Unipa en al Ma ke s o Con empo a y I alian Popula ion
Re eals De ails on I s P e-Roman He i age
F ancesca B isighelli
1,2,3.
, Vanesa A
´l a ez-Iglesias
1
, Manuel Fonde ila
1
, Alejand o Blanco-Ve ea
1
,
A
´ngel Ca acedo
1,4
, Vincenzo L. Pascali
2
, C is ian Capelli
3
, An onio Salas
1
*
.
1Unidade de Xene
´ ica, Facul ade de Medicina, Ins i u o de Medicina Legal, Uni e sidade de San iago de Compos ela, Galicia, Spain, 2Fo ensic Gene ics Labo a o y,
Ins i u e o Legal Medicine, Uni e si a
`Ca olica del Sac o Cuo e, Rome, I aly, 3Depa men o Zoology, Uni e si y o Ox o d, Ox o d, Uni ed Kingdom, 4Fundacio
´nPu
´blica
Galega de Medicina Xeno
´mica (FPGMX-SERGAS), CIBER en e medades a as, San iago de Compos ela, Galicia, Spain
Abs ac
Backg ound:
Acco ding o a chaeological eco ds and his o ical documen a ion, I aly has been a mel ing poin o
popula ions o di e en geog aphical and e hnic ma ices. Al hough I aly has been a a o i e subjec o nume ous
popula ion gene ic s udies, gene ic pa e ns ha e ne e been analyzed comp ehensi ely, including unipa en al and
au osomal ma ke s h oughou he coun y.
Me hods/P incipal Findings:
A o al o 583 indi iduals we e sampled om ac oss he I alian Peninsula, om en dis an (i
homogeneous by language) e hnic communi ies — and om wo linguis ic isola es (Ladins, G ecani Salen ini). All samples
we e i s yped o he mi ochond ial DNA (m DNA) con ol egion and selec ed coding egion SNPs (m SNPs). This da a
was pooled o analysis wi h 3,778 m DNA con ol- egion p o iles collec ed om he li e a u e. Secondly, a se o Y-
ch omosome SNPs and STRs we e also analyzed in 479 indi iduals oge he wi h a panel o au osomal ances y in o ma i e
ma ke s (AIMs) om 441 samples. The esul ing gene ic eco d e eals clines o gene ic equencies laid acco ding o he
la i ude slan along con inen al I aly – p obably gene a ed by demog aphical e en s da ing back o he Neoli hic. The
Ladins showed dis inc i e, i mo e ecen s uc u e. The Neoli hic con ibu ion was es ima ed o he Y-ch omosome as
14.5% and o m DNA as 10.5%. Y-ch omosome da a showed la ge di e en ia ion be ween No h, Cen e and Sou h han
m DNA. AIMs de ec ed a mino sub-Saha an componen ; his is howe e highe han o o he Eu opean non-Medi e anean
popula ions. The same signal o sub-Saha an he i age was also e iden in unipa en al ma ke s.
Conclusions/Signi icance:
I aly shows pa e ns o molecula a ia ion mi o ing o he Eu opean coun ies, al hough some
he e ogenei y exis s based on di e en analysis and molecula ma ke s. F om No h o Sou h, I aly shows clinal pa e ns ha
we e mos likely modula ed du ing Neoli hic imes.
Ci a ion: B isighelli F, A
´l a ez-Iglesias V, Fonde ila M, Blanco-Ve ea A, Ca acedo A
´, e al. (2012) Unipa en al Ma ke s o Con empo a y I alian Popula ion Re eals
De ails on I s P e-Roman He i age. PLoS ONE 7(12): e50794. doi:10.1371/jou nal.pone.0050794
Edi o : Da id Ca amelli, Uni e si y o Flo ence, I aly
Recei ed June 15, 2012; Accep ed Oc obe 24, 2012; Published Decembe 10, 2012
Copy igh : ß2012 B isighelli e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
Funding: The esea ch leading o hese esul s has ecei ed unding om he People P og amme (Ma ie Cu ie Ac ions) o he Eu opean Union’s Se en h
F amewo k P og amme FP7/2007–2013/unde REA g an ag eemen numbe 290344, and he Minis e io de Ciencia e Inno acio
´n (SAF2008-02971 and SAF2011-
26983)(AS). CC and FB we e pa ially unded by he B i ish Academy o he p ojec ‘‘The G eeks in he Wes : he gene ic legacy o he colonisa ion in Sou h I aly
and Sicily’’. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip .
Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis .
* E-mail: [email p o ec ed]s
.These au ho s con ibu ed equally o his wo k.
In oduc ion
I aly has his o ically been a con enien des ina ion o human
popula ions mig a ing om A ica, he Middle Eas and Eu opean
loca ions, in pa due o he geomo phological cha ac e is ics o
he I alian Peninsula [1]. These g oups se led p e e en ially on he
islands and coas al e i o ies [1] 500,000 yea s ago (ya), ha is,
along he Lowe Paleoli hic, he longes pe iod o human
p ehis o y, which was domina ed by he no able di usion o ools
made om laked s one [2]. Al hough ich in ools and animal
bones, only some o hese si es ha e p o ided a small quan i y o
human skele al emains esembling hose om he mo e ecen
si es o he Middle Paleoli hic, da ing o he Riss-Wu¨ m
in e glacial pe iod and pa o he succeeding Wu¨ m glacia ion
(ci ca 120,000 o 36,000 ya). These bones belong o a species
named Homo sapiens neande halensis. [2] In his long Paleoli hic
pe iod, na iga ion ac oss he Medi e anean was p obably a e
and some p esen -day islands we e accessible ac oss land b idges
la e co e ed by he ising sea [3]. Du ing he Uppe Paleoli hic,
om 36,000 o 10,000 ya, he icecap expansion o he La e Glacial
Maximum (LGM) pushed sou hwa d g oups o hun e s li ing in
Cen al Eu opean a eas [1], and he Neande hals ga e way o he
p esen species o man Homo sapiens sapiens du ing he inal phases
o he Wu¨ m glacia ion. The nume ous aces om his pe iod a e
pa icula ly ich in bu ials, animal bones and ools, he la e
ha ing been wo ked wi h inc eased p ecision [2]. In he ew
housand yea s o he ollowing Mesoli hic pe iod (ci ca 10,000 o
6,000 ya) he clima e con inued o g ow milde and si es om his
pe iod ha e been ound h oughou he en i e I alian peninsula,
being along he coas s in he plains and on he moun ains. Wi h
PLOS ONE | www.plosone.o g 1 Decembe 2012 | Volume 7 | Issue 12 | e50794
he Neoli hic pe iod, om ci ca 6,000 BC o 2,800 BC, he
adi ional hun ing and ga he ing economy was eplaced by he
in oduc ion o ag icul u e, s ock ea ing, wea ing and po e y.
