AMERICAN JOURNAL OF HUMAN BIOLOGY 13:576–589 (2001)

Mitochondrial DNA Sequence Analysis in Sicily

G. VONA,1* M.E. GHIANI,1 C.M. CALÒ,1 L. VACCA,1 M. MEMMÌ,2 AND L. VARESI2
1
Department of Experimental Biology, Section of Anthropological Sciences, University of
Cagliari, Monserrato, Italy
2
Faculty of Sciences and Technologies, University of Corsica, Corte, France

ABSTRACT This study reports data on the sequences of the first hypervariable segment of a
sample of the Sicilian population from Alia (Palermo, Italy). The results show the presence of 32
different haplotypes in the 49 individuals examined. The average number of pairwise nucleotide
differences was 4.04, i.e., 1.17% per nucleotide. The distribution of the nucleotide differences matches
the theoretical distribution and indicates only one major episode of expansion that occurred between
20,732 and 59,691 years ago, between the Middle Paleolithic and Upper Paleolithic. Compared with
the other populations, parameters of the Sicilian sample lie in an intermediate position between the
eastern and western Mediterranean populations. This is due to numerous contacts that Sicily has had
with the Mediterranean area since prehistoric times. At the same time, the singularity of some of the
haplotypes present in the sample studied indicates the persistence of some characteristics caused by
genetic drift and isolation that the population has endured in the course of its history. Am. J. Hum.
Biol. 13:576–589, 2001. © 2001 Wiley-Liss, Inc.

Mitochondrial DNA has been fully se-
quenced by Anderson et al. (1981). A hap-
loid DNA of 16,569 base pairs contains in-
formation for the codification of some
proteins and various types of ribosomal and
transfer RNA specific of mitochondrial me-
tabolism. The mtDNA molecule has a non-
coding part, called a D-loop, which contains
the origin of the replication and information
that controls it. One of the fundamental
characteristics of mtDNA is that it has a
mutation rate roughly 10 times higher than
the rate in nuclear DNA so that short-term
evolving phenomena can be analyzed (Fer-
ris et al., 1981; Cann et al., 1984; Vigilant et
al., 1989; Ward et al., 1991). This mutation
level is substantiated by an accumulation of
mutations transmitted through the mater-
nal line, since another basic property of
mtDNA is that it is inherited only mater-
nally. Mutations are most frequently shown
by base substitutions, while deletions and
insertions rarely appear. Transitions are al-
ways more numerous than transversions
(Brown et al., 1979).
Many analyses show a preference for seg-
ment I of the non-codifying area of mtDNA,
which seems to have the highest evolution
rate of the molecule. This is the area that
was analyzed in a sample from Sicily (Italy)
in the context of populations originating
mainly in the Mediterranean region.
HISTORICAL BACKGROUND
The most ancient traces of human pres-
ence in Sicily, the largest island in the west-
ern Mediterranean, date back to Paleolithic,
Neolithic, and the Iron Age and show influ-
ences of both the western and eastern areas
of the Mediterranean. This suggests that
even in prehistoric times Sicily was an im-
portant crossroads of culture and trade be-
tween the eastern and western Mediterra-
nean.
During the great Indo-European migra-
tions, the eastern part of Sicily was occupied
by the Siculi (1,200 B.C.), who pushed the
resident populations, the Sicans and the
Elimi, toward the central and western parts
of the island. The 8th century B.C. saw the
beginning of one the most intense coloniza-
tions: colonization by the Greeks affected
most parts of the coastal area, and the ex-
isting Phoenician presence was limited to
the northwestern area. Over the next few
centuries, Sicily became completely “Hellen-
ized.”
Another important time for Sicily was the
period under Roman rule. Confiscated lands
were given to veteran Roman soldiers, and
great estates were established where large
Contract grant sponsor: Ministero dell’Università e Ricerca
Scientifica e Tecnologica (MURST, Italy), Programmi Ricerca
Scientifica di Rilevante Interesse Nazionale (ex-40%); Contract
grant number: 9805557360-003.
*Correspondence to: Prof. Giuseppe Vona, Department of Ex-
perimental Biology, Section of Anthropological Sciences, Cit-
tadella Universitaria, S.S. 554, Km. 4,500, 09042 Monserrato
(Ca), Italy. E-mail: vona@vaxca1.unica.it
Received 26 May 2000; Revision received 3 January 2001; Ac-
cepted 7 January 2001

© 2001 Wiley-Liss, Inc.

PROD #M20045R2
 mtDNA IN SICILY
numbers of slaves were used to work the
fields. Both the landowners and slaves came
from all corners of the Roman domain. The
Romans, contrary to the Greeks, pushed
their occupation deeply inland, transform-
ing the island also ecologically. Intense de-
forestation took place, and the monoculture
of wheat started. Sicily in effect became the
granary of Rome.
After the passage of several other groups,
e.g., the Vandals from North Africa and the
Byzantines, Arab domination began in 827
A.D. with the landing in Gela of 10,000
Muslims from North Africa and Spain. This
transformed Sicily into the center of a wide
confederation of Muslim nations.
In 11th century A.D., Sicily was occupied
by the Normans and experienced an ex-
traordinary immigration of numerous
groups, particularly of Italians, French, and
Longobards. Spanish domination began in
1282, and Sicily went through one of the
most disastrous periods of its history. Span-
ish domination was aggravated by the
spread of banditry and the presence of a
large number of mercenaries. Return to
monoculture linked to deforestation and a
series of terrible events such as famine,
earthquakes, and outbreaks of plague and
cholera forced many of Sicilians to emigrate.
Before becoming part of the realm of Italy in
1860 Sicily was the stage for many wars,
which brought powerful armies of foreign
soldiers to the island. For example, the res-
toration of the Borbonnes (1820) cost Sicily
an invasion of 12,000 Austrian soldiers.
Sicily’s history over the last century was
characterized by a series of formidable and
irresolute problems, leading to a high level
of emigration which peaked in the years af-
ter the two World Wars; 1,500,000 Sicilians
abandoned the island after the First World
War.
