AMERICAN JOURNAL OF HUMAN BIOLOGY 00:00–00 (2016)
Original Research Article
Genetic Herita Genetic Heritage ge of Croati Croatians ans in the Southeastern Southeastern Europe European an Gene Pool—Y Chromosome Analysis of the Croatian Continental and Island Population 1 1 ´ ,1 DUBRAVKA HAVA S ´ ,2 MATE MUSTAC ´ ,3 JELENA SARAC, * TENA SARIC AUGUSTIN, NATALIJA NOVOKMET,1 NENAD VEKARIC ´ ,5 BRANIMIR NEVAJDA,6 ANTON GLASNOVI C ´ ,6 SA SA BLA ZENKA GRAHOVAC,4 MILJENKO KAPOVIC MISSONI,1,7 SIIRI ROOTSI,8 AND PAVAO RUDAN1,9 1 Institute for Anthropological Research, 10000 Zagreb, Croatia 2 Institute for Historical Sciences, Croatian Academy of Sciences and Arts, 20000 Dubrovnik, Croatia 3 Occupational Health Clinic, 23000 Zadar, Zadar, Croatia 4 Department of Pathology and Pathological Anatomy, School of Medicine, University of Rijeka, 51000 Rijeka, Croatia 5 Department of Biology and Medical Genetics, School of Medicine, University of Rijeka, 51000 Rijeka, Croatia 6 Dubrava University Hospital, 10000 Zagreb, Croatia 7 “Josip Juraj Strossmayer” University of Osijek, School of Medicine, Osijek, Croatia 8 Estonian Biocentre and Institute for Molecular and Cell Biology, Department of Evolutionary Biology, , University of Tartu, 51010 Tartu, Estonia 9 Anthropological Center of the Croatian Academy of Sciences and Arts, 10000 Zagreb, Croatia
The research objective of this study is to enlarge and deepen the Y chromosome research on the CroObjectives: The Objectives: atian population and enable additional insights into the population diversity and historic events that shaped the current genetic landscape of Croatia and Southeastern Europe (SEE). Materials and Methods: A Methods: A high-resolution phylogenetic and phylogeographic analysis of 66 biallelic (SNPs) and 17 microsatellite (STRs) markers of the Y chromosome was performed using 720 Croatian samples. The obtained results were wer e pla placed ced in a wid wider er Eur Europe opean an con contex textt by com compar pariso ison n wit with h 4450 445 0 sam sample pless fro from m a num number ber of oth other er Eur Europe opean an populations. A high diversity of haplogroups was observed in the overall Croatian sample, and all typical European Y Results: A Results: chromosome haplogroups with corresponding clinal patterns were observed. Three distinct genetic signals were identifiable in the Croatian paternal gene pool - I2a1b-M423, R1a1a1b1a*-M558, and E1b1b1a1b1a-V13 haplogroups. The analyses of the dominant and autochthonous I2a1b-M423 lineage ( >30%) suggest that SEE had a Discussion: The Discussion: significant role in the Upper Paleolithic, the R1a1a1b1a*-M558 lineage (19%) represents a signal from present day Slavic populations of Central Europe in the Croatian population, and the phylogeography of the E1b1b1a1b1a-V13 clade (around 9%) implies cultural diffusion of agriculture into Europe via the Balkan Peninsula. Am. J. Hum. Biol. C 2016 Wiley Periodicals, Inc. V 00:000–000, 2016.
