Mitochondrial DNA sequence analysis in Sicily

Mitochondrial DNA Sequence Analysis in SicilyG. VONA,1* M.E. GHIANI,1 C.M. CALO,1 L. VACCA,1 M. MEMMI,2 AND L. VARESI2

1Department of Experimental Biology, Section of Anthropological Sciences, University ofCagliari, Monserrato, Italy2Faculty of Sciences and Technologies, University of Corsica, Corte, France

ABSTRACT This study reports data on the sequences of the first hypervariable segment of asample of the Sicilian population from Alia (Palermo, Italy). The results show the presence of 32different haplotypes in the 49 individuals examined. The average number of pairwise nucleotidedifferences was 4.04, i.e., 1.17% per nucleotide. The distribution of the nucleotide differences matchesthe theoretical distribution and indicates only one major episode of expansion that occurred between20,732 and 59,691 years ago, between the Middle Paleolithic and Upper Paleolithic. Compared withthe other populations, parameters of the Sicilian sample lie in an intermediate position between theeastern and western Mediterranean populations. This is due to numerous contacts that Sicily has hadwith the Mediterranean area since prehistoric times. At the same time, the singularity of some of thehaplotypes present in the sample studied indicates the persistence of some characteristics caused bygenetic 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 someproteins and various types of ribosomal andtransfer RNA specific of mitochondrial me-tabolism. The mtDNA molecule has a non-coding part, called a D-loop, which containsthe origin of the replication and informationthat controls it. One of the fundamentalcharacteristics of mtDNA is that it has amutation rate roughly 10 times higher thanthe rate in nuclear DNA so that short-termevolving phenomena can be analyzed (Fer-ris et al., 1981; Cann et al., 1984; Vigilant etal., 1989; Ward et al., 1991). This mutationlevel is substantiated by an accumulation ofmutations transmitted through the mater-nal line, since another basic property ofmtDNA is that it is inherited only mater-nally. Mutations are most frequently shownby base substitutions, while deletions andinsertions 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 evolutionrate of the molecule. This is the area thatwas analyzed in a sample from Sicily (Italy)in the context of populations originatingmainly in the Mediterranean region.

HISTORICAL BACKGROUNDThe 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 areasof the Mediterranean. This suggests thateven 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 occupiedby the Siculi (1,200 B.C.), who pushed theresident populations, the Sicans and theElimi, toward the central and western partsof the island. The 8th century B.C. saw thebeginning of one the most intense coloniza-tions: colonization by the Greeks affectedmost parts of the coastal area, and the ex-isting Phoenician presence was limited tothe northwestern area. Over the next fewcenturies, Sicily became completely “Hellen-ized.”

Another important time for Sicily was theperiod under Roman rule. Confiscated landswere given to veteran Roman soldiers, andgreat estates were established where large

Contract grant sponsor: Ministero dell’Universita e RicercaScientifica e Tecnologica (MURST, Italy), Programmi RicercaScientifica di Rilevante Interesse Nazionale (ex-40%); Contractgrant 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: [email protected]

Received 26 May 2000; Revision received 3 January 2001; Ac-cepted 7 January 2001

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

© 2001 Wiley-Liss, Inc.

PROD #M20045R2

numbers of slaves were used to work thefields. Both the landowners and slaves camefrom all corners of the Roman domain. TheRomans, contrary to the Greeks, pushedtheir occupation deeply inland, transform-ing the island also ecologically. Intense de-forestation took place, and the monocultureof wheat started. Sicily in effect became thegranary of Rome.

After the passage of several other groups,e.g., the Vandals from North Africa and theByzantines, Arab domination began in 827A.D. with the landing in Gela of 10,000Muslims from North Africa and Spain. Thistransformed Sicily into the center of a wideconfederation of Muslim nations.