Many of these events had important de-
mographic implications, influencing the bio-
logic history of Sicily and shaping its genetic
structure (Vona et al., 2000). The effects of
one of the events, which dramatically af-
fected Sicily, was the discovery in 1995 of
about 300 skeletons of individuals who had
died of cholera in 1837 A.D. in the small
commune of Alia, in the eastern part of the
province of Palermo. Situated 800 meters
above sea level on the southwestern slopes
of the Madonie mountain chain, about 80
km from Palermo (Fig. 1), Alia today has a
577
population of about 4,000 inhabitants. In
1837 Alia, along with many other Sicilian
villages, was hit by an epidemic of cholera
and the population was reduced by at least
10%. Given the high number of deaths, the
bodies were mostly buried in a cave on the
outskirts of the village rather than in the
traditional cemetery. At the end of the epi-
demic, entrance to the cave was closed and
only in 1995 did an excavation bring the re-
mains to light. This study reports the re-
sults of the analysis of the sequences of the
control segment I of mitochondrial DNA
taken from a sample of the present-day
population of from Alia, Sicily.
MATERIALS AND METHODS
Population
The sample includes 49 unrelated, appar-
ently healthy, adults of both sexes from Alia
whose families were born and lived in the
same village for at least three generations.
With prior informed consent, a blood sample
was collected from each individual by veni-
puncture at the Municipal Outpatients
Clinic at Alia. The sample was put in a ster-
ile test tube containing EDTA, stored at
−20°C, and then transported to the Depart-
ment of Experimental Biology at Cagliari
University.
Amplification and sequencing of DNA
The DNA was subsequently extracted
from the whole blood using the QIAamp
Blood Kitt (QIAGEN). The DNA was ampli-
fied starting from 10 ml of the final lysatum.
The hypervariable segment I of the D-loop
was amplified by polymerase chain reaction.
The primer sequences were as follows:
L15996, 58-CTC-CAC-CAT-TAG-CAC-CCA-
AAG-C-38, and H16401, 58-TGA-TTT-CAC-
GGA-GGA-TGG-TG-38, thus producing an
amplified fragment of 425 bases.
PCR conditions were as follows: 15 min at
95°C in 2.5 U of HotStar Taq Polymeraset
for a total reaction volume of 50 ml (10 mM
each of dNTP, 25 mM di MgCl2, 15 ml of the
primer couples, 10 ml of DNA of the sample
to be amplified); 35 cycles at 94°C for 45 sec,
56°C for 1 min and 74°C for 1 min. Chain
elongation was continued after the last cycle
for 10 min at 72°C.
The products of the amplification were vi-
sualized by electrophoresis on agarose gels
containing 2% ethidium bromide. The prod-
ucts of the PCR were then purified in Cen-
578
Fig. 1. Geographical localization of the studied populations. 1 4 Sicily; 2 4 Sardinia; 3 4 Corsica; 4 4 Tuscany;
5 4 Basques; 6 4 Spain; 7 4 Berbers; 8 4 Turkey; 9 4 Middle East; 10 4 Asia; 11 4 Africa.
tricon-100t tubes (Perkin-Elmer). Subse-
quently, 10 ml of amplified and purified
mtDNA were sequenced by an automated
sequencer ABI 373. Each sample was se-
quenced with the H and L primers used for
the PCR. Some of the samples were se-
quenced twice, and some were sequenced
with the primer H or L. The results were
compared in order to avoid possible ambigu-
ities in readings. The sequences thus ob-
tained covered a total of 388 bases, from
base 16,023 to base 16,410, and were com-
pared with Anderson’s (1981) reference se-
quence through the CLUSTAL V program.
Statistical analysis
Internal genetic diversity. The statistical
analyses were carried out using the pack-
ages PHYLIP 3.51 C (Felsenstein, 1989)
and ARLEQUIN 1.1 (Schneider et al., 1997).
The phylogenetic analysis of the sequences
was performed in accordance with Kimura’s
(1980) two-parameter model. The tree rela-
G. VONA ET AL.
tive to the 49 sequences was drawn up after
100 permutations, according to the neigh-
bor-joining method (Saitou and Nei, 1987)
and the difference between clusters was
tested through the transformed pairwise
Fst method (Reynolds et al., 1983; Slatkin,
1995). The expected number of frequencies
and the effective size of the population were
estimated under the infinite allele model by
means of the number of individuals and se-
quences (Hartl and Clark, 1989). Variability
within the sample examined was analyzed
by Shannon’s diversity index in its stan-
dardized form H8 (Magurran, 1988).
Dating of expansion. The pairwise nucleo-
tide difference distribution was fitted to the
Rogers and Harpending (1992) model, and
the standard errors were calculated by
1,000 bootstrap iterations. The parameters
t and u were then obtained following the
two-parameter model of Harpending et al.
(1993). Expansion time was estimated in ac-
cordance with the model of Roger and
 mtDNA IN SICILY
Harpending (1992) through the parameter t
using mutation level calculations taken
from reported works (Vigilant et al., 1991;
Ward et al., 1991) and a generation period of
20 years.
Comparison between populations. The se-
quences of the Sicilian sample were com-
pared with those of the following popula-
tions: Basque (Bertranpetit et al., 1995),
Berber (Corte-Real et al., 1996), Corsican
(Varesi et al., 2000), Middle East (Di Rienzo
and Wilson, 1991), Sardinian (Di Rienzo
and Wilson, 1991), Spanish (Corte-Real et
al., 1996), Turkish (Comas et al., 1996), Tus-
can (Francalacci et al., 1996), African (Vigi-
lant et al., 1989), and Asian (Stoneking,
1993). A neighbor-joining tree (Saitou and
Nei, 1987) was constructed using the ge-
netic distances computed through the
method proposed by Rao (1982).