This new cul u al in luence came pa icula ly om he Eas e n
Medi e anean and he Nea Eas . Using he po e y p oduc ion
abo e all, i has been possible o econs uc ai ly accu a ely he
a ious phases o his complex pe iod [2]. Du ing he Coppe ,
B onze and I on ages, nume ous popula ion mo emen s occu ed
be ween he Medi e anean basin and he Middle Eas [4].
Exchange o me als would de e mine he ans o ma ion o he
i s social o ganiza ions in ancien ci iliza ions [4]. Sa dinia, Sicily
and Tuscany we e among he i s I alian e i o ies o be occupied
by humans due o hei s a egic loca ion and he p esence in hei
e i o ies o impo an me al esou ces [5].
Di e en cul u es, ecognized on he basis o di e en a che-
ological indings, se lemen s and bu ial adi ions, a ose in he
pe iod be ween he Mesoli hic and I on Age. Be o e he Roman
conques , ancien I aly was cha ac e ized only by he p esence o
Indo-Eu opean popula ions [6] li ing in he I alian Peninsula since
he second millennium BC, co esponding o he pe iod be ween
he I on Age and Romaniza ion [2]. Du ing all his pe iod he e
we e also inc easing con ac s wi h he Phoenician and G eek
colonis s: he o me being la gely p esen on he coas s o Sa dinia
and wes e n Sicily and he la e in Sou he n I aly. These colonies
had a conside able in luence on he de elopmen o local cul u es
( om he Picenian o Campano-Samni e and he Apulian o
B u io-Lucanian) [5].
The eco d o all he popula ions ha inhabi ed he I alian
e i o y du ing (p e)-his o y is incomple e; many eco ds we e o
unce ain loca ion and/o ambiguous denomina ion [6]. A he
beginning o he i s millennium BC he ollowing na i e ibes
could be dis inguished on he I alian e i o y: he Ligu es, on he
coas ha bea s hei name, in he no he n Apennine alleys, pa
o he p e-alpine alleys and he wes e n Po Valley; he Sicani, in
he in e io o Sicily; and he I ali, in p esen -day Calab ia ( om
whom comes he name ‘I aly’, which was o be ex ended o all he
e i o y o he peninsula). Besides he al eady men ioned
Te ama e ibe, on he sou he n edge o he Po Valley, and he
Villano ans, p obably om Eas e n Eu ope who se led h ough-
ou Cen al I aly, he e we e also he Umb ians o he eas o he
uppe basin o he Tibe . The Vene i, who occupied he e i o y
ha s ill bea s hei name, o iginally came om Illy ia as did he
Messapii (now mode n Salen o o Sou h Apulia) and Iapyges, who
se led in p esen -day Puglia (Apulia) [5]. Many o he popula ions
o Cen al-Sou he n I aly we e c ea ed by he mixing o local and
o eign elemen s da ing back o he p e ious millennium; i is he
case o he Sabines and La ini who se led in Lazio oge he wi h
Falisci, Aequi, Volsci, He nici and Ausones. The in e io o
Ab uzzo was domina ed by he Ves ini, Paeligni and Ma si, while
he cen al Ad ia ic coas was popula ed by Picen es, Ma ucini
and F en ani. The Apennine a ea o Molise and Basilica a was
peopled by he Samni es and Lucanians. In Calab ia and Sicily
he e we e also he B u ii and Siculi.
The Phoenician coloniza ion o he coas s o he Wes e n
Medi e anean we e mainly limi ed in I aly o Sa dinia and
wes e n Sicily and p eceded ha o he G eeks. I was ollowed by
Punic se lemen s (T apani, Pale mo, Caglia i) linked o he
ancien Phoenician colony o Ca hage.
A he ime o he Roman Empi e, a leas wo non-Indo-
Eu opean popula ions s ill inhabi ed I aly, namely, he Ligu es, in
he no hwes e n a ea, and he E uscans wi h se lemen s loca ed
in a eas a om he E u ia (Tuscany and High La ium), such as
he Po Plain and he coas o Campania. A he same ime, Sa dinia
expe ienced he lou ishing o a non-Indo-Eu opean Nu agic
ci iliza ion and, hen, he Phoenician coloniza ion.
Gene ics alone canno disen angle he ex emely complex
demog aphy o I aly h ough his o y. Some demog aphic mo e-
men s ha e howe e le signals on unipa en al and nuclea
ma ke s. Mos o he gene ic s udies a ge ed local, e.g. [7], o
egional, e.g. [8–11], I alian popula ions.
Fo he Y-ch omosome, some a emp s ha e been unde aken o
analyze I alian a ia ion o a mo e gene al scale [12–14]. Many
s udies ha e analyzed speci ic haplog oups in he Y-ch omosomes,
e.g. [15,16], o he m DNA, e.g. [8,9]. In gene al, he di e en
s udies indica e ha he gene ic s uc u e o he p esen I alian
popula ion seems o e lec , a leas in pa , he e hnic s a i ica ion
o p e-Roman imes [14]. S udies ca ied ou in he pas appea o
show a majo No h–Sou h cline consis en wi h a chaeological
es ima es o wo dis inc p ocesses: he i s coloniza ion o he a ea
du ing he Paleoli hic pe iod and he subsequen Neoli hic
expansion om he Middle Eas a e he las glacial [14]. The e
is some co espondence be ween pa e ns o a ia ion a he Y-
ch omosome and geog aphy. Thus, no he n I aly shows simila
equencies as he haplog oups o Cen al Eu ope, wi h p e alence
o he wes e n R1-M173 haplog oup compa e o he eas e n I-
M170. In he No h, E3b1-M35 and J2-M172 show low
equencies bu a e mo e p e alen in he Sou h, which has been
in e p e ed o be a signal o he gene low coming om Cen al
Eu opean Neoli hic a me s [17]. R1a1-M17 is a he a e, bo h
in he No h, whe e i p obably o igina es om eas e n Eu ope,
and in he Sou h, o possible G eek p o enience [17]. Occu ence
o J2-M172 Y-ch omosomes in Tuscany has been ela ed o he
E uscan he i age o he egion (see [17]). The wo I alian majo
islands, Sicily and Sa dinia, show a di e en demog aphic his o y.
The Y-ch omosome a iabili y o Sicily sha es a common his o y
wi h ha o sou he n I aly, en iched by an addi ional A ab
con ibu ion, bu also No h A ican and G eek in luences [18].
On he o he hand, Sa dinia has been conside ed o be a gene ic
ou lie wi hin Eu ope showing clea signals o ounde e ec s;
some schola s sugges ha i s peoples could be o ancien Ibe ian
o igin [19]; ecen gene ic s udies poin o gene ic con ibu ion
coming om sou he n F ance [20].