Southeastern Europe (SEE), has been inhabited since the Middl Middle e Paleol Paleolithic ithic according according to prehi prehistoric storic archaeological findings of “Proto-Aurignacian” culture in the Bulgarian cave Bacho Kiro 37–43 kya (Kozlowski et al., 1982; Kozlowski and Otte, 2000; Teyssandier et al., 2006; Hoffecker fec ker,, 200 2009). 9). A con consid sidera erable ble number number of mtD mtDNA NA and Y chromosome population-genetic studies of the SEE region reflectt the turbulent reflec turbulent and complex demographic demographic histo history ry of this region, influenced by gene flow from various parts of Eurasia and a long history of intermixing. It has been suggested that SEE has played a key role in the Upper Paleolithic recolonization of the wider European area, as well as in the Neolithic spread of agriculture from the Near East, serving as a gateway between the Middle East and the rest of Europe (Battaglia et al., 2009; Forenbaher and Miracle, 2005; Kovacˇ evic´ et al., 2014; Marjanovic´ et al., 2005; Mellars, 2004; Primorac et al., 2011; Semino et al., 2000, 2004). Croatia, as a SEE country, has been subject to extensive multidisciplinary anthropological researches for over four decades (Rudan et al., 2004 and the references therein) and is situated in a key region between the rest of Europ Europe e and the Near East (Kovacˇ evic´ et al., 2014). The research objective of this particular study is to better characterize the Y chrom chromosome osome diversity diversity of the Croatian population population and enable addit additional ional insights insights into the geneti geneticc specifi specifics cs and historic events that shaped the curre current nt genetic landscape of SEE and Croatia. The specific aims of this study C V
2016 Wiley Periodicals, Inc.
are to: (i) assess the genetic structure and variability of Croatian paternal genetic heritage; (ii) depict the phylogenetic and phylogeographic relationships inside the three dominant domin ant Y chrom chromosome osome haplogroups haplogroups (hgs) in Croat Croatia, ia, and (iii) place the Croatian paternal genetic heritage into a wider European context. MATERIALS MA TERIALS AND METHODS Sample The sample consists of 720 randomly chosen male individuals; 336 coming from the continental part of Croatia Contract grant sponsor: the Ministry of Science, Education and Sports of the Republ Republic ic of Croati Croatia; a; Contrac Contractt grant number number:: 196-1962766196-1962766-2751; 2751; Contract grant sponsor: the European Union European Regional Development Fund through the Centre of Excellence in Genomics, Estonian Basic Research; Contract grant number: SF 0270177s08; Contract grant sponsor: Estonian Science Foundation; Contract grant number: 7445; Contract grantt spo gran sponso nsor: r: Ins Instit tituti utiona onall Res Resear earch ch Fun Fundin ding g fro from m the Est Estoni onian an Research Council; Contract grant number: IUT24-1. *Corre *Co rrespo sponde ndence nce to: Jel Jelena ena Sara Sarac; c; Ins Instit titute ute for Ant Anthro hropol pologi ogical cal Research, Gajeva Street 32, 10000 Zagreb, Croatia. E-mail: jelena.sarac@ Research, jelena.sarac@ inantro.hr Additional Supporting Information may be found in the online version of this article.
Conflict of interest: None to declare. Received 24 August 2015; Revision received 22 March 2016; Accepted 10 May 2016 DOI: 10.1002/ajh 10.1002/ajhb.22876 b.22876 Publ Pu blis ishe hed d on onli line ne 00 Mo Mont nth h (wileyonlinelibrary.com).
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Fig. 1.
Map of Croatia with exact location of all the sampled subpopulations within the sample.
