Uniparental markers of contemporary italian population reveals details on its pre-roman heritage

Uniparental Markers of Contemporary Italian Population Reveals Details on Its Pre-Roman Heritage Francesca Brisighelli 1,2,3. , Vanesa A ´lvarez-Iglesias 1 , Manuel Fondevila 1 , Alejandro Blanco-Verea 1 , A ´ngel Carracedo 1,4 , Vincenzo L. Pascali 2 , Cristian Capelli 3 , Antonio Salas 1 * . 1Unidade de Xene ´tica, Facultade de Medicina, Instituto de Medicina Legal, Universidade de Santiago de Compostela, Galicia, Spain, 2Forensic Genetics Laboratory, Institute of Legal Medicine, Universita `Cattolica del Sacro Cuore, Rome, Italy, 3Department of Zoology, University of Oxford, Oxford, United Kingdom, 4Fundacio ´nPu ´blica Galega de Medicina Xeno ´mica (FPGMX-SERGAS), CIBER enfermedades raras, Santiago de Compostela, Galicia, Spain Abstract Background: According to archaeological records and historical documentation, Italy has been a melting point for populations of different geographical and ethnic matrices. Although Italy has been a favorite subject for numerous population genetic studies, genetic patterns have never been analyzed comprehensively, including uniparental and autosomal markers throughout the country. Methods/Principal Findings: A total of 583 individuals were sampled from across the Italian Peninsula, from ten distant (if homogeneous by language) ethnic communities — and from two linguistic isolates (Ladins, Grecani Salentini). All samples were first typed for the mitochondrial DNA (mtDNA) control region and selected coding region SNPs (mtSNPs). This data was pooled for analysis with 3,778 mtDNA control-region profiles collected from the literature. Secondly, a set of Y- chromosome SNPs and STRs were also analyzed in 479 individuals together with a panel of autosomal ancestry informative markers (AIMs) from 441 samples. The resulting genetic record reveals clines of genetic frequencies laid according to the latitude slant along continental Italy – probably generated by demographical events dating back to the Neolithic. The Ladins showed distinctive, if more recent structure. The Neolithic contribution was estimated for the Y-chromosome as 14.5% and for mtDNA as 10.5%. Y-chromosome data showed larger differentiation between North, Center and South than mtDNA. AIMs detected a minor sub-Saharan component; this is however higher than for other European non-Mediterranean populations. The same signal of sub-Saharan heritage was also evident in uniparental markers. Conclusions/Significance: Italy shows patterns of molecular variation mirroring other European countries, although some heterogeneity exists based on different analysis and molecular markers. From North to South, Italy shows clinal patterns that were most likely modulated during Neolithic times. Citation: Brisighelli F, A ´lvarez-Iglesias V, Fondevila M, Blanco-Verea A, Carracedo A ´, et al. (2012) Uniparental Markers of Contemporary Italian Population Reveals Details on Its Pre-Roman Heritage. PLoS ONE 7(12): e50794. doi:10.1371/journal.pone.0050794 Editor: David Caramelli, University of Florence, Italy Received June 15, 2012; Accepted October 24, 2012; Published December 10, 2012 Copyright: ß2012 Brisighelli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007–2013/under REA grant agreement number 290344, and the Ministerio de Ciencia e Innovacio ´n (SAF2008-02971 and SAF2011- 26983)(AS). CC and FB were partially funded by the British Academy for the project ‘‘The Greeks in the West: the genetic legacy of the colonisation in South Italy and Sicily’’. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]s .These authors contributed equally to this work. Introduction Italy has historically been a convenient destination for human populations migrating from Africa, the Middle East and European locations, in part due to the geomorphological characteristics of the Italian Peninsula [1]. These groups settled preferentially on the islands and coastal territories [1] 500,000 years ago (ya), that is, along the Lower Paleolithic, the longest period of human prehistory, which was dominated by the notable diffusion of tools made from flaked stone [2]. Although rich in tools and animal bones, only some of these sites have provided a small quantity of human skeletal remains resembling those from the more recent sites of the Middle Paleolithic, dating to the Riss-Wu¨rm interglacial period and part of the succeeding Wu¨rm glaciation (circa 120,000 to 36,000 ya). These bones belong to a species named Homo sapiens neanderthalensis. [2] In this long Paleolithic period, navigation across the Mediterranean was probably rare and some present-day islands were accessible across land bridges later covered by the rising sea [3]. During the Upper Paleolithic, from 36,000 to 10,000 ya, the icecap expansion of the Late Glacial Maximum (LGM) pushed southward groups of hunters living in Central European areas [1], and the Neanderthals gave way to the present species of man Homo sapiens sapiens during the final phases of the Wu¨rm glaciation. The numerous traces from this period are particularly rich in burials, animal bones and tools, the latter having been worked with increased precision [2]. In the few thousand years of the following Mesolithic period (circa 10,000 to 6,000 ya) the climate continued to grow milder and sites from this period have been found throughout the entire Italian peninsula, being