In 11th century A.D., Sicily was occupiedby the Normans and experienced an ex-traordinary immigration of numerousgroups, particularly of Italians, French, andLongobards. Spanish domination began in1282, and Sicily went through one of themost disastrous periods of its history. Span-ish domination was aggravated by thespread of banditry and the presence of alarge number of mercenaries. Return tomonoculture linked to deforestation and aseries of terrible events such as famine,earthquakes, and outbreaks of plague andcholera forced many of Sicilians to emigrate.Before becoming part of the realm of Italy in1860 Sicily was the stage for many wars,which brought powerful armies of foreignsoldiers to the island. For example, the res-toration of the Borbonnes (1820) cost Sicilyan invasion of 12,000 Austrian soldiers.

Sicily’s history over the last century wascharacterized by a series of formidable andirresolute problems, leading to a high levelof emigration which peaked in the years af-ter the two World Wars; 1,500,000 Siciliansabandoned the island after the First WorldWar.

Many of these events had important de-mographic implications, influencing the bio-logic history of Sicily and shaping its geneticstructure (Vona et al., 2000). The effects ofone of the events, which dramatically af-fected Sicily, was the discovery in 1995 ofabout 300 skeletons of individuals who haddied of cholera in 1837 A.D. in the smallcommune of Alia, in the eastern part of theprovince of Palermo. Situated 800 metersabove sea level on the southwestern slopesof the Madonie mountain chain, about 80km from Palermo (Fig. 1), Alia today has a

population of about 4,000 inhabitants. In1837 Alia, along with many other Sicilianvillages, was hit by an epidemic of choleraand the population was reduced by at least10%. Given the high number of deaths, thebodies were mostly buried in a cave on theoutskirts of the village rather than in thetraditional cemetery. At the end of the epi-demic, entrance to the cave was closed andonly in 1995 did an excavation bring the re-mains to light. This study reports the re-sults of the analysis of the sequences of thecontrol segment I of mitochondrial DNAtaken from a sample of the present-daypopulation of from Alia, Sicily.

MATERIALS AND METHODSPopulation

The sample includes 49 unrelated, appar-ently healthy, adults of both sexes from Aliawhose families were born and lived in thesame village for at least three generations.With prior informed consent, a blood samplewas collected from each individual by veni-puncture at the Municipal OutpatientsClinic 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 CagliariUniversity.

Amplification and sequencing of DNAThe DNA was subsequently extracted

from the whole blood using the QIAampBlood Kitt (QIAGEN). The DNA was ampli-fied starting from 10 ml of the final lysatum.The hypervariable segment I of the D-loopwas 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 anamplified fragment of 425 bases.

PCR conditions were as follows: 15 min at95°C in 2.5 U of HotStar Taq Polymerasetfor a total reaction volume of 50 ml (10 mMeach of dNTP, 25 mM di MgCl2, 15 ml of theprimer couples, 10 ml of DNA of the sampleto be amplified); 35 cycles at 94°C for 45 sec,56°C for 1 min and 74°C for 1 min. Chainelongation was continued after the last cyclefor 10 min at 72°C.

The products of the amplification were vi-sualized by electrophoresis on agarose gelscontaining 2% ethidium bromide. The prod-ucts of the PCR were then purified in Cen-

mtDNA IN SICILY 577

tricon-100t tubes (Perkin-Elmer). Subse-quently, 10 ml of amplified and purifiedmtDNA were sequenced by an automatedsequencer ABI 373. Each sample was se-quenced with the H and L primers used forthe PCR. Some of the samples were se-quenced twice, and some were sequencedwith the primer H or L. The results werecompared in order to avoid possible ambigu-ities in readings. The sequences thus ob-tained covered a total of 388 bases, frombase 16,023 to base 16,410, and were com-pared with Anderson’s (1981) reference se-quence through the CLUSTAL V program.