On he o he hand, mi ochond ial DNA s udies show ha I aly
does no di e oo much om o he Eu opean popula ions;
howe e , some popula ions ha e he same peculia i ies and
p ese e signals o he ancien pas demog aphic e en , such as
he Tuscans [8,9], o he Ladins [7,21,22]. Recen ly, pa e ns o
a ia ion obse ed in haplog oup U5b3 demons a ed o he i s
ime he exis ence o a No h I alian p e-his o ical human e uge
om he hos ile Cen al Eu opean egions co e ed by he ice o
he Las Glacial Maximum pe iod [20]; his a ea, as was also he
F anco-Can ab ian egion [23–26], se ed as a egion o
Eu opean epopula ion du ing he beginning o he Holocene.
The main aim o he p esen s udy was comp ehensi ely o
analyze he pa e ns o m DNA and Y-ch omosome a ia ion in
I aly. This s udy di e s om p e ious ones in ha : (1) i p o ides
m DNA da a om 12 new sample popula ions om I aly; (2) we
analyzed wo linguis ic isola es, Ladin and G ecani Salen ini, he
la e sampled o he i s ime in his s udy; (3) we analyzed a
sample popula ion om Luce a (Sou he n I aly) o he i s ime, a
popula ion ha acco ding o documen a ion ecei ed an impo -
an inpu o No h A ican immig an s du ing he hi een h
cen u y; (4) we analyzed he pa e ns o m DNA a ia ion in I aly
globally, ha is, by combining mo e han 3,700 con ol egion
p o iles om he li e a u e (41 popula ion samples in o al) coupled
wi h he mo e han 580 new p o iles p o ided he e; (5) Y-
ch omosome haplo ype and haplog oup pa e ns a e analyzed in
Pa e ns o m DNA Va ia ion in I aly
PLOS ONE | www.plosone.o g 2 Decembe 2012 | Volume 7 | Issue 12 | e50794
pa allel wi h he m DNA da a in o de o de e mine he possible
di e ences ha occu ed his o ically in he male e sus emale
demog aphic mo emen s; and (6) he in lux o mig an s om
A ica (No h and sub-Saha an) and o he egions is also analyzed
using phylogeog aphic in e ences, and also a model o admix u e
based on haplo ypic da a and a panel o ances y in o ma i e
ma ke s (AIMs).
Ma e ials and Me hods
E hics s a emen
W i en in o med consen was ob ained om all sample dono s.
Analysis o m DNA sequences was app o ed by he ins i u ional
e iew boa ds o he Uni e si a` Ca olica del Sac o Cuo e (Roma).
Mo eo e , he s udy con o ms o he Spanish Law o Biomedical
Resea ch (Law 14/2007- 3 o July).
Samples
A o al o 583 indi iduals we e sampled om along he I alian
Peninsula, ep esen ing 12 di e en popula ions (Figu e 1), wo o
hem (Ladin and G ecani Salen ini) being linguis ic isola es, and
he Luce a being a his o ical encla e o A abs coming om No h
A ica. A b ie desc ip ion o hese la e h ee popula ions is gi en
below.
In he I alian e i o y, he Alpine a c ep esen s one o he main
a eas o p esence o alloglo popula ions, some o hem biologically
isola ed o his o ical and geog aphic easons [27]. A he end o
he medie al pe iod (,1200 AD) and especially in he alley zone,
a i s coloniza ion o na i e peasan s began, s a ing wi h he use
o lands p e iously exploi ed only o pas u e and he lumbe .
Successi ely, wi h di e en modali ies and unde he con ol o laic
and ecclesias ical owne s, he coloniza ion p ocess in ol ed
mig an nuclei om he Ty ol, Ca in hian a ea and o he zones
[28]. Cu en ly, he Alpine a c popula ions a e di e en ia ed wi h
a ema kable cul u al di e si y ha is well ep esen ed by linguis ic
elemen s. Thus, besides he o icial main languages, nume ous
mino i y languages o dialec s a e also he cul u al pa imony o
linguis ic mino i ies [27,29]. Ladin is o en a ibu ed o be a elic
o ulga La in dialec s associa ed wi h Rhae o-Romance
languages. In he as mul i-e hnic Holy Roman Empi e, and
hen a e 1804 he Aus ian empi e, he Ladins we e le in
ela i e peace and we e allowed o con inue he use o hei
language and cul u e.
G ecani Salen ini is a Hellenic-speaking linguis ic island o
Salen o, si ua ed in sou he n Puglia, and consis ing o nine
municipali ies in which a neo-G eek dialec , also known as
G ecanic o G iko, is spoken. The o igins o his linguis ic island in
Salen ine G eece a e unce ain. The Ge man linguis G. Rohl s
p oposed i s o igin in he Magna G aecia egion; while O. Pa langeli
sugges s a Byzan ine de i a ion o he G iki o Salen o. G eek
esea che s (e.g. A. Ka anas asis) claim he inpu o Byzan ine
elemen s in he p e-exis ing Magna G aecia ma ix. The G eek
a i al in he Salen ine Peninsula occu ed bo h in he Magna
G aecia, and pos e io Byzan ine domina ions. The nume ous
illages o G ecani Salen ini had a G eek cul u e and language
and p ac iced he G eek-o hodox eligion. In he beginning o he
No man conques (ele en h cen u y), and mo e in ensi ely wi h
he a i al o di e en casa i (clans) (S e ian, Angioin, A agones,
e c), he ca holic cle gy supplan ed hose o he o hodox ai h
[30].
The Luce a popula ion has ecei ed an impo an in lux om
No h A ican A ab peoples (see [31]). Thus, a e he collapse o
he Roman Empi e in Eu ope, he A ab domina ion sp ead in o
he Medi e anean Basin. Re e ed o ei he as Moo s in Ibe ia o
Sa acens in Sou he n I aly and Sicily, A abs a i ed in Eu ope in
711 AD, and in 831 AD Ibe ia and Sicily we e almos comple ely
subjec ed o A ab domina ion [31]. In he hi een h cen u y,
F ede ick II mo ed he Sicilian A abs o he ci y o Luce a (No h
Apulia) [32]. This sample was geno yped o STRs and Y-
ch omosome SNPs in Capelli e al. [31]
To he bes o ou knowledge, all indi iduals collec ed in he
p esen s udy we e no ma e nally and pa e nally closely ela ed;
hey had di e en su names and all he dono s e e ed back a
leas wo gene a ions in he egion whe e he samples we e
collec ed.
All he samples we e analyzed o he con ol egion and
selec ed m SNPs (see below). A subse o he samples comp ised
un ela ed males (n= 292) ep esen ing se en di e en popula ions.
These samples we e geno yped o a panel o 17 Y-ch omosome
SNPs (see below), and we e p e iously geno yped o he Y ile
[33]. In addi ion, au osomal ances y in o ma i e ma ke s (AIMs)
we e geno yped in 441 indi iduals (see below).