along the coasts in the plains and on the mountains. With PLOS ONE | www.plosone.org 1 December 2012 | Volume 7 | Issue 12 | e50794 the Neolithic period, from circa 6,000 BC to 2,800 BC, the traditional hunting and gathering economy was replaced by the introduction of agriculture, stock rearing, weaving and pottery. This new cultural influence came particularly from the Eastern Mediterranean and the Near East. Using the pottery production above all, it has been possible to reconstruct fairly accurately the various phases of this complex period [2]. During the Copper, Bronze and Iron ages, numerous population movements occurred between the Mediterranean basin and the Middle East [4]. Exchange of metals would determine the transformation of the first social organizations in ancient civilizations [4]. Sardinia, Sicily and Tuscany were among the first Italian territories to be occupied by humans due to their strategic location and the presence in their territories of important metal resources [5]. Different cultures, recognized on the basis of different archeological findings, settlements and burial traditions, arose in the period between the Mesolithic and Iron Age. Before the Roman conquest, ancient Italy was characterized only by the presence of Indo-European populations [6] living in the Italian Peninsula since the second millennium BC, corresponding to the period between the Iron Age and Romanization [2]. During all this period there were also increasing contacts with the Phoenician and Greek colonists: the former being largely present on the coasts of Sardinia and western Sicily and the latter in Southern Italy. These colonies had a considerable influence on the development of local cultures (from the Picenian to Campano-Samnite and the Apulian to Bruttio-Lucanian) [5]. The record of all the populations that inhabited the Italian territory during (pre)-history is incomplete; many records were of uncertain location and/or ambiguous denomination [6]. At the beginning of the first millennium BC the following native tribes could be distinguished on the Italian territory: the Ligures, on the coast that bears their name, in the northern Apennine valleys, part of the pre-alpine valleys and the western Po Valley; the Sicani, in the interior of Sicily; and the Itali, in present-day Calabria (from whom comes the name ‘Italy’, which was to be extended to all the territory of the peninsula). Besides the already mentioned Terramare tribe, on the southern edge of the Po Valley, and the Villanovans, probably from Eastern Europe who settled throughout Central Italy, there were also the Umbrians to the east of the upper basin of the Tiber. The Veneti, who occupied the territory that still bears their name, originally came from Illyria as did the Messapii (now modern Salento or South Apulia) and Iapyges, who settled in present-day Puglia (Apulia) [5]. Many other populations of Central-Southern Italy were created by the mixing of local and foreign elements dating back to the previous millennium; it is the case of the Sabines and Latini who settled in Lazio together with Falisci, Aequi, Volsci, Hernici and Ausones. The interior of Abruzzo was dominated by the Vestini, Paeligni and Marsi, while the central Adriatic coast was populated by Picentes, Marrucini and Frentani. The Apennine area of Molise and Basilicata was peopled by the Samnites and Lucanians. In Calabria and Sicily there were also the Bruttii and Siculi. The Phoenician colonization of the coasts of the Western Mediterranean were mainly limited in Italy to Sardinia and western Sicily and preceded that of the Greeks. It was followed by Punic settlements (Trapani, Palermo, Cagliari) linked to the ancient Phoenician colony of Carthage. At the time of the Roman Empire, at least two non-Indo- European populations still inhabited Italy, namely, the Ligures, in the northwestern area, and the Etruscans with settlements located in areas far from the Etruria (Tuscany and High Latium), such as the Po Plain and the coast of Campania. At the same time, Sardinia experienced the flourishing of a non-Indo-European Nuragic civilization and, then, the Phoenician colonization. Genetics alone cannot disentangle the extremely complex demography of Italy through history. Some demographic movements have however left signals on uniparental and nuclear markers. Most of the genetic studies targeted local, e.g. [7], or regional, e.g. [8–11], Italian populations. For the Y-chromosome, some attempts have been undertaken to analyze Italian variation to a more general scale [12–14]. Many studies have analyzed specific haplogroups in the Y-chromosomes, e.g. [15,16], or the mtDNA, e.g. [8,9]. In general, the different studies indicate that the genetic structure of the present Italian population seems to reflect, at least in part, the ethnic stratification of pre-Roman times [14]. Studies carried out in the past appear to show a major North–South cline consistent with archaeological estimates of two distinct processes: the first colonization of the area during the Paleolithic period and the subsequent Neolithic expansion from the Middle East after the last glacial [14]. There is some correspondence between patterns of variation at the Y- chromosome and geography. Thus, northern Italy shows similar frequencies as the haplogroups of Central Europe, with prevalence of the western R1-M173 haplogroup compare to the eastern I- M170. In the