Statistical analysisInternal 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 sequenceswas performed in accordance with Kimura’s(1980) two-parameter model. The tree rela-

tive to the 49 sequences was drawn up after100 permutations, according to the neigh-bor-joining method (Saitou and Nei, 1987)and the difference between clusters wastested through the transformed pairwiseFst method (Reynolds et al., 1983; Slatkin,1995). The expected number of frequenciesand the effective size of the population wereestimated under the infinite allele model bymeans of the number of individuals and se-quences (Hartl and Clark, 1989). Variabilitywithin the sample examined was analyzedby Shannon’s diversity index in its stan-dardized form H8 (Magurran, 1988).

Dating of expansion. The pairwise nucleo-tide difference distribution was fitted to theRogers and Harpending (1992) model, andthe standard errors were calculated by1,000 bootstrap iterations. The parameterst and u were then obtained following thetwo-parameter model of Harpending et al.(1993). Expansion time was estimated in ac-cordance with the model of Roger and

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.

578 G. VONA ET AL.

Harpending (1992) through the parameter tusing mutation level calculations takenfrom reported works (Vigilant et al., 1991;Ward et al., 1991) and a generation period of20 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 Rienzoand Wilson, 1991), Sardinian (Di Rienzoand Wilson, 1991), Spanish (Corte-Real etal., 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 andNei, 1987) was constructed using the ge-netic distances computed through themethod proposed by Rao (1982).

RESULTSSequence diversity

Table 1 shows the variable sites in thecontrol region of mtDNA compared with thereference sequence. The 49 individualsshow 32 different sequences with 36 vari-able sites and 108 mutations. With the soleexception of transversion C→A, the substi-tutions are all transitions. The transition/transversion ratio is, therefore, 36:1; muchhigher than that reported by Tamura andNei (1993) for human populations. Most ofthe transitions concern the pyrimidines andare 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, whileindividual SA34 showed 7. The site with thehighest number of substitutions is 16,126with a T→C transition in 12 individuals(24.49%), followed by sites 16,311 and16,189 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 individualsand the number of sequences, it was pos-sible to calculate the expected number of se-quences (Hartl and Clark, 1989). The valuetaken of u in the sample of 49 individualsand 32 different sequences is 38.93, whichcorresponds to an estimate of 128.33 se-quences per 1,000 individuals. This value ofu is much closer to that calculated in Corsi-

cans (38.52) (Varesi et al., 2000) and inBasques (27.58) (Bertranpetit et al., 1995)than that shown by Tuscans (99.38) (Fran-calacci et al., 1996).

Phylogenetic analysisSequence variability as expressed

through the index of diversity H8 (Table 2) is0.961. If the Asian sample is excluded, thisvalue is one of the highest reported in Table2. Values vary between the 0.891 in Berbersand 0.991 in the Middle East.

The average number of pairwise differ-ences in nucleotides between all possiblecouples of individuals is 4.040 ± 0.845, i.e.,1.17% per nucleotide. The number of stepsneeded to construct the parsimony tree is1.122.

Phylogenetic analysis of the sequencescarried out with the DNADIST program ofPHYLIP 3.51C and using the method pro-posed by Kimura (1980) allowed the con-struction of a neighbor-joining tree from thedistances (Saitou and Nei, 1987). The treeshown in Figure 2 shows how the sequencessplit into three well-defined clusters. Thesequences that do not show variations fromthe Anderson et al. (1981) sequence are incluster B together with other sequences,which have a lower number of transitionsand with the only sequence carrying atransversion. 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 seemsquite random, and analysis of the varianceshows 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, hasone of the lowest values among those re-ported for the populations in Table 2. Thissuggests that, from a phylogenetic point ofview, the sequences obtained are closely cor-related.

Nucleotide difference distributionAnalysis 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 onepeak at 4.04 (variance 4 4.943) (Table 2).The distribution matches the theoretical

mtDNA IN SICILY 579

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distribution based on the model of Rogersand Harpending (1992) (x2 4 18.421; df 411; P 4 0.072). This suggests that only onemajor episode of population expansion inthe past which would have increased an ini-tial population of roughly 300–900 to some-thing between 12,000 and 35,000 individu-als (Table 3).