RESULTS
Sequence diversity
Table 1 shows the variable sites in the
control region of mtDNA compared with the
reference sequence. The 49 individuals
show 32 different sequences with 36 vari-
able sites and 108 mutations. With the sole
exception of transversion C→A, the substi-
tutions are all transitions. The transition/
transversion ratio is, therefore, 36:1; much
higher than that reported by Tamura and
Nei (1993) for human populations. Most of
the transitions concern the pyrimidines and
are equally distributed between the two pos-
sible types. Only 6 individuals have a se-
quence identical to that of Anderson et al.
(1981).
The highest number of variable sites pre-
sent in any one individual (SA23) is 8, while
individual SA34 showed 7. The site with the
highest number of substitutions is 16,126
with a T→C transition in 12 individuals
(24.49%), followed by sites 16,311 and
16,189 with the same type of transition in 8
(16.33%) and 7 (14.29%) individuals, respec-
tively.
Under the assumption of the infinite al-
lele model, using the number of individuals
and the number of sequences, it was pos-
sible to calculate the expected number of se-
quences (Hartl and Clark, 1989). The value
taken of u in the sample of 49 individuals
and 32 different sequences is 38.93, which
corresponds to an estimate of 128.33 se-
quences per 1,000 individuals. This value of
u is much closer to that calculated in Corsi-
579
cans (38.52) (Varesi et al., 2000) and in
Basques (27.58) (Bertranpetit et al., 1995)
than that shown by Tuscans (99.38) (Fran-
calacci et al., 1996).
Phylogenetic analysis
Sequence variability as expressed
through the index of diversity H8 (Table 2) is
0.961. If the Asian sample is excluded, this
value is one of the highest reported in Table
2. Values vary between the 0.891 in Berbers
and 0.991 in the Middle East.
The average number of pairwise differ-
ences in nucleotides between all possible
couples of individuals is 4.040 ± 0.845, i.e.,
1.17% per nucleotide. The number of steps
needed to construct the parsimony tree is
1.122.
Phylogenetic analysis of the sequences
carried out with the DNADIST program of
PHYLIP 3.51C and using the method pro-
posed by Kimura (1980) allowed the con-
struction of a neighbor-joining tree from the
distances (Saitou and Nei, 1987). The tree
shown in Figure 2 shows how the sequences
split into three well-defined clusters. The
sequences that do not show variations from
the Anderson et al. (1981) sequence are in
cluster B together with other sequences,
which have a lower number of transitions
and with the only sequence carrying a
transversion. From the Fst calculation,
clusters A and C appear the most similar
(Fst 4 0.053), whereas clusters B and C re-
sult the most differentiated (Fst 4 0.1256).
Nevertheless, the association of the se-
quences in the different clusters seems
quite random, and analysis of the variance
shows that there is no significant heteroge-
neity between the three clusters (F 4 0,089;
df 4 2; 46).
In the Alia sample, the number of steps,
which is influenced by the number of differ-
ent sequences present in a population, has
one of the lowest values among those re-
ported for the populations in Table 2. This
suggests that, from a phylogenetic point of
view, the sequences obtained are closely cor-
related.
Nucleotide difference distribution
Analysis of the nucleotide difference dis-
tribution shows that the distribution curve
(Fig. 3) is bell-shaped, similar to that de-
scribed for other populations, with only one
peak at 4.04 (variance 4 4.943) (Table 2).
The distribution matches the theoretical
 TABLE 1. Variable sites of the control region found in Sicilian individualsa
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
0 0 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3
SICILY 6 9 1 2 2 6 6 6 7 8 8 9 9 0 2 2 3 5 6 6 6 7 7 7 8 9 9 9 9 9 0 1 1 2 5 6
N 4 49 9 3 4 6 9 3 8 9 2 6 9 2 3 9 3 4 5 6 1 4 5 0 4 8 9 2 4 5 6 8 4 1 9 0 5 2
AND C T C T G A C C T C T C C T C T A C C C A C G C A C C C C T T T G C C T
SA15, SA21, SA64 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C - - - - -
SA32 - - - - - - - - - - - - - - - C - - - - - - - - - - - - - - - C - - - -
SA35, SA19 - - - - - - - - - - - - - - - - - - - - - - A - - - - - - - - - - - - -
SA37 - - - - - - - - - - - - - - - - - T - - - T - - - - - - - - - - - - - -
SA38 T C - C - - - - - - - - - - - - - - - - - - - T - - - - - - - - - - - -
SA39 - - - - - - - - - - - - - C - - - - - - - - - - - - - - - - - - - - T -
SA40, SA26 - - - C - G - - - T C - - - - - - - - - - - - - - - T - - - - - - - - -
SA41 - - T - A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
SA42, SA9 - C - - - - - - - - - - - - - - - - - - G - - - - - - - - - - - - - - -
SA43, SA52, SA11, - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
SA13, SA18, SA27
SA45, SA46, SA49 T - - C - - - - - - - - - - - - - - - - - - - - - - - - - - - - A - - -
SA51, SA14 - - - - - - - - - - - - - - - - - - - - - - - T - - - - - - - - - - - -
SA53 - - - - - - - A - - - T - - - - G - - - - T - - - - - - - - - - - - - -
SA57 - - - - - - - - - - C - - - T - - - - - - - - - - - - - - - - - - - - -
SA58 T - - C - - - - - - C - - - - - - - - - - - - - - - - - - - - - - - - -
SA62 - - - C - - - - - - - - - - - - - - - - - - - - - T T - T - C - - - - -
SA67, SA1, SA7 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C - - - -
SA25, SA6 - - - - - - - - - - C - - - - - - - - - - - - - - - - - - - - - - - - -
SA44 - - - - - - - - - - C - - - T - - - - - - - - T - - - - - - - - - - - -
SA10 - - - - - - - - - - - - - - T - - - - - - - - - - T - - - - - - A - - -
SA22 - - - - - - - - - - - - - - T - - - - - - - - - - - - - - - - - - T - -
SA24 - - - - - - - T - - - - - - - - - - - - - - - - - - - - - - - - - - - C
SA28 - - - - - - - - C - - - - - - - - - - - - - - - - - - - - - C - - - - -
SA2 - - - - A - - - C - - - - - T - - - - - - - - - - - - - - - - C - - - -
SA33, SA3 - - - - - - - - - - - - - - - - - - - - - - - T - - - - - - - C - - - -
SA34 - - - C - - T - - - - - - - - - - - - T - - - - - T - T - - - - A - - C
SA36 T - - C - - - - - - - - T - - - - - - - - - - - - - - - - - - - - - - -
SA4 - - - C - - - - - - - - - - - - - - - - - - - - - - T - T - - - - - - -
SA5 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C
SA8 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C - C - - - -
SA23 - C - C - - T - - - - - - - - - - - - T - - - - - T - T - - - - A - - C
SA12 - - - - - - - - - - - T - - - - - - T - - - - - G - - - - - - - - - - -
a
Sequences are given in comparison to the references sequence of Anderson (1981). Only nucleotides differeing from the reference are shown.