DNA ex ac ion
Blood ex ac ion was pe o med wi h a sal ing-ou me hod [34],
modi ied and e-adap ed o buccal cells. Swabs we e incuba ed in
500 ml o 0.2 sodium ace a e, 35 ml o 10% SDS and 20 mlo
20 mg/ml P o einase K o 16 hou s a 56uC. They we e hen
emo ed and 500 ml o 3 M NaCl solu ion was added. P o eins
we e emo ed by cen i uga ion, and he DNA p ecipi a ed by
adding 1 ml o e hanol 100% a 220uC o a ew hou s. A e
cen i uga ion, he DNA pelle was wice washed wi h e hanol
70%, d ied and e-suspended in wa e . Fo he blood samples,
aliquo s o 500 ml each we e hawed and ed cells selec i ely lysed
by a 16lysis bu e . A e h ee washes wi h he lysis bu e , whi e
cells we e pelle ed and he DNA ex ac ed using he sal ing-ou
p o ocol. All he samples we e quan i ied by di ec compa ison
wi h s anda d on aga ose 1% minigels (1 g o aga ose in 100 ml o
TBE 1X- om he 1:10 dilui ion o TBE 10X).
PCR and m DNA con ol egion sequencing
M DNA has been sequenced o he comple e con ol egion,
om posi ion 16024 (in HVS-I) o 569 (in HVS-II). The i s and
second hype a iable egions (HVS-I/II) we e ampli ied ia he
polyme ase chain eac ion (PCR) and using p ime s epo ed by
A
´l a ez-Iglesias e al. [35].
PCR was ca ied ou in a 25 ml eac ion mix wi h 16 eac ion
bu e (20 mM T is-HCl, ph 8.0, 0.1 mM EDTA, 1 mM DDT,
50% ( / ) glyce ol), 1.5 mM MgCl
2
, 200 mM each dNTP,
0.4 mM each p ime , 2.5 U (Uni s). Taq polyme ase and 0.1–
1 ng DNA empla e was added o he eac ion mix u e (Taq DNA
Polyme ase, ecombinan . INVITROGENHCo po a ion). Am-
pli ica ion was ca ied ou in a GENE AMPHPCR SYSTEM
9700 (Applied Biosys ems, Fos e Ci y, Cali o nia,U.S.A.) using a
ho s a a 95uC o 1 min, ollowed by 36 cycles a 95uC o
30 sec, 55uC o 60 sec, and 72uC o 30 sec and a inal ex ension
a 72uC o 15 min. Be o e he sequencing eac ion, PCR p oduc s
we e checked by elec opho esis in polyac ylamide non-dena u -
ing gel (T9, C5), and subsequen ly he gel was s ained wi h sil e
ni a e. PCR p oduc s we e hen pu i ied wi h a Mul iSc eenH
PCR
m96
Pla e (Millipo e, Bed o d, Ma 01730, U.S.A), 96-well
de ice.The acuum-based, size exclusion sepa a ion e ec i ely
and quickly emo ed he con aining sal s, uninco po a ed dNTPs
and p ime s om PCR eac ions. Cycle sequencing was
pe o med on bo h s ands in a GENE AMPHPCR SYSTEM
9700 (AB) he mal cycle using he ABI P ismHdRhodamine
Te mina o Cycle Sequencing Ready Reac ion Ki (AB). This ki
consis s o a eac ion mix composed o : DNA-modi ied and
Pa e ns o m DNA Va ia ion in I aly
PLOS ONE | www.plosone.o g 3 Decembe 2012 | Volume 7 | Issue 12 | e50794
e mos able polyme ase, Bu e T is-HCl (pH 9.0), MgCl
2
,
dNTPs, dichlo o hodamine-ma ked ddNTPs. An aliquo e o
30 ng amplicon and 3.2 M p ime s we e added o a 2 ml
eac ion mix. Sequencing was ca ied ou using a ho s a a 96uC
o 4 min, ollowed by 36 cycles a 96uC o 15 sec, 50uC o
10 sec, 60uC o 2 sec and a inal ex ension a 60uC o 10 min.
The emo al o excess dideoxy e mina o s, p ime s and bu e
was accomplished wi h an alcoholic pu i ica ion.
The sequence p oduc s we e dena u ed wi h deionized o m-
amide and analyzed by capilla y elec opho esis on an ABI
PRISM 3130HGene ic Analyze (AB).The esul ing da a we e
analyzed wi h PE/ABD so wa e Sequencing Analysis 5.2 and
sequences we e aligned and compa ed wi h he Camb idge
sequence [36] om posi ion 16024 o16569 o HVS-I and om
posi ion 1 o 600 o HVS-II by he SeqScape .2.0 (AB).
Analysis o m DNA coding egion SNPs
Biallelic ma ke s we e geno yped using a mul iplex app oach
[37]. The selec ed SNPs we e combined in o wo mul iplex
eac ions. Mul iplex 1 included a selec ion o SNPs de ining
common Eu opean haplog oups [38]. Mul iplex 2 included
exclusi ely polymo phisms de ining sub-lineages inside hap-
log oup H. P ime s we e designed in o de o adjus he annealing
empe a u es and amplicon leng hs o allow analysis in mul iplex
eac ions [37]. The sizes o he PCR p oduc s anged om 80 o
224 bp.
Bo h mul iplexes we e pe o med using 10 ng o DNA empla e
in a 25 ml eac ion olume comp ising 16Taq Gold Bu e (AB),
200 mM o each dNTP, 2 mM MgCl
2
and 0.5 U o AmpliTaq
Gold Polyme ase (AB). Fo he p ime concen a ions, see [37].
Ampli ica ion was ca ied ou using a GENE AMPHPCR
SYSTEM 9700 (AB) he mocycle . A e a 95uC p e-incuba ion
s ep o 11 min, PCR was pe o med o a o al o 32 cycles using
he ollowing condi ions: 94uC dena u a ion o 30 sec, annealing
a 60uC o 30 sec and ex ension a 72uC o 1 min, ollowed by a
15 min inal ex ension a 72uC. PCR p oduc s we e checked by
polyac ylamide gel elec opho esis (T9, C5) isualized by sil e
s aining.