North, E3b1-M35 and J2-M172 show low frequencies but are more prevalent in the South, which has been interpreted to be a signal of the gene flow coming from Central European Neolithic farmers [17]. R1a1-M17 is rather rare, both in the North, where it probably originates from eastern Europe, and in the South, of possible Greek provenience [17]. Occurrence of J2-M172 Y-chromosomes in Tuscany has been related to the Etruscan heritage of the region (see [17]). The two Italian major islands, Sicily and Sardinia, show a different demographic history. The Y-chromosome variability of Sicily shares a common history with that of southern Italy, enriched by an additional Arab contribution, but also North African and Greek influences [18]. On the other hand, Sardinia has been considered to be a genetic outlier within Europe showing clear signals of founder effects; some scholars suggest that its peoples could be of ancient Iberian origin [19]; recent genetic studies point to genetic contribution coming from southern France [20]. On the other hand, mitochondrial DNA studies show that Italy does not differ too much from other European populations; however, some populations have the same peculiarities and preserve signals of the ancient past demographic event, such as the Tuscans [8,9], or the Ladins [7,21,22]. Recently, patterns of variation observed in haplogroup U5b3 demonstrated for the first time the existence of a North Italian pre-historical human refuge from the hostile Central European regions covered by the ice of the Last Glacial Maximum period [20]; this area, as was also the Franco-Cantabrian region [23–26], served as a region of European repopulation during the beginning of the Holocene. The main aim of the present study was comprehensively to analyze the patterns of mtDNA and Y-chromosome variation in Italy. This study differs from previous ones in that: (1) it provides mtDNA data from 12 new sample populations from Italy; (2) we analyzed two linguistic isolates, Ladin and Grecani Salentini, the latter sampled for the first time in this study; (3) we analyzed a sample population from Lucera (Southern Italy) for the first time, a population that according to documentation received an important input of North African immigrants during the thirteenth century; (4) we analyzed the patterns of mtDNA variation in Italy globally, that is, by combining more than 3,700 control region profiles from the literature (41 population samples in total) coupled with the more than 580 new profiles provided here; (5) Y- chromosome haplotype and haplogroup patterns are analyzed in Patterns of mtDNA Variation in Italy PLOS ONE | www.plosone.org 2 December 2012 | Volume 7 | Issue 12 | e50794 parallel with the mtDNA data in order to determine the possible differences that occurred historically in the male versus female demographic movements; and (6) the influx of migrants from Africa (North and sub-Saharan) and other regions is also analyzed using phylogeographic inferences, and also a model of admixture based on haplotypic data and a panel of ancestry informative markers (AIMs). Materials and Methods Ethics statement Written informed consent was obtained from all sample donors. Analysis of mtDNA sequences was approved by the institutional review boards of the Universita` Cattolica del Sacro Cuore (Roma). Moreover, the study conforms to the Spanish Law for Biomedical Research (Law 14/2007- 3 of July). Samples A total of 583 individuals were sampled from along the Italian Peninsula, representing 12 different populations (Figure 1), two of them (Ladin and Grecani Salentini) being linguistic isolates, and the Lucera being a historical enclave of Arabs coming from North Africa. A brief description of these latter three populations is given below. In the Italian territory, the Alpine arc represents one of the main areas of presence of alloglot populations, some of them biologically isolated for historical and geographic reasons [27]. At the end of the medieval period (,1200 AD) and especially in the valley zone, a first colonization of native peasants began, starting with the use of lands previously exploited only for pasture and the lumber. Successively, with different modalities and under the control of laic and ecclesiastical owners, the colonization process involved migrant nuclei from the Tyrol, Carinthian area and other zones [28]. Currently, the Alpine arc populations are differentiated with a remarkable cultural diversity that is well represented by linguistic elements. Thus, besides the official main languages, numerous minority languages or dialects are also the cultural patrimony of linguistic minorities [27,29]. Ladin is often attributed to be a relic of vulgar Latin dialects associated with Rhaeto-Romance languages. In the vast multi-ethnic Holy Roman Empire, and then after 1804 the Austrian empire, the Ladins were left in relative peace and were allowed to continue the use of their language and culture. Grecani Salentini is a Hellenic-speaking linguistic island of Salento, situated in southern Puglia, and consisting of nine municipalities in which a neo-Greek dialect, also known as Grecanic or Griko, is spoken. The origins of this linguistic island in Salentine Greece are uncertain. The German linguist G. Rohlfs proposed its origin in the Magna Graecia region; while O. Parlangeli suggests a Byzantine derivation of the Griki of Salento. Greek researchers (e.g. A. Karanastasis) claim the input of Byzantine elements in the pre-existing Magna Graecia matrix. The Greek arrival in the Salentine Peninsula occurred both in the Magna Graecia, and posterior Byzantine dominations. The numerous villages of Grecani Salentini had a Greek culture and language and practiced the Greek-orthodox religion. In the beginning of the Norman conquest (eleventh century), and more intensively with the arrival of different casati (clans) (Svevian, Angioin, Aragones, etc), the catholic clergy supplanted those of the orthodox faith [30]. The Lucera population has received an important influx from North African Arab peoples (see [31]). Thus, after the collapse of the Roman Empire in Europe, the Arab domination spread into the Mediterranean Basin. Referred to either as Moors in Iberia or Saracens in Southern Italy and Sicily, Arabs arrived in Europe in 711 AD, and in 831 AD Iberia and Sicily were almost completely subjected to Arab domination [31]. In the thirteenth century, Frederick II moved the Sicilian Arabs to the city of Lucera (North Apulia) [32]. This sample was genotyped for STRs and Y- chromosome SNPs in Capelli et al. [31] To the best of our knowledge, all individuals collected in the present study were not maternally and paternally closely related; they had different surnames and all the donors referred back at least two generations in the region where the samples were collected. All the samples were analyzed for the control region and selected mtSNPs (see below). A subset of the samples comprised unrelated males (n= 292) representing seven different populations. These samples were genotyped for a panel of 17 Y-chromosome SNPs (see below), and were previously genotyped for the Yfiler [33]. In addition, autosomal ancestry informative markers (AIMs) were genotyped in 441 individuals (see below). DNA extraction Blood extraction was performed with a salting-out method [34], modified and re-adapted to buccal cells. Swabs were incubated in 500 ml of 0.2 sodium acetate, 35 ml of 10% SDS and 20 mlof 20 mg/ml Proteinase K for 16 hours at 56uC. They were then removed and 500 ml of 3 M NaCl solution was added. Proteins were removed by centrifugation, and the DNA precipitated by adding 1 ml of ethanol 100% at 220uC for a few hours. After centrifugation, the DNA pellet was twice washed with ethanol 70%, dried and re-suspended in water. For the blood samples, aliquots of 500 ml each were thawed and red cells selectively lysed by a 16lysis buffer. After three washes with the lysis buffer, white cells were pelleted and the DNA extracted using the salting-out protocol. All the samples were quantified by direct comparison with standard on agarose 1% minigels (1 g of agarose in 100 ml of TBE 1X- from the 1:10 diluition of TBE 10X). PCR and mtDNA control region sequencing MtDNA has been sequenced for the complete control region, from position 16024 (in HVS-I) to 569 (in HVS-II). The first and second hypervariable regions (HVS-I/II) were amplified via the polymerase chain reaction (PCR) and using primers reported by A ´lvarez-Iglesias et al. [35]. PCR was carried out in a 25 ml reaction mix with 16reaction buffer (20 mM Tris-HCl, ph 8.0, 0.1 mM EDTA, 1 mM DDT, 50% (v/v) glycerol), 1.5 mM MgCl 2 , 200 mM each dNTP, 0.4 mM each primer, 2.5 U (Units). Taq polymerase and 0.1– 1 ng DNA template was added to the reaction mixture (Taq DNA Polymerase, recombinant. INVITROGENHCorporation). Amplification was carried out in a GENE AMPHPCR SYSTEM 9700 (Applied Biosystems, Foster City, California,U.S.A.) using a hot start at 95uC for 1 min, followed by 36 cycles at 95uC for 30 sec, 55uC for 60 sec, and 72uC for 30 sec and a final extension at 72uC for 15 min. Before the sequencing reaction, PCR products were checked by electrophoresis in polyacrylamide non-denatur- ing gel (T9, C5), and subsequently the gel was stained with silver nitrate. PCR products were then purified with a MultiScreenH PCR m96 Plate (Millipore, Bedford, Ma 01730, U.S.A), 96-well device.The vacuum-based, size exclusion separation effectively and quickly removed the containing salts, unincorporated dNTPs and primers from PCR reactions. Cycle sequencing was performed on both strands in a GENE AMPHPCR SYSTEM 9700 (AB) thermal cycler using the ABI PrismHdRhodamine Terminator Cycle Sequencing Ready Reaction Kit (AB). This kit consists of a reaction mix composed of: DNA-modified and Patterns of mtDNA Variation in Italy PLOS ONE | www.plosone.org 3 December 2012 | Volume 7 | Issue 12 | e50794 termostable polymerase, Buffer Tris-HCl (pH 9.0), MgCl 2 , dNTPs, dichlororhodamine-marked ddNTPs. An aliquote of 30 ng amplicon and 3.2 rM primers were added to a 2 ml reaction mix. Sequencing was carried out using a hot start at 96uC for 4 min, followed by 36 cycles at 96uC for 15 sec, 50uC for 10 sec, 60uC for 2 sec and a final extension at 60uC for 10 min. The removal of excess dideoxy terminators, primers and buffer was accomplished with an alcoholic purification. The sequence products were denatured with deionized formamide and analyzed by capillary electrophoresis on an ABI PRISM 3130HGenetic Analyzer (AB).The resulting data were analyzed with PE/ABD software Sequencing Analysis 5.2 and sequences were aligned and compared with the Cambridge sequence [36] from position 16024 to16569 for HVS-I and from position 1 to 600 for HVS-II by the SeqScape v.2.0 (AB). Analysis of mtDNA coding region SNPs