Dating expansionThe estimation of dating expansion of the

population was calculated by the method ofRogers and Harpending (1992) andHarpending et al. (1993). From the valuesobtained for the population of Alia for theparameters t 4 3.089, q0 4 0.951, q1 436.401, and the different mutation rates re-ported in literature, expansion would haveoccurred between 20,732 and 59,691 yearsago with an intermediate value of 39,679years (Table 3).

Comparison with other populations

All of the variable sites found in the Aliapopulation have already been described inother populations. Therefore, the Sicilianpopulation has no specific substitutions ofits own.

The substitution frequency in site 16,126obtained for Sicily lies midway between theeastern and western comparison popula-tions (Table 4). On the contrary, variabilityof site 16,311 does not appear to be distrib-uted according to a gradient as indicated byComas et al. (1996). The lowest frequency isfound among the Basques (11.11%) and thehighest among the Tuscans (21.15%). Forsite 16,311 Sicily has a frequency similar tothe Berbers, Sardinians, and Turks. An-other two mutated sites, 16,069 (12.24%)

and 16,223 (10.20%), both with a C→T tran-sition, have a frequency in Sicily midwaybetween the east and west. The two sitesalso appear to vary with a decreasing gra-dient from east to west. For 16,069 the Si-cilian frequency is closer to that of the Tus-cans and Turks than to those of the otherwestern populations, where the highest fre-quency (6.90%) is found in the Spanish. Thereverse is true for the substitution fre-quency of site 16,223, which, in Sicily, is ofthe western type. Substitution at site16,261, which has the highest frequency inthe Middle East (19.51%) and is absent inCorsica and Sardinia, barely reaches 2.04%in Sicily.

The comparison of pairwise differences inAlia and other populations (Fig. 4) showsthat the peak in Alia falls to the left side,having one of the lowest average values ofpairwise differences among those shown inTable 2. This position indicates that Alia’sexpansion time is one of the most recentcompared to the comparison populations.

The Sicilian sample was compared withthe other populations by the genetic dis-tances of Rao (1982). The genetic tree in Fig-ure 5, drawn from the matrix of distances inTable 5, is of the neighbor-joining type ob-tained with 100 iterations. The two samplesfrom Asia and Africa, which lie in two sepa-rate branches, are clearly differentiatedfrom the Euro-Mediterranean populations,which are all found in another branch. Thisstretches from the populations of the MiddleEast on one side, near the fork that sepa-rates Asia and Africa, to the Basques andCorsicans on the opposite side. The samplefrom Sicily lies between Tuscany andSardinia but closer to the former.

Table 6 shows the haplotype frequenciesthat emerge from the sequencing and thatSicily has in common with the other com-parison populations. While there are nocommon haplotypes between Sicily and theAfrican sample, there is one common se-quence with Asia (SA32). The most frequenthaplotype in Sicily is sequence SA43, whichis identical to the reference sequence ofAnderson et al. (1981), with an intermediatefrequency to those of the comparison popu-lations. The lowest values are shown byeastern populations and the highest by theCorsicans and Basques. This haplotype wasnot seen in the Middle East sample.