A e ampli ica ion, PCR p oduc s equi ed pu i ica ion o
emo e p ime s and uninco po a ed dNTPs. Pos -PCR pu i ica-
ion was pe o med wi h ExoSapIT (Ame shan Pha macia
Bio ech): 1 ml o PCR p oduc was incuba ed wi h 0.5 mlo
Figu e 1. Map showing he loca ion o he samples analyzed in he p esen s udy and hose collec ed om he li e a u e (see
Table 1). Pie cha s on he le display he dis ibu ion o m DNA haplog oup equencies, and hose on he igh he Y-ch omosome haplog oup
equencies.
doi:10.1371/jou nal.pone.0050794.g001
Pa e ns o m DNA Va ia ion in I aly
PLOS ONE | www.plosone.o g 4 Decembe 2012 | Volume 7 | Issue 12 | e50794
ExoSapIT o 15 min a 37uC ollowed by 15 min a 80uC o
enzyme inac i a ion. The minisequencing eac ion was pe o med
in a GENE AMPHPCR SYSTEM 9700 (AB) he mocycle
ollowing he ecommenda ions o he manu ac u e : 2 mlo
SNaPsho eady eac ion mix, 0.2 mM o ex ension p ime o
each SNP (see [37]) and 1 ml o bo h pu i ied PCR p oduc s in a
o al olume o 7 ml. The eac ion mix u e was subjec ed o 25
single base ex ension cycles o dena u a ion a 96uC o 10 sec,
annealing a 50uC o 5 sec and wi h an ex ension a 60uC du ing
30 sec. A e minisequencing eac ions, a pos -ex ension ea men
o emo e he 59-phospho yl g oup o ddNTPs aided he
p e en ion o co-mig a ion o uninco po a ed ddNTPs wi h
ex ended p ime s and p oduc ion o a high backg ound signal.
The inal olume (7 ml) was ea ed wi h 0.7 ml o SAP (Ame sham
Biosciences) o 60 min a 37uC, ollowed by 15 min a 80uC o
enzyme inac i a ion.
The minisequencing p oduc s (1.5 ml) we e mixed wi h 10 mlo
HiDi
TM
o mamide and 0.2 ml o GeneScan-120 LIZ size
s anda d (AB) and elec o o esis was pe o med on an ABI
PRISM 3130HGene ic Analyse (AB). The esul ing da a was
analyzed wi h Gene Mappe ID.
Minisequencing o SNPs cha ac e izing addi ional ypical
Eu opean haplog oups
Samples ha we e de e mined (using he SNP panel abo e) as
being de i ed om J/T (T14766C; C7028T; T4216C), U
(T14766C; C7028T; A12308G) and he U-subclade K
(T14766C; C7028T; A12308G; A10398G), we e u he geno-
yped using an addi ional se o 14 haplog oup-speci ic SNP
ma ke s ha iden i y he ollowing sub-b anches: J1 (G3010A), J1b
(G3010A; C13879T), J1c (G3010A; C114798T), J2 (G15257A),
T2a (A14687G), T2b (G5147A), U5a (A14793G), U5a1
(A14793G; A15218G), U5b (A7768G), U5b1 (A7768G;
A5656G), U5b2 (A7768G; C1721T), K1 (T14798C; T1189C),
K1a (T14798C; T1189C; C0497T) and K2 (T14798C; T1189C;
T9716C). PCR and minisequencing eac ions we e pe o med as
desc ibed abo e. Fo PCR and minisequencing p ime concen-
a ions, see Table S1.
Geno yping o Y-SNPs
Biallelic ma ke s we e geno yped using a mul iplex app oach
[39]. A se o 30 SNPs was es ed, allowing assigna ion o he
analyzed Y-ch omosome o haplog oups (Hg), ollowing he
nomencla u e and he phylogene ic ela ionships de ined om
he Y Ch omosome Conso ium [40]. The selec ed me hod o
allele disc imina ion was a single base ex ension eac ion using he
SNaPsho mul iplex ki (AB). We added he M269 ma ke o he
i s o he ou mul iplexes, in o de be e o dissec he sub-
haplog oup R1b (R1b3). The p ime s o his ma ke we e M269-F
59-TCA TGC CTA GCC TCA TTC CT-39and M269-R 59-
TCT TTT GTG TGC CTT CTG AGG-39, and he minisequen-
cing p ime 59-GGA ATG ATC AGG GTT TGG TTA AT-39.
Geno yping o AIMs
A panel o 52 AIMs we e geno yped acco ding o Sa´nchez e al.
[41] in a subse o 441 indi iduals. Se e al o he popula ion
da ase s we e used o in e -popula ion compa isons. This da a
co esponded o he CEPH panel (h p://www.cephb. /en/
cephdb/) as epo ed in HapMap (h p://hapmap.ncbi.nlm.nih.
go /) and was collec ed using he da a-mining ool SPSma
[42,43]; i includes popula ion samples om all o e he wo ld
(A ica, Eu ope, Asia, e c.); see legend o Figu e 2 o mo e
in o ma ion.
S a is ical analysis
A o al o 42 I alian popula ion samples we e analyzed o
m DNA in he p esen s udy. Compa a i e in e -popula ion
analyses we e also ca ied ou o he HVS-I segmen anging
om 16024 o 16365, since his is he analyzed segmen common
o all o hem. Haplo ype (H) and nucleo ide di e si y (p) and o he
di e si y indices [44–46] we e compu ed using DnaSP 4.10.3
so wa e [47]. P oblema ic a ia ion loca ed a ound 16189,
usually associa ed o leng h he e oplasmy e.g. 16182C o
16183C, was igno ed. Analysis o molecula a iance (AMOVA)
was ca ied ou using A lequin 3.5. [48]. Nomencla u e o m DNA
lineages ollowed p e ious s udies e.g. [23,25,38,49,50]; see
Phylo ee o a compila ion o he wo ldwide phylogeny and an
upda e o he nomencla u e based on en i e m DNA genomes
[51]. Geno yping and documen a ion e o s we e moni o ed
ollowing he phylpogene ic p inciples p e iously applied e.g. [52–
59].
Mi ochond ial DNA and Y-ch omosome da a was collec ed
om he li e a u e. The m DNA da a gene a ed in he p esen
s udy was analyzed oge he wi h 3,834 m DNA HVS-I I alian
p o iles collec ed om he li e a u e (Table S2; 76 sample
popula ions). The Y-SNPs we e analyzed oge he wi h 1,251
I alian p o iles epo ed in he li e a u e (16 popula ion samples). A
ull lis o e e ences o all he da a used in he p esen s udy is
gi en in Table S2.
Haplog oup equencies we e es ima ed by ch omosome
coun ing. S a is ical di e ences in haplog oup equencies we e
e alua ed using a Pea son’s chi- squa e es and by se ing up he
nominal signi ican alue aas 0.05.
Finally, classi ica ion o m DNA sequences in o haplog oups
was pe o med ollowing phylogene ic c i e ia (Phylo ee Build 14,
h p://www.phylo ee.o g/) and using bo h he con ol egion
sequence p o ile and m SNPs.
Resul s
Molecula di e si y o m DNA and Y-ch omosome I alian
p o iles
Di e si y indices we e compu ed o all he popula ions
analyzed in he p esen s udy and also in hose I alian popula ions
samples epo ed in he li e a u e (Tables 1 and 2). Popula ion
samples we e also g ouped in main egions (No h, Cen al, Sou h,
Wes , and Eas ) in o de o in es iga e he ole o geog aphy in he
dis ibu ion o m DNA a ia ion.