 mtDNA IN SICILY 581

TABLE 2. Sequences divergence in Sicilian and in the and 16,223 (10.20%), both with a C→T tran-
comparison populationsa sition, have a frequency in Sicily midway
N k a b d H8 between the east and west. The two sites
Sicily 49 32 37 4.040 ± 0.845 1.122 0.961 also appear to vary with a decreasing gra-
Sardinia 69 46 52 4.221 ± 0.558 1.826 0.930 dient from east to west. For 16,069 the Si-
Corsica 47 31 40 3.712 ± 0.822 1.774 0.903 cilian frequency is closer to that of the Tus-
Tuscany 52 40 54 5.032 ± 0.543 2.025 0.899
Basques 45 27 32 3.152 ± 0.721 1.481 0.912 cans and Turks than to those of the other
Spain 58 52 62 5.083 ± 0.994 1.846 0.980 western populations, where the highest fre-
Berbers 86 30 37 4.782 ± 0.872 1.700 0.891 quency (6.90%) is found in the Spanish. The
Turkey 45 38 56 5.386 ± 0.728 2.001 0.987 reverse is true for the substitution fre-
Middle East 42 38 59 7.083 ± 1.228 2.211 0.991
Asia 23 23 59 8.283 ± 1.011 3.261 1.000 quency of site 16,223, which, in Sicily, is of
Africa 22 12 34 3.194 ± 0.898 4.500 0.946 the western type. Substitution at site
a
N 4 number of individuals; k 4 number of different sequences;
16,261, which has the highest frequency in
a 4 number of variable nucleotides; b 4 mean nucleotide pair- the Middle East (19.51%) and is absent in
wise differences; d 4 mean number of steps per sequence in a Corsica and Sardinia, barely reaches 2.04%
maximum parsimony tree; H8 4 index of Shannon.
in Sicily.
The comparison of pairwise differences in
distribution based on the model of Rogers Alia and other populations (Fig. 4) shows
and Harpending (1992) (x2 4 18.421; df 4 that the peak in Alia falls to the left side,
11; P 4 0.072). This suggests that only one having one of the lowest average values of
major episode of population expansion in pairwise differences among those shown in
the past which would have increased an ini- Table 2. This position indicates that Alia’s
tial population of roughly 300–900 to some- expansion time is one of the most recent
thing between 12,000 and 35,000 individu- compared to the comparison populations.
als (Table 3). The Sicilian sample was compared with
the other populations by the genetic dis-
Dating expansion tances of Rao (1982). The genetic tree in Fig-
The estimation of dating expansion of the ure 5, drawn from the matrix of distances in
population was calculated by the method of Table 5, is of the neighbor-joining type ob-
Rogers and Harpending (1992) and tained with 100 iterations. The two samples
Harpending et al. (1993). From the values from Asia and Africa, which lie in two sepa-
obtained for the population of Alia for the rate branches, are clearly differentiated
parameters t 4 3.089, q0 4 0.951, q1 4 from the Euro-Mediterranean populations,
36.401, and the different mutation rates re- which are all found in another branch. This
ported in literature, expansion would have stretches from the populations of the Middle
occurred between 20,732 and 59,691 years East on one side, near the fork that sepa-
ago with an intermediate value of 39,679 rates Asia and Africa, to the Basques and
years (Table 3). Corsicans on the opposite side. The sample
from Sicily lies between Tuscany and
Comparison with other populations Sardinia but closer to the former.
All of the variable sites found in the Alia Table 6 shows the haplotype frequencies
population have already been described in that emerge from the sequencing and that
other populations. Therefore, the Sicilian Sicily has in common with the other com-
population has no specific substitutions of parison populations. While there are no
its own. common haplotypes between Sicily and the
The substitution frequency in site 16,126 African sample, there is one common se-
obtained for Sicily lies midway between the quence with Asia (SA32). The most frequent
eastern and western comparison popula- haplotype in Sicily is sequence SA43, which
tions (Table 4). On the contrary, variability is identical to the reference sequence of
of site 16,311 does not appear to be distrib- Anderson et al. (1981), with an intermediate
uted according to a gradient as indicated by frequency to those of the comparison popu-
Comas et al. (1996). The lowest frequency is lations. The lowest values are shown by
found among the Basques (11.11%) and the eastern populations and the highest by the
highest among the Tuscans (21.15%). For Corsicans and Basques. This haplotype was
site 16,311 Sicily has a frequency similar to not seen in the Middle East sample.
the Berbers, Sardinians, and Turks. An- Other than the Sicilian sample, haplo-
other two mutated sites, 16,069 (12.24%) types of the sequences SA33 and SA25 were
582 G. VONA ET AL.
Fig. 2. Neighbor-joining tree of the sequences in the Sicilian samples.
only found in the Basques, while haplotypes
SA36 and SA37 are also present in the
Spanish. The haplotype represented by se-
quence SA67, absent in the Middle East and
Tuscan samples, has its highest frequency
in the Berbers, with the Sicilian frequency
located between those of the Berbers and
the western populations. For sequence
SA40, which was not found in Tuscany,
Sardinia, Corsica, or the Basques, Sicily has
a frequency very similar to that of the east-
ern countries and the Berbers. Sequences
SA53 and SA58 seem to be exclusive to the
Sardinians and Sicilians, while haplotype
SA2 is shared only with the Tuscans. The
haplotypes that characterize the remaining
(44.9%) sequences of Alia are not found in
the comparison populations.