Other than the Sicilian sample, haplo-types of the sequences SA33 and SA25 were

TABLE 2. Sequences divergence in Sicilian and in thecomparison populationsa

N k a b d H8

Sicily 49 32 37 4.040 ± 0.845 1.122 0.961Sardinia 69 46 52 4.221 ± 0.558 1.826 0.930Corsica 47 31 40 3.712 ± 0.822 1.774 0.903Tuscany 52 40 54 5.032 ± 0.543 2.025 0.899Basques 45 27 32 3.152 ± 0.721 1.481 0.912Spain 58 52 62 5.083 ± 0.994 1.846 0.980Berbers 86 30 37 4.782 ± 0.872 1.700 0.891Turkey 45 38 56 5.386 ± 0.728 2.001 0.987Middle East 42 38 59 7.083 ± 1.228 2.211 0.991Asia 23 23 59 8.283 ± 1.011 3.261 1.000Africa 22 12 34 3.194 ± 0.898 4.500 0.946aN 4 number of individuals; k 4 number of different sequences;a 4 number of variable nucleotides; b 4 mean nucleotide pair-wise differences; d 4 mean number of steps per sequence in amaximum parsimony tree; H8 4 index of Shannon.

mtDNA IN SICILY 581

only found in the Basques, while haplotypesSA36 and SA37 are also present in theSpanish. The haplotype represented by se-quence SA67, absent in the Middle East andTuscan samples, has its highest frequencyin the Berbers, with the Sicilian frequencylocated between those of the Berbers andthe western populations. For sequenceSA40, which was not found in Tuscany,Sardinia, Corsica, or the Basques, Sicily hasa frequency very similar to that of the east-ern countries and the Berbers. SequencesSA53 and SA58 seem to be exclusive to theSardinians and Sicilians, while haplotypeSA2 is shared only with the Tuscans. Thehaplotypes that characterize the remaining(44.9%) sequences of Alia are not found inthe comparison populations.

DISCUSSION

The vast literature concerning mtDNAhighlights its importance in the study ofevolutionary processes in humans. Se-quence variation analysis of a control regionseems to be useful and highly effective inthe study of a population’s history, not onlyon a global level but also regionally.

Many genetic studies have been done onthe Sicilian population; they have concernedmainly proteins, and only recently has themolecular 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 geneticrelations that have marked the island’s

Fig. 2. Neighbor-joining tree of the sequences in the Sicilian samples.

582 G. VONA ET AL.

evolutionary history. This study shows thesequences of the first hypervariable seg-ment of mtDNA in a sample of the Sicilianpopulation from Alia in the province of Pal-ermo (Italy).

The results for Alia reveal several fea-

tures of the region of the first hypervariablesegment of mtDNA, some of which havebeen reported in other studies. A prevalenceof transitions over transversions is notedbut in a much higher proportion than foundin other populations. There is also a marked

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

mtDNA IN SICILY 583

predominance of pyrimidine transitionsover purine. The analysis shows that Sicil-ians, as far as the sequencing of mtDNA isconcerned, have some characteristics thatcan be considered midway among thosefound in comparison populations. This isparticularly evident in the cline of the fre-quencies of some substitutions, on the aver-age, and in the distribution of the nucleotidepairwise differences. The position taken inthe genetic tree of the populations also leadsto the same conclusion. The tree shows ap-proximately an east to west gradient withthe Middle East at one extreme and theBasques and Corsicans at the other.

The mutation at site 16,126, which ismost frequent in the Sicilian sample, showsa decreasing east–west variation of frequen-cies with extreme values in the Middle East(46.3%) and in the Basques (6.7%). The fre-quencies of the substitutions relative tosites 16,069 and 16,223 also appear to havean east→west gradient, the Sicilians havingan intermediate frequency between the twoextremes.

The distribution of the nucleotide pair-wise differences reflects the history of the

ancient population, especially the expan-sions and the periods during which they oc-curred. Rogers and Harpending (1992) dem-onstrated that the rapid growth of apopulation is signaled by a smooth mis-match distribution that has only one peak.The mismatch distribution in Alia samplesuggests that the main episode of expansiontook place between 20,732 and 59,691 yearsago, with an intermediate indication of39,679 years ago. The comparison popula-tions also show bell-shaped distributions,all denoting a series of expansions that tookplace later. Expansions occurred first in theeast and then in the west. This is consistentwith an east to west migration.