Mi ochond ial DNA haplo ypes o he samples analyzed in he
p esen s udy a e epo ed in Table S3.Table 1 shows he
molecula di e si y alues based on m DNA da a o 41 I alian
popula ion samples. The alues indica e ha he Isle o Elba is, by
a , he I alian popula ion sample ha shows he lowes di e si y
o all he indices compu ed, p obably as a consequence o i s
ela i e isola ion om he coun y. I has been epo ed ha his
was a well-known encla e o E uscan in luence, and some
m DNA pa icula i ies ha e been desc ibed be o e [8,9]. Al e na-
i ely, low molecula di e si y could be due o low sample sizes,
al hough his ac is mi o ed in he s anda d de ia ion o he
di e en es ima es. Excluding he Isle o Elba, haplo ype di e si y
in I aly anges om 0.834 o 1, nucleo ide di e si y om 0.01003
o 0.02409, and he a e age alue o nucleo ide di e ences om
3.4 o 8.19 (a alue ha is co ela ed wi h he nucleo ide di e si y).
In gene al, I aly shows some le el o he e ogenei y when examined
o di e si y alues.
When g ouping popula ions by main geog aphical egions, i
can be obse ed ha Cen al I aly has sligh ly lowe alues han
No h and Sou h I aly o all he indices compu ed (Table 1). The
Pa e ns o m DNA Va ia ion in I aly
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highe di e si y alues we e ound in Sou h I aly. Di e si y alues
a e howe e e y simila when examining popula ions loca ed in
Wes I aly e sus hose in he Eas . The inclusion o Sicily (as pa o
Sou h I aly) in he compu a ion does no subs an ially change hese
es ima es (Table 1).
Y-SNP da a we e ob ained o all he samples analyzed in he
p esen s udy (Table S4). Table 2 shows he di e si y indices o
he Y-SNPs in di e en I alian popula ions. The Y-STR di e si y
alues o he samples analyzed in he p esen s udy and o he
I alian and Eu opean samples ha e al eady been epo ed in
B isighelli e al. [33]. As expec ed, di e si y alues o Y-SNP
haplog oup pa e ns a e lowe han hose ob ained o he m DNA
haplo ypes gi en ha he indices a e based on haplog oup and no
on Y-STR haplo ypes. In ac , alues based on Y-STR p o iles
(minimum o ex ended Y ile p o iles) [33] a e highe han hose
obse ed o he HVS-I p o iles. Ladins a e among he popula ions
wi h he lowes Y-SNP di e si y alues, while he G ecani
Salen ini show di e si y alues ha a e compa able o o he
I alian samples. Modena shows ema kable low haplo ype
di e si y alues.
Phylogeog aphy
The m DNA haplog oup make-up o I aly as obse ed in ou
samples i s well wi h expec a ions in a ypical Eu opean
popula ion. Thus, mos o he I alian m DNAs (,89%) could be
a ibu ed o Eu opean haplog oups H (,40%), I (,3%), J (,9%),
T(,11%), U (,20%; U minus U6), V (,3%), X (,2%) and W
(,1%); Figu e 1. The e a e howe e impo an di e ences in
haplog oup equencies when examining hem by main geog aph-
ical egions. Thus, o ins ance, haplog oup H is 59% in he
No h, 46% in he Cen e , and decays o ,33% in he Sou h;
mo eo e , hese egional di e ences a e s a is ically signi ican :
No h s Sou h (Pea son’s chi-squa e, unadjus ed-P al-
ue,0.00003), and Cen e s Sou h (Pea son’s chi-squa e, unad-
jus ed-P alue,0.03724).
Mi ochond ial DNA haplo ypes o A ican o igin a e mainly
ep esen ed by haplog oups M1 (0.3%), U6 (0.8%) and L (1.2%);
om he e onwa ds, L will be used o e e o all m DNA lineages,
excluding he non-A ican b anches N and M [60,61].
A o al o 282 Y-ch omosomes we e analyzed o a se o Y-
SNPs and we e classi ied in o 22 di e en haplog oups (Figu e 3).
Two haplog oups we e no ound, e en hough ma ke s de ining
hese clades we e es ed: N3 and R1a1. Fi e haplog oups
ep esen ed 76.71% o he o al ch omosomes: R1b3, J2,
I(xI1b2), E3b1 and G. The equencies a e aged ac oss popula-
ions we e 26%, 21.2%, 10.2%, 9.9% and 9.2%, espec i ely. The
emaining haplog oups sum o 23.2% in he o al sample, and
ne e abo e 4% in single popula ion samples.
R1b3 equency was ound o be highe in he no he n pa o
he coun y, while he Y-ch omosome haplog oups G and E3b1,
J2 and I(xI1b2) equencies we e highe in he sou h and in he
cen al pa o he coun y, espec i ely (Figu e 1).
Regional di e ences a e subs an ially highe in he Y-ch omo-
some han in he m DNA. Thus, o ins ance, haplog oup R in he
Y-ch omosome was 54% in he No h, 18% in he Cen e , and
31% in he Sou h. F equency di e ences we e s a is ically
signi ican be ween No h s Cen e (Pea son’s chi-squa e,
unadjus ed-P alue = 0.0014), and No h s Sou h (Pea son’s chi-
Figu e 2. Analysis o AIMs in I alian popula ions
e sus
o he con inen al popula ion g oups. (A) PCA o I alian popula ions di ided in o
he main egions No h, Cen e and Sou h (as analyzed in he p esen s udy) and o he Eu opean popula ions; (B) he same I alian popula ions plus
sub-Saha an A ican, and Asian popula ions; (C) iangle plo as ob ained using STRUCTURE analysis o I alian, Eu opean, sub-Saha an, and Asian
popula ions; (D) ba plo o ances al membe ship alues as ob ained using STRUCTURE analysis o he same popula ions used in (C). Popula ion
codes: 1: Angola; 2: Kenya-Ban u NE; 3: Mozambique; 4: Namibia-San; 5: Nige ia-Yo uba; 6: Senegal-Mandenka; 7: Sou h A ica-Ban u; 8: Uganda; 9:
B i ain; 10: Denma k; 11: F ench; 12: Ge many; 13: I eland; 14*: NW Spain; 15*: Po ugal; 16: Slo enia; 17: China-Dai; 18: China-Da u; 19: China-Han; 20:
China-Hezhen; 21: Japanese; 22: Mongolia; 23: Taiwan; 24: Thailand. Geno ypes we e downloaded using he me hod in [43,83] and belong o he
CEPH panel. An as e isk indica es Medi e anean popula ions.
doi:10.1371/jou nal.pone.0050794.g002
Pa e ns o m DNA Va ia ion in I aly
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Table 1. Di e si y indices compu ed o di e en I alian egions based on HVS-I da a (sequence segmen 16090–16365).