Biallelic markers were genotyped using a multiplex approach [37]. The selected SNPs were combined into two multiplex reactions. Multiplex 1 included a selection of SNPs defining common European haplogroups [38]. Multiplex 2 included exclusively polymorphisms defining sub-lineages inside haplogroup H. Primers were designed in order to adjust the annealing temperatures and amplicon lengths to allow analysis in multiplex reactions [37]. The sizes of the PCR products ranged from 80 to 224 bp. Both multiplexes were performed using 10 ng of DNA template in a 25 ml reaction volume comprising 16Taq Gold Buffer (AB), 200 mM of each dNTP, 2 mM MgCl 2 and 0.5 U of AmpliTaq Gold Polymerase (AB). For the primer concentrations, see [37]. Amplification was carried out using a GENE AMPHPCR SYSTEM 9700 (AB) thermocycler. After a 95uC pre-incubation step for 11 min, PCR was performed for a total of 32 cycles using the following conditions: 94uC denaturation for 30 sec, annealing at 60uC for 30 sec and extension at 72uC for 1 min, followed by a 15 min final extension at 72uC. PCR products were checked by polyacrylamide gel electrophoresis (T9, C5) visualized by silver staining. After amplification, PCR products required purification to remove primers and unincorporated dNTPs. Post-PCR purification was performed with ExoSapIT (Amershan Pharmacia Biotech): 1 ml of PCR product was incubated with 0.5 mlof Figure 1. Map showing the location of the samples analyzed in the present study and those collected from the literature (see Table 1). Pie charts on the left display the distribution of mtDNA haplogroup frequencies, and those on the right the Y-chromosome haplogroup frequencies. doi:10.1371/journal.pone.0050794.g001 Patterns of mtDNA Variation in Italy PLOS ONE | www.plosone.org 4 December 2012 | Volume 7 | Issue 12 | e50794 ExoSapIT for 15 min at 37uC followed by 15 min at 80uC for enzyme inactivation. The minisequencing reaction was performed in a GENE AMPHPCR SYSTEM 9700 (AB) thermocycler following the recommendations of the manufacturer: 2 mlof SNaPshot ready reaction mix, 0.2 mM of extension primer for each SNP (see [37]) and 1 ml of both purified PCR products in a total volume of 7 ml. The reaction mixture was subjected to 25 single base extension cycles of denaturation at 96uC for 10 sec, annealing at 50uC for 5 sec and with an extension at 60uC during 30 sec. After minisequencing reactions, a post-extension treatment to remove the 59-phosphoryl group of ddNTPs aided the prevention of co-migration of unincorporated ddNTPs with extended primers and production of a high background signal. The final volume (7 ml) was treated with 0.7 ml of SAP (Amersham Biosciences) for 60 min at 37uC, followed by 15 min at 80uC for enzyme inactivation. The minisequencing products (1.5 ml) were mixed with 10 mlof HiDi TM formamide and 0.2 ml of GeneScan-120 LIZ size standard (AB) and electroforesis was performed on an ABI PRISM 3130HGenetic Analyser (AB). The resulting data was analyzed with Gene Mapper ID. Minisequencing of SNPs characterizing additional typical European haplogroups Samples that were determined (using the SNP panel above) as being derived from J/T (T14766C; C7028T; T4216C), U (T14766C; C7028T; A12308G) and the U-subclade K (T14766C; C7028T; A12308G; A10398G), were further genotyped using an additional set of 14 haplogroup-specific SNP markers that identify the following sub-branches: 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 reactions were performed as described above. For PCR and minisequencing primer concentrations, see Table S1. Genotyping of Y-SNPs Biallelic markers were genotyped using a multiplex approach [39]. A set of 30 SNPs was tested, allowing assignation of the analyzed Y-chromosome to haplogroups (Hg), following the nomenclature and the phylogenetic relationships defined from the Y Chromosome Consortium [40]. The selected method for allele discrimination was a single base extension reaction using the SNaPshot multiplex kit (AB). We added the M269 marker to the first of the four multiplexes, in order better to dissect the subhaplogroup R1b (R1b3). The primers of this marker were M269-F 59-TCA TGC CTA GCC TCA TTC CT-39and M269-R 59- TCT TTT GTG TGC CTT CTG AGG-39, and the minisequencing primer 59-GGA ATG ATC AGG GTT TGG TTA AT-39. Genotyping of AIMs A panel of 52 AIMs were genotyped according to Sa´nchez et al. [41] in a subset of 441 individuals. Several other population datasets were used for inter-population comparisons. This data corresponded to the CEPH panel (http://www.cephb.fr/en/ cephdb/) as reported in HapMap (http://hapmap.ncbi.nlm.nih. gov/) and was collected using the data-mining tool SPSmart [42,43]; it includes population samples from all over the world (Africa, Europe, Asia, etc.); see legend of Figure 2 for more information. Statistical analysis A total of 42 Italian population samples were analyzed for mtDNA in the present study. Comparative inter-population analyses were also carried out for the HVS-I segment ranging from 16024 to 16365, since this is the analyzed segment common to all of them. Haplotype (H) and nucleotide diversity (p) and other diversity indices [44–46] were computed using DnaSP 4.10.3 software [47]. Problematic variation located around 16189, usually associated to length heteroplasmy e.g. 16182C or 16183C, was ignored. Analysis of molecular variance (AMOVA) was carried out using Arlequin 3.5. [48]. Nomenclature