The population with the oldest expansionwould be the Middle East, roughly between47,000 and 135,000 years ago. On the con-trary, the most recent expansion wouldhave taken place among the Basques, about14,000–41,000 years ago. The observationsnoted from the various analyses seem to beconsistent with an east–west migration (Co-mas et al., 1996; Francalacci et al., 1996).

Barbujani et al. (1995), in a study ofmtDNA in Italy, suggested that the time ofexpansion of the Italian populations, includ-ing Sicily, was somewhere between 8,200and 20,525 years ago, and the size of thepopulation was estimated between 1,160and 2,326 females. The authors also pro-posed two possible expansions: one after themaximum of the last glaciation in the UpperPaleolithic and the other at the beginning ofthe Neolithic. The data in the present studyonly partly agree with Barbujani et al.(1995), both in terms of the time of expan-sion and the increase of population num-bers. As indicated by the calculated dataand in Figure 4, Sicily seems to be amongthe populations with a more recent expan-sion, but this unique event falls during theMiddle and Upper Paleolithic, and its tem-poral position seems to agree with archaeo-logical dates (Chilardi et al., 1996). Further,the estimated increase in population ismuch more substantial than that indicatedby Barbujani et al. (1995).

The first presence of modern populationsis placed in the Near East from where theyexpanded toward Europe (Stringer, 1989;Mellars, 1993) replacing the Neanderthals.Other expansions successively occurred,such as in the Neolithic, which stretchedfrom western Asia toward Europe and in-cluded Sicily. Other invasions also occurred

TABLE 3. Demographic parameter estimates for theSicilian population according to the model of Roger

and Harpending (1992) for different proposedmutation ratesa

m N0 N1

Times ofdivergence

(years)1.490 × 10−3

(Ward et al., 1991)319 12,215 20,732

7.785 × 10−4

(Vigilant et al., 1991)610 23,380 39,679

5.175 × 10−4

(Vigilant et al., 1991)919 35,170 59,691

aN0 and N1 are the effective population sizes before and after theexpansion episode.

TABLE 4. Most frequent substitution in the Siciliansample in comparison with other populations

Site position16,069 16,126 16,223 16,261 16,311

Sicily 12.24 24.49 10.20 2.04 16.33Sardinia 5.80 17.39 7.25 0.00 17.39Corsica 2.13 8.51 8.51 0.00 19.15Tuscany 13.46 23.08 13.46 5.77 21.15Basques 4.44 6.67 4.44 2.22 11.11Spain 6.90 18.97 10.34 3.45 12.07Berbers 3.50 8.20 17.60 0.00 16.50Turkey 15.56 26.67 28.89 8.89 17.78Middle East 19.51 46.34 26.83 19.51 14.63Asia 0.00 9.52 47.62 9.52 19.05Africa 0.00 0.00 86.67 0.00 100.00

584 G. VONA ET AL.

in Sicily, which, as already noted, became acrossroads of human, cultural, and commer-cial traffic. Thus, a question of interest ishow Sicily’s numerous contacts with other

populations influenced the genetic structurewith regard to mtDNA.

There is no trace of all these events in themismatch distribution. Most of the popula-

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

mtDNA IN SICILY 585

tions considered show a bell-shaped distri-bution of the pairwise differences. Some ofthe populations, e.g., Sardinia and Corsica,were to some degree invaded by the samepopulations that invaded Sicily, but in thesecases, too, no effects attributable to theseinvasions are evident in any of the curves.Nevertheless, it should be remembered thatthe genetic diversity found at the mtDNAlevel in contemporary human populations isdue to the differences accumulated both be-fore and after their expansion (Relethford,1988).

When the frequencies of substitutionsfound in the sequences analysis and thehaplotypes present in the Sicilian popula-tion are examined, it is evident that the Si-cilian gene pool has been influenced byother Mediterranean basin populations. Agene contribution from the Near East ap-pears unquestionable. Contributions to theSicilian gene pool also appear to come fromother 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 bypopulations passing through the island. Inmost cases these haplotypes seem to date tothe Paleolithic and Neolithic.