DISCUSSION
The vast literature concerning mtDNA
highlights its importance in the study of
evolutionary processes in humans. Se-
quence variation analysis of a control region
seems to be useful and highly effective in
the study of a population’s history, not only
on a global level but also regionally.
Many genetic studies have been done on
the Sicilian population; they have concerned
mainly proteins, and only recently has the
molecular point of view been discussed
(Vona et al., 2000).
The results suggest several genetic par-
ticularities of the Sicilian population de-
termined by complex cultural and genetic
relations that have marked the island’s
 mtDNA IN SICILY 583

Fig. 3. Pairwise differences distribution of Sicilians and other comparison populations.

evolutionary history. This study shows the
sequences of the first hypervariable seg-
ment of mtDNA in a sample of the Sicilian
population from Alia in the province of Pal-
ermo (Italy).
The results for Alia reveal several fea-
tures of the region of the first hypervariable
segment of mtDNA, some of which have
been reported in other studies. A prevalence
of transitions over transversions is noted
but in a much higher proportion than found
in other populations. There is also a marked
584 G. VONA ET AL.

TABLE 3. Demographic parameter estimates for the ancient population, especially the expan-
Sicilian population according to the model of Roger sions and the periods during which they oc-
and Harpending (1992) for different proposed
mutation ratesa curred. Rogers and Harpending (1992) dem-
onstrated that the rapid growth of a
Times of
divergence population is signaled by a smooth mis-
m N0 N1 (years) match distribution that has only one peak.
1.490 × 10−3 319 12,215 20,732 The mismatch distribution in Alia sample
(Ward et al., 1991) suggests that the main episode of expansion
7.785 × 10−4 610 23,380 39,679 took place between 20,732 and 59,691 years
(Vigilant et al., 1991) ago, with an intermediate indication of
5.175 × 10−4 919 35,170 59,691
(Vigilant et al., 1991) 39,679 years ago. The comparison popula-
a
tions also show bell-shaped distributions,
N0 and N1 are the effective population sizes before and after the
expansion episode. all denoting a series of expansions that took
place later. Expansions occurred first in the
east and then in the west. This is consistent
TABLE 4. Most frequent substitution in the Sicilian
sample in comparison with other populations
with an east to west migration.
The population with the oldest expansion
Site position would be the Middle East, roughly between
16,069 16,126 16,223 16,261 16,311 47,000 and 135,000 years ago. On the con-
Sicily 12.24 24.49 10.20 2.04 16.33 trary, the most recent expansion would
Sardinia 5.80 17.39 7.25 0.00 17.39 have taken place among the Basques, about
Corsica 2.13 8.51 8.51 0.00 19.15
Tuscany 13.46 23.08 13.46 5.77 21.15 14,000–41,000 years ago. The observations
Basques 4.44 6.67 4.44 2.22 11.11 noted from the various analyses seem to be
Spain 6.90 18.97 10.34 3.45 12.07 consistent with an east–west migration (Co-
Berbers 3.50 8.20 17.60 0.00 16.50 mas et al., 1996; Francalacci et al., 1996).
Turkey 15.56 26.67 28.89 8.89 17.78
Middle East 19.51 46.34 26.83 19.51 14.63 Barbujani et al. (1995), in a study of
Asia 0.00 9.52 47.62 9.52 19.05 mtDNA in Italy, suggested that the time of
Africa 0.00 0.00 86.67 0.00 100.00 expansion of the Italian populations, includ-
ing Sicily, was somewhere between 8,200
and 20,525 years ago, and the size of the
predominance of pyrimidine transitions population was estimated between 1,160
over purine. The analysis shows that Sicil- and 2,326 females. The authors also pro-
ians, as far as the sequencing of mtDNA is posed two possible expansions: one after the
concerned, have some characteristics that maximum of the last glaciation in the Upper
can be considered midway among those Paleolithic and the other at the beginning of
found in comparison populations. This is the Neolithic. The data in the present study
particularly evident in the cline of the fre- only partly agree with Barbujani et al.
quencies of some substitutions, on the aver- (1995), both in terms of the time of expan-
age, and in the distribution of the nucleotide sion and the increase of population num-
pairwise differences. The position taken in bers. As indicated by the calculated data
the genetic tree of the populations also leads and in Figure 4, Sicily seems to be among
to the same conclusion. The tree shows ap- the populations with a more recent expan-
proximately an east to west gradient with sion, but this unique event falls during the
the Middle East at one extreme and the Middle and Upper Paleolithic, and its tem-
Basques and Corsicans at the other. poral position seems to agree with archaeo-
The mutation at site 16,126, which is logical dates (Chilardi et al., 1996). Further,
most frequent in the Sicilian sample, shows the estimated increase in population is
a decreasing east–west variation of frequen- much more substantial than that indicated
cies with extreme values in the Middle East by Barbujani et al. (1995).
(46.3%) and in the Basques (6.7%). The fre- The first presence of modern populations
quencies of the substitutions relative to is placed in the Near East from where they
sites 16,069 and 16,223 also appear to have expanded toward Europe (Stringer, 1989;
an east→west gradient, the Sicilians having Mellars, 1993) replacing the Neanderthals.
an intermediate frequency between the two Other expansions successively occurred,
extremes. such as in the Neolithic, which stretched
The distribution of the nucleotide pair- from western Asia toward Europe and in-
wise differences reflects the history of the cluded Sicily. Other invasions also occurred
 mtDNA IN SICILY 585

Fig. 4. Neighbor-joining tree relating eleven populations, according to the distance matrix shown in Table 5.