of mtDNA lineages followed previous studies e.g. [23,25,38,49,50]; see Phylotree for a compilation of the worldwide phylogeny and an update of the nomenclature based on entire mtDNA genomes [51]. Genotyping and documentation errors were monitored following the phylpogenetic principles previously applied e.g. [52– 59]. Mitochondrial DNA and Y-chromosome data was collected from the literature. The mtDNA data generated in the present study was analyzed together with 3,834 mtDNA HVS-I Italian profiles collected from the literature (Table S2; 76 sample populations). The Y-SNPs were analyzed together with 1,251 Italian profiles reported in the literature (16 population samples). A full list of references for all the data used in the present study is given in Table S2. Haplogroup frequencies were estimated by chromosome counting. Statistical differences in haplogroup frequencies were evaluated using a Pearson’s chisquare test and by setting up the nominal significant value aas 0.05. Finally, classification of mtDNA sequences into haplogroups was performed following phylogenetic criteria (Phylotree Build 14, http://www.phylotree.org/) and using both the control region sequence profile and mtSNPs. Results Molecular diversity of mtDNA and Y-chromosome Italian profiles Diversity indices were computed for all the populations analyzed in the present study and also in those Italian populations samples reported in the literature (Tables 1 and 2). Population samples were also grouped in main regions (North, Central, South, West, and East) in order to investigate the role of geography in the distribution of mtDNA variation. Mitochondrial DNA haplotypes for the samples analyzed in the present study are reported in Table S3.Table 1 shows the molecular diversity values based on mtDNA data for 41 Italian population samples. The values indicate that the Isle of Elba is, by far, the Italian population sample that shows the lowest diversity for all the indices computed, probably as a consequence of its relative isolation from the country. It has been reported that this was a well-known enclave of Etruscan influence, and some mtDNA particularities have been described before [8,9]. Alternatively, low molecular diversity could be due to low sample sizes, although this fact is mirrored in the standard deviation of the different estimates. Excluding the Isle of Elba, haplotype diversity in Italy ranges from 0.834 to 1, nucleotide diversity from 0.01003 to 0.02409, and the average value of nucleotide differences from 3.4 to 8.19 (a value that is correlated with the nucleotide diversity). In general, Italy shows some level of heterogeneity when examined for diversity values. When grouping populations by main geographical regions, it can be observed that Central Italy has slightly lower values than North and South Italy for all the indices computed (Table 1). The Patterns of mtDNA Variation in Italy PLOS ONE | www.plosone.org 5 December 2012 | Volume 7 | Issue 12 | e50794 higher diversity values were found in South Italy. Diversity values are however very similar when examining populations located in West Italy versus those in the East. The inclusion of Sicily (as part of South Italy) in the computation does not substantially change these estimates (Table 1). Y-SNP data were obtained for all the samples analyzed in the present study (Table S4). Table 2 shows the diversity indices for the Y-SNPs in different Italian populations. The Y-STR diversity values for the samples analyzed in the present study and other Italian and European samples have already been reported in Brisighelli et al. [33]. As expected, diversity values of Y-SNP haplogroup patterns are lower than those obtained for the mtDNA haplotypes given that the indices are based on haplogroup and not on Y-STR haplotypes. In fact, values based on Y-STR profiles (minimum or extended Yfiler profiles) [33] are higher than those observed for the HVS-I profiles. Ladins are among the populations with the lowest Y-SNP diversity values, while the Grecani Salentini show diversity values that are comparable to other Italian samples. Modena shows remarkable low haplotype diversity values. Phylogeography The mtDNA haplogroup make-up of Italy as observed in our samples fits well with expectations in a typical European population. Thus, most of the Italian mtDNAs (,89%) could be attributed to European haplogroups H (,40%), I (,3%), J (,9%), T(,11%), U (,20%; U minus U6), V (,3%), X (,2%) and W (,1%); Figure 1. There are however important differences in haplogroup frequencies when examining them by main geographical regions. Thus, for instance, haplogroup H is 59% in the North, 46% in the Center, and decays to ,33% in the South; moreover, these regional differences are statistically significant: North vs South (Pearson’s chi-square, unadjusted-Pval- ue,0.00003), and Center vs South (Pearson’s chi-square, unadjusted-Pvalue,0.03724). Mitochondrial DNA haplotypes of African origin are mainly represented by haplogroups M1 (0.3%), U6 (0.8%) and L (1.2%); from here onwards, L will be used to refer to all mtDNA lineages, excluding the non-African branches N and M [60,61]. A total of 282 Y-chromosomes were analyzed for a set of Y- SNPs and were classified into 22 different haplogroups (Figure 3). Two haplogroups were not found, even though markers defining these clades were tested: N3 and R1a1. Five haplogroups represented 76.71% of the