Popula ion Region Pop ID Re e ence N k k/n S h
P
M
Ligu i NW 1 p.s. 50 40 0.8 53 0.96260.021 0.0142660.0145 4.875
To ino NW 5 [84] 50 45 0.9 49 0.99360.007 0.0148360.0011 5.056
Ladin NE 2, 13 p.s. [22,62,63,66] 504 170 0.3 106 0.96060.005 0.0125160.0004 4.252
Pa ia NE 6 [84] 47 35 0.7 44 0.96960.017 0.0131660.0012 4.502
Udine NE 3 p.s. 51 32 0.6 38 0.90360.038 0.0123160.0135 4.19858
A ezzo/Chiusi CW 1 [9] 14 14 1 22 1.00060.027 0.0148860.0129 5.088
Casen ino CW 15 [8] 122 77 0.6 167 0.97960.007 0.0240960.0082 8.190
Colle ecchio/Magliano Sabino CW 3 [9] 12 11 0.9 14 0.98560.040 0.0120160.0015 4.106
Elba CW 2 [9] 16 6 0.4 11 0.68360.120 0.0085360.0017 2.908
Fi enze CW 9 [84] 48 40 0.8 54 0.98060.014 0.0133260.0012 4.556
Jenne CW 22 [85] 103 34 0.3 47 0.83460.036 0.0100660.0360 3.440
La ini CW 5 p.s. 48 29 0.6 35 0.90260.039 0.0100360.0010 3.429
La ium CW 20 [86] 52 37 0.7 48 0.95960.019 0.0131360.0014 4.492
Mu lo CW 16 [8] 86 60 0.7 68 0.97660.010 0.0132760.0009 4.524
Roma CW 12 [84] 58 49 0.8 55 0.98760.008 0.0143360.0011 4.901
Te ni CW 11 [84] 29 20 0.7 33 0.94160.034 0.0120160.0014 4.108
Tuscany CW 4 [9,10,87] 127 86 0.7 77 0.98260.007 0.0130560.0075 4.464
Vallepie a CW 21 [85] 21 8 0.4 17 0.87160.044 0.0128160.0014 4.381
Vol e a CW 14 [8] 114 57 0.5 62 0.95560.013 0.0119360.0007 4.057
Ab uzzo CE 17 [86,88] 61 53 0.8 62 0.99060.007 0.0150060.0010 5.131
Ancona CE 10 [84] 73 55 0.7 59 0.96360.017 0.0137960.0010 4.717
Bologna CE 7 [84,89] 146 79 0.5 64 0.97060.008 0.0125060.0006 4.278
Cen e Eas CE 23 [90] 83 62 0.7 60 0.97460.012 0.0135260.0009 4.625
C oa ian I alians CE 19 [86] 41 28 0.7 46 0.97060.015 0.0152460.0017 5.213
Modena CE 8 [84] 44 33 0.7 43 0.95860.023 0.0113960.0012 3.895
Molise CE 18 [86] 62 41 0.6 58 0.93860.025 0.0126060.0013 4.309
Piceni CE 4 p.s. 53 43 0.8 56 0.98560.009 0.0130660.0011 4.414
Bel ede e SW 10 p.s. 50 41 0.8 44 0.98060.013 0.0132060.0010 4.532
Calab ia SW 27 [91,92] 389 213 0.5 128 0.98360.003 0.0152160.0004 5.203
Campania SW 30 [86] 48 41 0.8 59 0.98060.014 0.0151960.0014 5.166
Ca ania SW 11 p.s. 40 35 0.9 45 0.99060.010 0.0146060.0012 4.979
Sicily SW 28 [38,93–96] 558 240 0.4 125 0.95860.006 0.0128960.0004 4.343
T apani SW 12 p.s. 40 30 0.7 36 0.97760.013 0.0131360.0013 4.465
Apulia SE 26 [86] 26 24 0.9 43 0.99160.015 0.0155060.0022 5.304
Basilica a SE 25 [91] 92 65 0.7 70 0.98360.007 0.0129060.0008 4.428
G ecani Salen ini SE 8 p.s. 47 37 0.8 44 0.98960.007 0.0131060.0011 4.480
Luce a SE 6 p.s. 60 42 0.7 55 0.97660.011 0.0134560.0011 4.586
Sou h Apulia SE 9 p.s. 53 38 0.7 49 0.97360.014 0.0157960.0010 5.401
Sanni i SE 7 p.s. 50 41 0.8 49 0.98860.008 0.0142060.0013 4.843
Sa dinia – 29 [38,87,97] 351 171 0.4 98 0.95060.009 0.0118360.0004 4.033
Geog aphical egion
No h I aly – – 702 267 0.4 126 0.96360.004 0.0128260.0004 4.295
Cen al I aly – – 1413 500 0.4 216 0.95860.004 0.0124360.0002 4.113
Sou h I aly – – 1453 569 0.4 183 0.97360.002 0.0136860.0002 4.541
Wes I aly (wi hou Sicily) – – 1437 578 0.4 232 0.96960.003 0.0131560.0002 4.405
Wes I aly (wi h Sicily) – – 2075 709 0.3 236 0.96360.003 0.0126060.0002 4.133
Eas I aly – – 1493 520 0.3 165 0.96460.003 0.0127760.0002 4.200
NW = No h-Wes ; NE = No h-Eas ; CW = Cen e -Wes ; CE = Cen e -Eas ; SW = Sou h-Wes ; SE = Sou h-Eas ; N= sample size; k = numbe o di e en haplo ypes;
S = seg ega ing si es; h = haplo ype di e si y; p= nucleo ide di e si y; M = a e age numbe o nucleo ide di e ences.
doi:10.1371/jou nal.pone.0050794. 001
Pa e ns o m DNA Va ia ion in I aly
PLOS ONE | www.plosone.o g 7 Decembe 2012 | Volume 7 | Issue 12 | e50794
squa e, unadjus ed-P alue,0.00004). Haplog oup J2 also e-
ealed impo an egional di e ences; i added o 9% in he No h,
37% in he Cen e , and 22% in he Sou h, wi h s a is ically
signi ican di e ences be ween he No h s Cen e (Pea son’s chi-
squa e, unadjus ed-P alue,0.00002), No h s Sou h (Pea son’s
chi-squa e, unadjus ed-P alue,0.00148), and in he limi o
signi icance Cen e s Sou h (Pea son’s chi-squa e, unadjus ed-P
alue,0.049).