in Sicily, which, as already noted, became a populations influenced the genetic structure
crossroads of human, cultural, and commer- with regard to mtDNA.
cial traffic. Thus, a question of interest is There is no trace of all these events in the
how Sicily’s numerous contacts with other mismatch distribution. Most of the popula-
586 G. VONA ET AL.

TABLE 5. Pairwise differences among the Sicilians and the comparison populationsa
Sicily Sardinia Corsica Tuscany Basques Spain Berbers Turkey M. East Asia Africa
Sicily 4.04 4.14 3.98 5.54 3.68 4.56 5.09 4.73 5.80 6.52 11.18
Sardinia 0.009 4.22 4.02 4.63 3.77 4.69 5.13 4.92 5.96 6.69 10.68
Corsica 0.104 0.059 3.71 4.45 3.51 4.51 5.16 4.69 5.94 6.48 11.34
Tuscany 0.000 0.003 0.082 5.03 4.21 5.09 5.55 5.21 6.21 6.98 10.75
Basques 0.087 0.083 0.080 0.115 3.15 4.19 4.80 4.54 5.69 6.21 11.72
Spain 0.000 0.038 0.117 0.033 0.076 5.08 5.55 5.28 6.31 6.89 10.66
Berbers 0.681 0.630 0.916 0.644 0.836 0.618 4.78 5.80 6.61 7.21 10.41
Turkey 0.018 0.116 0.142 0.003 0.272 0.047 0.719 5.38 6.33 6.99 10.53
Middle East 0.238 0.309 0.549 0.158 0.576 0.232 0.683 0.097 7.08 7.91 10.26
Asia 0.358 0.437 0.483 0.326 0.493 0.211 0.678 0.163 0.230 8.28 10.43
Africa 7.562 6.978 7.890 6.635 8.554 6.528 6.434 6.249 5.124 4.695 3.19
a
Below the diagonal: intermatches-based genetic distances, D 4 Dij − (Di + Dj/2) (RAO). Above the diagonal: intermatches between
pairs of populations.
Diagonal: mismatches within populations (i.e., parwise differences).

tions considered show a bell-shaped distri-
bution of the pairwise differences. Some of
the populations, e.g., Sardinia and Corsica,
were to some degree invaded by the same
populations that invaded Sicily, but in these
cases, too, no effects attributable to these
invasions are evident in any of the curves.
Nevertheless, it should be remembered that
the genetic diversity found at the mtDNA
level in contemporary human populations is
due to the differences accumulated both be-
fore and after their expansion (Relethford,
1988).
When the frequencies of substitutions
found in the sequences analysis and the
haplotypes present in the Sicilian popula-
tion are examined, it is evident that the Si-
cilian gene pool has been influenced by
other Mediterranean basin populations. A
gene contribution from the Near East ap-
pears unquestionable. Contributions to the
Sicilian gene pool also appear to come from
other regions of the Mediterranean. In fact,
some haplotypes that the Sicilian popula-
tion has in common with the other compari-
son populations can be seen as traces left by
populations passing through the island. In
most cases these haplotypes seem to date to
the Paleolithic and Neolithic.
In view of Sicily’s prehistoric and historic
past, it is difficult to say whether these ex-
isted prior to the population expansion, if
they were from the Paleolithic and Neolithic
migrations, or if they reached the island in
more recent times through groups that had
retained them in their gene pool.
Further, the haplotype frequencies that
the Sicilians have in common with each in-
dividual population are low and lead us to
the suggestion that the external influences
of each population are of little importance.
Analysis of the Sicilian mtDNA haplo-
types in relation to the recent work of Rich-
ards et al. (2000) on founder lineages of
mtDNA in Europe suggests that about 70%
of the haplotypes from the sample of pre-
sent-day Alia could date back to the Paleo-
lithic, whereas about 10% could have origi-
nated during the Neolithic. These values
are very close to those that Richards et al.
(2000) have indicated for the central Medi-
terranean: around 80% for the Paleolithic
component and 10% for the Neolithic.
On the basis of these observations, it
seems likely, therefore, that the various im-
migrations which took place in Sicily did
have as much impact on the mtDNA genetic
structure, as that which resulted after the
population expansion about 26,000 years
ago.
About 45% of the sequences found in the
Alia sample are of haplotypes that are not
present in the comparison populations. This
peculiarity could be linked to the partial iso-
lation that Alia experienced in some periods
and to genetic drift. For example, for the
first part of the 19th century A.D., the popu-
lation of Alia was characterized by a pro-
gressive process of isolation, interrupted
only after the unification of Italy (1860). In
1901 the number of inhabitants from Alia
who had emigrated to the United States
amounted to more than 3,000. Analysis of
surnames reveals, particularly in the
middle part of the 19th century, an ex-
tremely low number of different surnames
in Alia: roughly 13 out of 100 (Bigazzi,
2000). Thus, between 1830 and 1844, the
period of the cholera epidemic, of the 219
surnames of the previous period, Alia re-
tained only 127 different surnames. Fur-
ther, Alia, situated on the edge of the main
 TABLE 6. Absolute and relative frequencies of the haplotypes common to Sicily and other populations
SICILY SARDINIA CORSICA TUSCANY BASQUES SPAIN BERBERS TURKEY M. EAST ASIA AFRICA
Haplotype n 4 49 n 4 69 n 4 47 n 4 52 n 4 45 n 4 58 n 4 85 n 4 45 n 4 41 n 4 21 n 4 15