total chromosomes: R1b3, J2, I(xI1b2), E3b1 and G. The frequencies averaged across populations were 26%, 21.2%, 10.2%, 9.9% and 9.2%, respectively. The remaining haplogroups sum to 23.2% in the total sample, and never above 4% in single population samples. R1b3 frequency was found to be higher in the northern part of the country, while the Y-chromosome haplogroups G and E3b1, J2 and I(xI1b2)frequencies were higher in the south and in the central part of the country, respectively (Figure 1). Regional differences are substantially higher in the Y-chromo- some than in the mtDNA. Thus, for instance, haplogroup R in the Y-chromosome was 54% in the North, 18% in the Center, and 31% in the South. Frequency differences were statistically significant between North vs Center (Pearson’s chi-square, unadjusted-Pvalue = 0.0014), and North vs South (Pearson’s chi- Figure 2. Analysis of AIMs in Italian populations versus other continental population groups. (A) PCA of Italian populations divided into the main regions North, Center and South (as analyzed in the present study) and other European populations; (B) the same Italian populations plus sub-Saharan African, and Asian populations; (C) triangle plot as obtained using STRUCTURE analysis of Italian, European, sub-Saharan, and Asian populations; (D) bar plot of ancestral membership values as obtained using STRUCTURE analysis of the same populations used in (C). Population codes: 1: Angola; 2: Kenya-Bantu NE; 3: Mozambique; 4: Namibia-San; 5: Nigeria-Yoruba; 6: Senegal-Mandenka; 7: South Africa-Bantu; 8: Uganda; 9: Britain; 10: Denmark; 11: French; 12: Germany; 13: Ireland; 14*: NW Spain; 15*: Portugal; 16: Slovenia; 17: China-Dai; 18: China-Daru; 19: China-Han; 20: China-Hezhen; 21: Japanese; 22: Mongolia; 23: Taiwan; 24: Thailand. Genotypes were downloaded using the method in [43,83] and belong to the CEPH panel. An asterisk indicates Mediterranean populations. doi:10.1371/journal.pone.0050794.g002 Patterns of mtDNA Variation in Italy PLOS ONE | www.plosone.org 6 December 2012 | Volume 7 | Issue 12 | e50794 Table 1. Diversity indices computed for different Italian regions based on HVS-I data (sequence segment 16090–16365). Population Region Pop ID Reference N k k/n S h P M Liguri NW 1 p.s. 50 40 0.8 53 0.96260.021 0.0142660.0145 4.875 Torino 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 Pavia 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 Arezzo/Chiusi CW 1 [9] 14 14 1 22 1.00060.027 0.0148860.0129 5.088 Casentino CW 15 [8] 122 77 0.6 167 0.97960.007 0.0240960.0082 8.190 Collevecchio/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 Firenze 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 Latini CW 5 p.s. 48 29 0.6 35 0.90260.039 0.0100360.0010 3.429 Latium CW 20 [86] 52 37 0.7 48 0.95960.019 0.0131360.0014 4.492 Murlo 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 Terni 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 Vallepietra CW 21 [85] 21 8 0.4 17 0.87160.044 0.0128160.0014 4.381 Volterra CW 14 [8] 114 57 0.5 62 0.95560.013 0.0119360.0007 4.057 Abruzzo 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 Center East CE 23 [90] 83 62 0.7 60 0.97460.012 0.0135260.0009 4.625 Croatian Italians 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 Belvedere SW 10 p.s. 50 41 0.8 44 0.98060.013 0.0132060.0010 4.532 Calabria 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 Catania 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 Trapani 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 Basilicata SE 25 [91] 92 65 0.7 70 0.98360.007 0.0129060.0008 4.428 Grecani Salentini SE 8 p.s. 47 37 0.8 44 0.98960.007 0.0131060.0011 4.480 Lucera SE 6 p.s. 60 42 0.7 55 0.97660.011 0.0134560.0011 4.586 South Apulia SE 9 p.s. 53 38 0.7 49 0.97360.014 0.0157960.0010 5.401 Sanniti SE 7 p.s. 50 41 0.8 49 0.98860.008 0.0142060.0013 4.843 Sardinia – 29 [38,87,97] 351 171 0.4 98 0.95060.009 0.0118360.0004 4.033 Geographical region North Italy – – 702 267 0.4 126 0.96360.004 0.0128260.0004 4.295 Central Italy – – 1413 500 0.4 216 0.95860.004 0.0124360.0002 4.113 South Italy – – 1453 569 0.4 183 0.97360.002 0.0136860.0002 4.541 West Italy (without Sicily) – – 1437 578 0.4 232 0.96960.003 0.0131560.0002 4.405 West Italy (with Sicily) – – 2075 709 0.3 236 0.96360.003 0.0126060.0002 4.133 East Italy – – 1493 520 0.3 165 0.96460.003 0.0127760.0002 4.200 NW = North-West; NE = North-East; CW = Center-West; CE = Center-East; SW = South-West; SE = South-East; N= sample size; k = number of different haplotypes; S = segregating sites; h = haplotype diversity; p= nucleotide diversity; M = average number of nucleotide differences. doi:10.1371/journal.pone.0050794.t001 Patterns of mtDNA Variation in Italy PLOS ONE | www.plosone.org 7 December 2012 | Volume 7 | Issue 12 | e50794 square, unadjusted-Pvalue,0.00004). Haplogroup J2 also revealed important regional differences; it added to 9% in the North, 37% in the Center, and 22% in the South, with statistically significant differences between the North vs Center (Pearson’s chisquare, unadjusted-Pvalue,0.00002), North vs South (Pearson’s chi-square, unadjusted-Pvalue,0.00148), and in the limit of significance Center vs South (Pearson’s chi-square, unadjusted-P value,0.049). Autosomal ancestry in Italy A panel of 52 AIMs was genotyped in 435 Italian individuals in order to estimate the proportion of ancestry from a three-way differentiation: sub-Saharan