figure 3
Pairwise genetic distances (FST × 1,000) and corrected mean pairwise
sequence differences between the Etruscans and modern populations of
Europe and of the Mediterranean shores. The values referring to the
current population of Tuscany are underlined. (more ...)

The mean pairwise sequence differences between the Etruscans and other
European populations range from 3.79 to 6.26 substitutions, with a
global average of 4.64. The Tuscans differ from the Etruscans by 4.65
substitutions-that is, almost exactly the value that would be expected
with a random population from the database. However, these values are
also affected by the internal diversity of both samples being
compared. If we subtract the internal diversity, thus obtaining Nei's
(1987) dAB distance, the genetic distance between Etruscans and
Tuscans becomes the shortest, with other southern and eastern
Mediterranean populations also showing relatively close genetic
relationships (fig. 3). In the MDS plot (fig. 4), the Etruscans fall
out of an unstructured cluster comprising most European and Caucasus
populations, including the Turks.

To better compare the Etruscan gene pool with those of contemporary
Italy, we treated these populations as hybrids among four potential
parental populations, from the four corners of the area considered in
this study (table 2). The likely contributions of each parental
population, or admixture coefficients, are similar for the three
modern Italian populations, but Etruscans differ in two aspects: they
show closer relationships both to North Africans and to Turks than any
contemporary population. In particular, the Turkish component in their
gene pool appears three times as large as in the other populations.
These admixture estimates are not to be taken at their face value, for
numerous assumptions underlie their estimation. Here they only serve
to show that, with respect to modern Italian gene pools, the Etruscan
one contains an excess of haplotypes suggesting evolutionary ties with
the populations of the southern and eastern Mediterranean shores.
Table 2 Table 2
Estimates of Admixture Rates in the Etruscan and in Modern Italian
Populations[Note]

Discussion

The first question we wanted to address is whether there is any
evidence that the Etruscans were not a biological population, but
rather an assemblage of biologically heterogeneous people who shared
many cultural traits. Neither their internal genetic diversity, which
is less than in comparable modern populations, nor the insignificant
heterogeneity among Etruscan sites or time periods supports that view.
In agreement with dental evidence (Moggi-Cecchi et al. 1997), the
genetic data of this study suggest that either the people whom we call
"Etruscans" shared a set of ancestors (and therefore can be considered
a biological population as much as can current European populations)
or they were mixed with people whose mitochondrial features did not
differ from theirs.

As for the second question, which concerned the genetic relationships
between the Etruscans and modern populations, various tests show that
the Tuscans are the Etruscans' closest neighbors in terms of genetic
distances. Despite that broad similarity, however, Etruscans and
Tuscans share only two haplotypes. This finding is difficult to
interpret in the absence of data on any other European population of
the preclassical period. One possible interpretation is that all or
most European populations of that time period were as different from
their modern counterparts as the Etruscans appear to be. This would
imply either extensive gene flow or a high rate of extinction of
mitochondrial haplotypes, both processes causing a drastic change of
the mitochondrial pool in the last 2,500 years. More importantly, a
result of that kind would force us to reconsider the universally held
assumption that patterns in the DNA of modern individuals reflect the
evolutionary processes affecting their prehistoric ancestors (see,
e.g., Piazza et al. 1988; Sokal 1991; von Haeseler et al. 1995;
Richards et al. 2000, 2002; Semino et al. 2000). Alternatively, should
other ancient populations prove similar to comparable modern ones, one
should conclude that the Etruscans' mitochondrial sequences underwent
extinction at a particularly high rate and look for an explanation for
that. Until more ancient DNA data become available, both scenarios
will remain possible, although we favor the latter.

Social structure may have affected these results. All skeletons we
typed were found in tombs containing artifacts that could be
attributed with confidence to the Etruscan culture. Those tombs
typically belong the social elites (Barker and Rasmussen 1998), and so
the individuals we studied may represent a specific social group, the
upper classes. We do not know whether that group differed genetically
from the rest of the population, which might be the case when a
foreign elite imposes its rule, and often its language, over a region
(Renfrew 1989). If the upper class had indeed somewhat distinct DNAs,
our results could mean that this elite class became largely extinct,
while the rest of the population, whose DNA we do not know, may well
have contributed to the modern gene pool of Tuscany. This would be the
likely effect of a process of assimilation, from which the social
elites were excluded, more or less deliberately.

To summarize, only a few Etruscan sequences find an exact match in the
modern database, but all belong to lineages that are still present in
Europe. Some genetic affinities with modern people from western Europe
reflect the sharing of lineages (5AM, 6AM, and 19M) that are
widespread over the whole continent, and that therefore do not seem to
point to any migrational contact but rather to a common origin of
various European gene pools. On the contrary, the similarity between
the Etruscan and Turkish gene pools may indeed reflect some degree of
gene flow. Commercial exchanges are documented between the Etruscan
harbours and Asia Minor (Tykot 1994) and trading is often accompanied
by interbreeding, ultimately leading to detectable levels of genetic
affinity (see Relethford and Crawford 1995). Thus, the present study
suggests that gene flow from the eastern (and possibly southern)
Mediterranean shores, not necessarily from Lydia as proposed by
Herodotus, left a mark in the Etruscan gene pool, above and beyond
what is observed in contemporary Italy.

The limited genealogical continuity between the Etruscans (be they
representative of the upper class or of the entire population) and
their modern counterparts of Tuscany calls for an explanation. An
approximate estimate of the Etruscan population size in the 6th
century b.c. is probably somewhere between 150,000 and 250,000 women
(Rasmussen 2004). In the future, we plan to quantify the probability
of extinction of all mitochondrial haplotypes but two in a subdivided
population of that size, by simulating both neutral evolution and a
disadvantage representing ethnic discrimination after the Roman
assimilation. We shall also ask whether reasonable rates of gene flow
for 2,500 years can cause a dramatic replacement of mitochondrial
lineages in a population of that size. But a clearer picture is likely
to emerge only when genetic data on other European populations of the
same age become available for comparison.

For the time being, however, this is the first large-scale study of a
pre-Roman European population in which all the strictest criteria for
the validation of ancient DNA sequences have been followed. Within the
limits imposed by the sample size (although our sample is large for
ancient DNA studies, compared with 12 samples in Endicott et al. 2003,
19 samples in Lalueza-Fox et al. 2001, and 17 samples in Lalueza-Fox
et al. 2003), the Etruscan sites appear to have rather homogeneous
genetic characteristics. Their mitochondrial haplotypes are very
similar, but rarely identical, to those commonly observed in
contemporary Italy and suggest that the links between the Etruscans
and eastern Mediterranean region were in part associated with genetic,
and not only cultural, exchanges.

Acknowledgments

This study was supported by funds from the Universities of Ferrara and
Florence, the Italian Ministry of Universities (MIUR [FISR and COFIN
2003]), the Fondazione Cassa di Risparmio di Ferrara, and the
Fondazione Dino Terra. We are grateful to Robert Tykot, Vincent
Macaulay, Peter Forster, Jaume Bertranpetit, Claudio Bravi, George van
Driem, Graeme Barker, and Tom Rasmussen, for several suggestions and
for critical reading of a preliminary manuscript; to S. Sanna and S.
Conti, for their technical contribution; and to E. Pacciani, who
provided us with the Magliano, Marsiliana, and Tarquinia samples.

.