SA43 6 15 11 9 9 5 8 2 0 0 0
0.122 0.217 0.234 0.173 0.200 0.086 0.094 0.044 0.000 0.000 0.000
SA67 3 3 3 0 2 2 7 1 0 0 0
0.061 0.043 0.064 0.000 0.044 0.034 0.082 0.022 0.000 0.000 0.000
SA44 1 0 0 0 1 0 0 0 2 0 0
0.020 0.000 0.000 0.000 0.022 0.000 0.000 0.000 0.049 0.000 0.000
SA33 2 0 0 0 2 0 0 0 0 0 0
0.041 0.000 0.000 0.000 0.044 0.000 0.000 0.000 0.000 0.000 0.000
SA15 3 1 0 1 3 0 0 2 0 0 0
0.061 0.014 0.000 0.019 0.067 0.000 0.000 0.044 0.000 0.000 0.000
SA25 2 0 0 0 1 0 0 0 0 0 0
0.041 0.000 0.000 0.000 0.022 0.000 0.000 0.000 0.000 0.000 0.000
SA40 2 0 0 0 0 1 4 2 2 0 0
0.041 0.000 0.000 0.000 0.000 0.017 0.047 0.044 0.049 0.000 0.000
SA36 1 0 0 0 0 1 0 0 0 0 0
0.020 0.000 0.000 0.000 0.000 0.017 0.000 0.000 0.000 0.000 0.000
SA37 1 0 0 0 0 1 0 0 0 0 0
0.020 0.000 0.000 0.000 0.000 0.017 0.000 0.000 0.000 0.000 0.000
SA32 1 1 0 1 0 0 0 0 0 1 0
0.020 0.014 0.000 0.019 0.000 0.000 0.000 0.000 0.000 0.048 0.000
SA4 1 0 0 0 0 0 0 1 1 0 0
0.020 0.000 0.000 0.000 0.000 0.000 0.000 0.022 0.024 0.000 0.000
SA53 1 3 0 0 0 0 0 0 0 0 0
0.020 0.043 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
SA58 1 1 0 0 0 0 0 0 0 0 0
0.020 0.014 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
SA2 1 0 0 1 0 0 0 0 0 0 0
0.020 0.000 0.000 0.019 0.000 0.000 0.000 0.000 0.000 0.000 0.000
588 G. VONA ET AL.
trade routes and never having much to offer
economically, has always been in a mar-
ginal and isolated position compared to
flows within the island.
The middle position occupied by the popu-
lation of Sicily confirms its ancient origin,
on the one hand, and the conservation of its
genetic identity, on the other hand. The ge-
netic divergence that emerges in some of the
parameters analyzed and in the peculiarity
of some sequences may have been caused by
the most recent expansions, which in-
creased local mitochondrial variability,
which, in turn, has since been maintained
by isolation. During the period of cholera
about 30% of the surnames disappeared
from Alia, but in the following periods a
similar number of new surnames, mainly
from the surrounding areas, were intro-
duced.
The characteristics of mtDNA offered by
the population of Alia indicate, on the one
hand, the influences of contacts which the
island has had with various populations of
the Mediterranean area, and on the other
hand, some particularities which seem to
have been shaped by the effects of genetic
drift and isolation. These particularities are
also evident in studies of the genetic struc-
ture of the Sicilian population, in general,
and of Alia (Vona et al., 2000). This analysis
also confirms a position for Sicily midway
between the European and Middle East
populations (Rickards et al., 1992).
In work carried out with restriction en-
zymes on mtDNA in a sample of Sicilians,
Semino et al. (1989) indicated the presence
(4.4%) of the African complex HpaI-3/
AvaII-3 (40% in Senegal and in the Bantu of
South Africa). The authors hypothesized a
migration of genes from Africa to Sicily, es-
timated at about 10%, which was intro-
duced into the Sicilian gene pool by Black
slaves brought by the Phoenicians and the
Romans and/or by Arab migrations. Results
at the mtDNA sequencing level, however,
show no Black African influence in the Si-
cilian population. The haplotypes that
emerge from the sequences, some of which
common to both Sicilians and Berbers, sug-
gest the possibility of a certain amount of
contact with the populations of north Africa.
Results of the analysis of mtDNA se-
quences indicate that it is difficult to iden-
tify in the characteristics of the present-day
population of Sicily genetic influences that
could be attributed to any single group that
invaded the island and settled there for
shorter or longer periods. Further study of
mtDNA by applying the restriction enzymes
to the present and earlier (from the time of
the 1837 cholera outbreak) populations
could shed light on the interactions between
the Sicilian population and other popula-
tions of the Mediterranean area which
needs to be examined more thoroughly.
ACKNOWLEDGMENTS
We are grateful to the mayor of Alia, Dr.
G. D’Andrea, and the whole population for
their helpfulness.
LITERATURE CITED
Anderson S, Bankier AT, Barrel BG, De Bruijn MHL,
Coulson AP, Drouin J, Eperon IC, Nierlich DP, Roe
BA, Sanger F, Schreier PH, Smith AJH, Staden R,
Young IG. 1981. Sequence and organisation of the hu-
man mitochondrial genome. Nature 290:457–465.
Barbujani G, Bertorelle G, Capitani G, Scozzari R.
1995. Geographical structuring in the mtDNA of Ital-
ian. Proc Natl Acad Sci U S A 92:9171–9175.
Bertanpetit J, Sala J, Calafell F, Underhill PA, Moral P,
Comas D. 1995. Human mitochondrial DNA variation
and the origin of Basques. Ann Hum Genet 59:63–81.
Bigazzi R. 2000. Analisi microevolutive sulla struttura
biologica e demografica della popolazione di Alia (PA)
del XIX secolo. Tesi di Dottorato di Ricerca in Scienze
Antropologiche, Università di Firenze.
Brown WM, Geroge JRM, Wilson AC. 1979 Rapid evo-
lution of animal mitochondrial DNA. Proc Natl Acad
Sci U S A 76:1967–1971.
Cann RL, Brown WM, Wilson RC. 1984. Polymorphic
sites and the mechanism of evolution in human mito-
chondrial DNA. Genetics 106:479–499.
Chilardi S, Frayer DW, Gioia P, Macchiarelli R, Mussi
M. 1996. Fontana Nuova di Ragusa (Sicily, Italy):
southernmost Aurignatian site in Europe. Antiquity
70:553–563.
Comas D, Calafell F, Mateu E, Pérez-Lezaun A, Ber-
tranpetit J. 1996. Geographic variation in human mi-
tochondrial DNA control region sequence: the popu-
lation history of Turkey and its relationship to the
European populations. Mol Biol Evol 13:1067–1077.
Corte Real HBSM, Macaulay VA, Richards MB, Hariti
G, Issard MS, Cambon-Thomsen A, Papiha S. 1996.