Africa, Europe and Asia. Structure analyses allowed us to infer membership proportions in population samples, and these proportions can be graphically displayed, as in Figure 2. This analysis indicated that Italians have a basal proportion of sub-Saharan ancestry that is higher (9.2%, on average) than other central or northern European populations (1.5%, on average). The amount of African ancestry in Italians is however more comparable to (but slightly higher than) the average in other Mediterranean countries (7.1%). Figure 2 shows in a triangle plot the relationships of Italians compared to other European, African and Asian populations. PCA observations confirmed the results from Structure analysis, clustering Italian profiles tightly with other European ones. Thus, PCA indicated that North, Central and South Italy do not show differences between them, nor from other European populations (Figure 2). PCA also indicated clear-cut differences between Italians, Africans and Asians (Figure 2). AMOVA AMOVA analyses were carried out following different grouping schemes. The samples were pooled into a single population, but also by considering main Italian regions. Analyses were carried out over haplogroups and haplotypes of the Y-chromosome and the mtDNA (Table 3). AMOVA indicated that, among populations, variance was more strongly stratified for the Y-chromosome than for the Table 2. Diversity indices computed for different Italian regions based on Y-SNPs. Population Region Reference N k k/n Gene Diversity Liguria NW Present study 46 9 0.19 0.766260.0502 Ladin NE [14] 34 6 0.17 0.534860.0979 Udine NE Present study 47 10 0.21 0.776160.0441 Central Tuscany CW [14] 40 8 0.20 0.739760.0616 Elba Island CW [14] 94 7 0.07 0.674260.0445 Latini CW Present study 44 11 0.25 0.825660.0395 Latium CW [14] 43 9 0.20 0.802660.0388 Tuscany-Latium border CW [14] 76 7 0.09 0.755460.0350 Central Marche CE [14] 59 7 0.11 0.729460.0364 Marche CE [11] 162 13 0.08 0.848960.0152 Marche-Appennine CE [14] 25 7 0.28 0.803360.0514 Modena CE [98] 62 8 0.12 0.532060.0743 Piceni CE Present study 38 9 0.23 0.820860.0450 Rimini-Val Marecchia CE [99] 163 12 0.35 0.699060.0308 Belvedere SW Present study 27 9 0.33 0.854760.0477 East Campania SW [14] 46 7 0.15 0.687060.0618 Sicily SW Present study 57 12 0.21 0.832760.0311 West Campania SW [14] 80 10 0.12 0.844660.0224 West Calabria SW [14] 57 7 0.12 0.752560.0307 Sanniti SE Present study 30 10 0.33 0.864460.0409 Grecani Salentini SE Present study 47 7 0.14 0.812260.0242 Lucera SE [31] 60 9 0.15 0.836560.0236 South Apulia SE [14] 49 9 0.18 0.852960.0237 Sardinia [100] 336 14 0.04 0.809860.0136 Geographical region North Italy – – 127 14 0.11 0.840060.0189 Central Italy – – 806 21 0.03 0.887060.0053 South Italy – – 453 20 0.04 0.890960.0060 West Italy (without Sicily) – – 553 17 0.03 0.856760.0094 West Italy (with Sicily) – – 610 20 0.03 0.870560.0078 East Italy – – 776 22 0.02 0.903460.0037 Codes are as in Table 1. doi:10.1371/journal.pone.0050794.t002 Patterns of mtDNA Variation in Italy PLOS ONE | www.plosone.org 8 December 2012 | Volume 7 | Issue 12 | e50794 mtDNA; the difference was much more marked for the analysis based on haplogroups (14.39% vs 1.17%) than for the analysis based on haplotypes (2.34% vs 0.79%). Among population variance was very low when analyzing main geographical regions; however, it was the latitude (North vs Center vs South) that appeared to account for higher values of among-population variance rather than longitude (West vs East), with the exception of the Y-chromosome haplogroups (although the values are below 1%); Table 3. Again, the Y-chromosome showed slightly higher values of among-population variance than did the mtDNA. For the Y-chromosome, a significant proportion of the withinpopulation variance moved to among-population within-groups variance, probably due to the fact that all population samples had a very high proportion of singleton Yfiler haplotypes, elevating the maximum values of haplogroup diversity for all of them [33]. Linguistic isolates: Ladin and Grecani Salentini Two linguistic isolates are represented in the samples analyzed in the present study: the Ladin and the Grecani Salentini. Other population samples of the Ladin have already been analyzed in the literature [22,62,63]. We here sampled 41 new Figure 3. Phylogeny of Y-chromosome SNPs and haplogroup frequencies in different Italian populations. doi:10.1371/journal.pone.0050794.g003 Table 3. AMOVA analysis of main Italian regions (Permutations: 20000; P-value,0.0000) for the mtDNA control region data and the Y-chromosome STRs and SNPs. All populations (%) North vs Center vs South (%) West vs East (%) HAPLOTYPES mtDNA (48 populations) Among pops 0.79 0 0 Within pops 99.21 99.25 99.21 Among pops within groups – 0.75 0.79 Y-chromosome (15 populations) Among pops 2.34 1.18 0 Within pops 97.66 97.32 97.85 Among pops within groups – 1.50 2.15 HAPLOGROUPS mtDNA (19 populations) Among pops 1.17 0.36 0 Within pops 98.83 98.72 98.83 Among pops within groups – 0.92 1.17 Y-chromosome (24 populations) Among pops 13.92 0.07 0.83 Within pops 86.08 86.06 85.74 Among pops within groups – 13.87 13.44 Sardinians were not included in the analysis. References for population samples are given in Table S2. doi:10.1371/journal.pone.0050794.t003 Patterns of mtDNA Variation in Italy PLOS ONE | www.plosone.org 9 December 2012 | Volume 7 | Issue 12 | e50794