In view of Sicily’s prehistoric and historicpast, it is difficult to say whether these ex-isted prior to the population expansion, ifthey were from the Paleolithic and Neolithicmigrations, or if they reached the island inmore recent times through groups that hadretained them in their gene pool.

Further, the haplotype frequencies thatthe Sicilians have in common with each in-dividual population are low and lead us tothe suggestion that the external influencesof 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 ofmtDNA 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 valuesare very close to those that Richards et al.(2000) have indicated for the central Medi-terranean: around 80% for the Paleolithiccomponent and 10% for the Neolithic.

On the basis of these observations, itseems likely, therefore, that the various im-migrations which took place in Sicily didhave as much impact on the mtDNA geneticstructure, as that which resulted after thepopulation expansion about 26,000 yearsago.

About 45% of the sequences found in theAlia sample are of haplotypes that are notpresent in the comparison populations. Thispeculiarity could be linked to the partial iso-lation that Alia experienced in some periodsand to genetic drift. For example, for thefirst part of the 19th century A.D., the popu-lation of Alia was characterized by a pro-gressive process of isolation, interruptedonly after the unification of Italy (1860). In1901 the number of inhabitants from Aliawho had emigrated to the United Statesamounted to more than 3,000. Analysis ofsurnames reveals, particularly in themiddle part of the 19th century, an ex-tremely low number of different surnamesin Alia: roughly 13 out of 100 (Bigazzi,2000). Thus, between 1830 and 1844, theperiod of the cholera epidemic, of the 219surnames of the previous period, Alia re-tained only 127 different surnames. Fur-ther, Alia, situated on the edge of the main

TABLE 5. Pairwise differences among the Sicilians and the comparison populationsa

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

586 G. VONA ET AL.

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trade routes and never having much to offereconomically, has always been in a mar-ginal and isolated position compared toflows 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 itsgenetic identity, on the other hand. The ge-netic divergence that emerges in some of theparameters analyzed and in the peculiarityof some sequences may have been caused bythe most recent expansions, which in-creased local mitochondrial variability,which, in turn, has since been maintainedby isolation. During the period of choleraabout 30% of the surnames disappearedfrom Alia, but in the following periods asimilar number of new surnames, mainlyfrom the surrounding areas, were intro-duced.

The characteristics of mtDNA offered bythe population of Alia indicate, on the onehand, the influences of contacts which theisland has had with various populations ofthe Mediterranean area, and on the otherhand, some particularities which seem tohave been shaped by the effects of geneticdrift and isolation. These particularities arealso evident in studies of the genetic struc-ture of the Sicilian population, in general,and of Alia (Vona et al., 2000). This analysisalso confirms a position for Sicily midwaybetween the European and Middle Eastpopulations (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 ofSouth Africa). The authors hypothesized amigration of genes from Africa to Sicily, es-timated at about 10%, which was intro-duced into the Sicilian gene pool by Blackslaves brought by the Phoenicians and theRomans and/or by Arab migrations. Resultsat the mtDNA sequencing level, however,show no Black African influence in the Si-cilian population. The haplotypes thatemerge from the sequences, some of whichcommon to both Sicilians and Berbers, sug-gest the possibility of a certain amount ofcontact 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-daypopulation of Sicily genetic influences thatcould be attributed to any single group that

invaded the island and settled there forshorter or longer periods. Further study ofmtDNA by applying the restriction enzymesto the present and earlier (from the time ofthe 1837 cholera outbreak) populationscould shed light on the interactions betweenthe Sicilian population and other popula-tions of the Mediterranean area whichneeds to be examined more thoroughly.

ACKNOWLEDGMENTSWe are grateful to the mayor of Alia, Dr.

G. D’Andrea, and the whole population fortheir helpfulness.

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Mitochondrial DNA sequence analysis in Sicily

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