Genetic diversity in the Iberian Peninsula deter-
mined from mitochondrial sequence analysis. Ann
Hum Genet 60:331–350.
Culotta P. 1995. L’architettura della Gurfa. In: La
Gurfa e il Mediterraneo. Alia: Ed. Comune di Alia.
Cumbo G. 1995. Le grotte della Gurfa ed altre emer-
genze archeologiche nella Sicilia centrale, zona cusci-
netto, tra le idrovie Platani e Torto. Alia: Ed. Comune
di Alia.
Di Rienzo A, Wilson AC. 1991. Branching Pattern in the
Evolutionary Tree for Human Mitochondrial DNA.
Proc Natl Acad Sci U S A 88:1597–1601.
Felsenstein J. 1989. PHYLIP-phylogeny inference pack-
age (version 3.2). Cladistics 5:164–166.
Ferris S, Brown WM, Davidson WS, Wilson AC. 1981.
Extensive polymorphism in the mitochondrial DNA of
apes. Proc Natl Acad Sci U S A 78:6319–6323.
Francalacci P, Bertranpetit J, Calafell F, Underi-Ill PA.
1996. Sequence diversity of the control region of mi-
tochondrial DNA in Tuscany and its implications for
 mtDNA IN SICILY
the peopling of Europe. Am J Phys Anthropol 100:
443–460.
Harpending HC, Sherry ST, Rogers AR, Stoneking M.
1993. The genetic structure of ancient human popu-
lations. Curr Anthropol 34:483–496.
Hartl DL, Clark AG. 1989. Principles of population ge-
netics. Sunderland, MA: Sinauer Associates.
Kimura M. 1980. A simple method for estimating evo-
lutionary rates of base substitutions through com-
parative studies of nucleotide sequences. J Mol Evol
16:111–120.
Magurran AE. 1988. Ecological diversity and its mea-
sure. Princeton: Princeton University Press.
Mellars P. 1993. Archeology and modern human origins
in Europe. Proc Br Acad 82:1–35.
Rao CR. 1982. Diversity and dissimilarity coefficients: a
unified approach. Theor Pop Biol 21:24–43.
Reynolds J, Weir BS, Cockerham CC. 1983. Estimation
for the coancestry coefficient: basis for short-term ge-
netic distances. Genetics 105:767–779.
Relethford JH. 1988. Mitochondrial DNA and ancient
population growth. Am J Phys Anthropol 105:1–7.
Richards M, Macaulay V, Hickey E, Vega E, Sykes B,
Guida V, Rengo C, Sellitto D, Cruciani F, Kivisild T,
Villems R, Thomas M, Rychkov S, Rychkov O, Rych-
kov Y, Gölge M, Dimitrov D, Hill E, Bradley D, Ro-
mano V, Calı̀ F, Vona G, Demaine A, Papiha S, Tri-
antaphyllidis C, Stefanescu G, Hatina J, Belledi M, Di
Rienzo A, Novalletto A, Oppenheim A, Nørby S, Al-
Zahari N, Satachiara Benerecetti S, Scozzari R, Tor-
roni A, Bandelt H-J. 2000. Tracing European lineages
in the Near Eastern mtDNA Pool. Am J Hum Genet
67:1251–1276.
Rickards O, Biondi G, De Stefano GF, Vecchi F, Walter
H. 1992. Genetic structure of the population of Sicily.
Am J Phys Anthropol 87:395–406.
Rogers AR, Harpending H. 1992. Population growth
makes waves in the distribution of pairwise genetic
distances. Mol Biol Evol 9:552–569.
Saitou N, Nei M. 1987. The neighbor-joining method: a
new method for reconstructing phylogenetic trees.
Mol Biol Evol 4:406–425.
Schneider S, Kueffer JM, Roessli D, Excoffier L. 1997.
589
Arlequin 1.1. Software program for population genetic
data analysis, User Manual Genetic and Biometry
Lab., University of Geneva.
Semino O, Torroni A, Scozzari R, Brega A, De Benedic-
tis G, Santachiara Benerecetti AS. 1989. Mitochon-
drial DNA polymorphisms in Italy. III. Population
data from Sicily: a possible quantitation of maternal
African ancestry. Ann Hum Genet 53:193–202.
Slatkin M. 1995. A measure of population subdivision
based on microsatellite allele frequencies. Genetics
139:457–462.
Stoneking M. 1993. DNA and recent human evolution.
Evol Anthropol. 2:60-73.
Stringer CB. 1989. The origin of the early modern hu-
mans: a comparison of the European and non-
European evidence. In: Mellars P, Stringer C, editors.
The human revolution: behavioural and biological
perspectives on the origin of modern humans. Prince-
ton: Princeton University Press. p 232–244.
Tamura A, Nei M. 1993. Estimation of the number of
nucleotide substitutions in the control region of
mtDNA im humans and chimpanzees. Mol Biol Evol
10:512–526.
Vigilant L, Pennington R, Harpending H, Kocher TD,
Wilson AC. 1989. Mitochondrial DNA sequences in
single hairs from Southern African Population. Evo-
lution 86:9350–9354.
Vigilant L, Stoneking M, Harpending H, Hawkes K,
Wilson AC. 1991. African populations and the evolu-
tion of mitochondrial DNA. Science 253:1503–1507.
Varesi L, Memmı̀ M, Cristofari MC, Mameli GE, Calò
CM, Vona G. 2000. Mitochondrial control-region se-
quence variation in the Corsican population, France.
Am J Hum Biol 12:339–351.
Vona G, Chiarelli B, Ghiani ME, Sineo L. 2000. Genetic
structure of Sicily: a review. In: Susanne C, Bodzsar
EB, editors. Human population genetics in Europe,
Vol 1. Budapest: Eotvos University Press. p 63–78.
Ward RH, Frazier BL, Dew-Jager K, Pääbo S. 1991.
Extensive mitochondrial diversity within a single
Amerindian tribe. Proc Natl Acad Sci U S A 88:8720–
8724.