Au osomal ances y in I aly
A panel o 52 AIMs was geno yped in 435 I alian indi iduals in
o de o es ima e he p opo ion o ances y om a h ee-way
di e en ia ion: sub-Saha an A ica, Eu ope and Asia. S uc u e
analyses allowed us o in e membe ship p opo ions in popula ion
samples, and hese p opo ions can be g aphically displayed, as in
Figu e 2. This analysis indica ed ha I alians ha e a basal
p opo ion o sub-Saha an ances y ha is highe (9.2%, on
a e age) han o he cen al o no he n Eu opean popula ions
(1.5%, on a e age). The amoun o A ican ances y in I alians is
howe e mo e compa able o (bu sligh ly highe han) he a e age
in o he Medi e anean coun ies (7.1%). Figu e 2 shows in a
iangle plo he ela ionships o I alians compa ed o o he
Eu opean, A ican and Asian popula ions.
PCA obse a ions con i med he esul s om S uc u e analysis,
clus e ing I alian p o iles igh ly wi h o he Eu opean ones. Thus,
PCA indica ed ha No h, Cen al and Sou h I aly do no show
di e ences be ween hem, no om o he Eu opean popula ions
(Figu e 2). PCA also indica ed clea -cu di e ences be ween
I alians, A icans and Asians (Figu e 2).
AMOVA
AMOVA analyses we e ca ied ou ollowing di e en g ouping
schemes. The samples we e pooled in o a single popula ion, bu
also by conside ing main I alian egions. Analyses we e ca ied ou
o e haplog oups and haplo ypes o he Y-ch omosome and he
m DNA (Table 3).
AMOVA indica ed ha , among popula ions, a iance was
mo e s ongly s a i ied o he Y-ch omosome han o he
Table 2. Di e si y indices compu ed o di e en I alian egions based on Y-SNPs.
Popula ion Region Re e ence N k k/n Gene Di e si y
Ligu ia NW P esen s udy 46 9 0.19 0.766260.0502
Ladin NE [14] 34 6 0.17 0.534860.0979
Udine NE P esen s udy 47 10 0.21 0.776160.0441
Cen al Tuscany CW [14] 40 8 0.20 0.739760.0616
Elba Island CW [14] 94 7 0.07 0.674260.0445
La ini CW P esen s udy 44 11 0.25 0.825660.0395
La ium CW [14] 43 9 0.20 0.802660.0388
Tuscany-La ium bo de CW [14] 76 7 0.09 0.755460.0350
Cen al Ma che CE [14] 59 7 0.11 0.729460.0364
Ma che CE [11] 162 13 0.08 0.848960.0152
Ma che-Appennine CE [14] 25 7 0.28 0.803360.0514
Modena CE [98] 62 8 0.12 0.532060.0743
Piceni CE P esen s udy 38 9 0.23 0.820860.0450
Rimini-Val Ma ecchia CE [99] 163 12 0.35 0.699060.0308
Bel ede e SW P esen s udy 27 9 0.33 0.854760.0477
Eas Campania SW [14] 46 7 0.15 0.687060.0618
Sicily SW P esen s udy 57 12 0.21 0.832760.0311
Wes Campania SW [14] 80 10 0.12 0.844660.0224
Wes Calab ia SW [14] 57 7 0.12 0.752560.0307
Sanni i SE P esen s udy 30 10 0.33 0.864460.0409
G ecani Salen ini SE P esen s udy 47 7 0.14 0.812260.0242
Luce a SE [31] 60 9 0.15 0.836560.0236
Sou h Apulia SE [14] 49 9 0.18 0.852960.0237
Sa dinia [100] 336 14 0.04 0.809860.0136
Geog aphical egion
No h I aly – – 127 14 0.11 0.840060.0189
Cen al I aly – – 806 21 0.03 0.887060.0053
Sou h I aly – – 453 20 0.04 0.890960.0060
Wes I aly (wi hou Sicily) – – 553 17 0.03 0.856760.0094
Wes I aly (wi h Sicily) – – 610 20 0.03 0.870560.0078
Eas I aly – – 776 22 0.02 0.903460.0037
Codes a e as in Table 1.
doi:10.1371/jou nal.pone.0050794. 002
Pa e ns o m DNA Va ia ion in I aly
PLOS ONE | www.plosone.o g 8 Decembe 2012 | Volume 7 | Issue 12 | e50794
m DNA; he di e ence was much mo e ma ked o he analysis
based on haplog oups (14.39% s 1.17%) han o he analysis
based on haplo ypes (2.34% s 0.79%). Among popula ion
a iance was e y low when analyzing main geog aphical egions;
howe e , i was he la i ude (No h s Cen e s Sou h) ha
appea ed o accoun o highe alues o among-popula ion
a iance a he han longi ude (Wes s Eas ), wi h he excep ion o
he Y-ch omosome haplog oups (al hough he alues a e below
1%); Table 3. Again, he Y-ch omosome showed sligh ly highe
alues o among-popula ion a iance han did he m DNA. Fo
he Y-ch omosome, a signi ican p opo ion o he wi hin-
popula ion a iance mo ed o among-popula ion wi hin-g oups
a iance, p obably due o he ac ha all popula ion samples had
a e y high p opo ion o single on Y ile haplo ypes, ele a ing he
maximum alues o haplog oup di e si y o all o hem [33].
Linguis ic isola es: Ladin and G ecani Salen ini
Two linguis ic isola es a e ep esen ed in he samples analyzed
in he p esen s udy: he Ladin and he G ecani Salen ini.
O he popula ion samples o he Ladin ha e al eady been
analyzed in he li e a u e [22,62,63]. We he e sampled 41 new
Figu e 3. Phylogeny o Y-ch omosome SNPs and haplog oup equencies in di e en I alian popula ions.
doi:10.1371/jou nal.pone.0050794.g003
Table 3. AMOVA analysis o main I alian egions (Pe mu a ions: 20000; P- alue,0.0000) o he m DNA con ol egion da a and
he Y-ch omosome STRs and SNPs.
All popula ions (%)
No h s Cen e s Sou h
(%) Wes s Eas (%)
HAPLOTYPES
m DNA (48 popula ions)
Among pops 0.79 0 0
Wi hin pops 99.21 99.25 99.21
Among pops wi hin g oups – 0.75 0.79
Y-ch omosome (15 popula ions)
Among pops 2.34 1.18 0
Wi hin pops 97.66 97.32 97.85
Among pops wi hin g oups – 1.50 2.15
HAPLOGROUPS
m DNA (19 popula ions)
Among pops 1.17 0.36 0
Wi hin pops 98.83 98.72 98.83
Among pops wi hin g oups – 0.92 1.17
Y-ch omosome (24 popula ions)
Among pops 13.92 0.07 0.83
Wi hin pops 86.08 86.06 85.74
Among pops wi hin g oups – 13.87 13.44
Sa dinians we e no included in he analysis. Re e ences o popula ion samples a e gi en in Table S2.
doi:10.1371/jou nal.pone.0050794. 003
Pa e ns o m DNA Va ia ion in I aly
PLOS ONE | www.plosone.o g 9 Decembe 2012 | Volume 7 | Issue 12 | e50794