TABLE 1. Genetic diversity, mean number of pairwise differences and average gene diversity over loci Haplogroup I2a1b-M423 R1a1a1b1aa-M558 E1b1b1a1b1a-V13 TOTAL (STRs) TOTAL (SNPs)a
N
No. of haplotypes
Haplotype diversity 6 SD
MNPD
Average GD over loci
57 48 21 126 720
56 44 17 114
0,999 6 0,003 0,995 6 0,005 0,9816 2,580 0,998 6 0,001 0,993
5,514 6 2,690 5,895 6 2,864 5,109 6 1,627 10,833 6 4,961
0,324 6 0,175 0,310 6 0,167 0,268 6 0,151 0,637 6 0,323
a
Nei’ genetic diversity measure
and 384 from eight Croatian islands (Fig. 1 and Supporting Information Table 1). Samples were taken from the DNA bank of the Institute for Anthropological Research, Zagreb. The blood for DNA analyses was collected during fieldwork carried out by the Institute for Anthropological Research over a 12-year span (1997–2009), following the approval of the ethical committee of the Institute for Anthropologic al Research and School of Medicine, University of Zagreb, Croatia. Participants gave their informed consent to participate in the research. Siblings and male relatives were avoided in the selection of the samples for DNA analysis based on obtained genealogical data, as well as examinees without at least two generations of ancestors living in the sampled subpopulation. All of the subsequen t laboratory and molecular analyses were performed at the Institute for Anthropological Research in Zagreb and at the Estonian Biocentre Tartu, Estonia. American Journal of Human Biology
Genotyping The samples were surveyed for a total of 66 different biallelic markers using either restriction fragment length polymorphism (RFLP) analysis, in/del assays or direct Sanger sequencing (Fig. 2). The nomenclature used in this study is adopted from the International Society of Genetic Genealogy (2015). Y-DNA Haplogroup Tree 2015, Version: 10.102, Date: 9 December 2015, http://www. isogg.org/tree /[Date of access: December 9 th, 2015]. The order of marker genotyping was based on the known haplogroup hierarchy (www.isogg.org/tree; Karafet et al., 2008) and previously observed haplogroup composition and frequencies in this part of Europe. Each sample was tested for SNPs after the initial amplification using polymerase chain reaction and primer sets designed for each marker. List of sequences and primers for each marker are listed in Supporting Information Tables 2 and 3.
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Fig. 2.
Phylogenetic tree with all the tested markers and haplogroup frequencies.
A subset of 126 samples designated as haplogroup I2a1b-M423 ( N 5 57), R1a1a1b1a*-M558 ( N 5 48) or E1b1b1a1b-V13 ( N 5 21) was typed for 17 short tandem repeats (Y-STRs) using the Applied Biosystems AmpFlSTR Yfiler Kit and according to manufacturer recommendations (Supporting Information Table 4). The named three haplogroups were chosen for further STR analysis, because they harbored > 60% of the total paternal genetic varation in Croatia. For the multicopy STR DYS389I,II the DYS389b value was DYS389I subtracted from DYS389II (Cooper et al., 1996). The fragments were run on a ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems) and the results were analyzed using the GeneMapper 4.0 program (Applied Biosystems). Phylogenetic analyses Y-STR haplotypes were used to construct phylogenetic networks for haplogroups I2a1b-M423, R1a1a1b1a*-M558 and E1b1b1a1b-V13, using the program Network 4.6.0.0 and Network Publisher program (Fluxus-Engineering, Suffolk, England, UK) and applying the median-joining algorithm (Bandelt et al., 1999). STR loci were weighted according to Helgason et al. (2000) and the sizes of the nodes are proportional to the number of individuals. The networks were performed utilizing eight STR loci to enable comparisons with a significant number of published data collections. After reviewing the currently published literature, we found that the Battaglia et al. (2009) paper was still the most relevant source of comparable Southeastern European datasets (eight STR loci) for I2a1 and E-V13 clade. Although there are studies with a higher number of STRs available, there are often problems with the compatibility of STR sets used across studies or with a
lack of haplogroup defining SNPs, which makes comparisons difficult. Statistical analyses A pairwise difference method based on molecular differences between the haplotypes was used for calculating pairwise Fst genetic distances between populations. They were obtained from Y-STR haplotypes using Arlequin 3.5 software (Excoffier and Lischer, 2010). Principal Component Analysis (PCA) and Multidimensional Scaling (MDS) were used for visual representation of genetic distances and differences in haplogroup diversity and frequency between the populations. PCA plots were constructed using the free software POPSTR (http//harpending.huamnevo.utah.edu/popstr/) based on biallelic markers and MDS plots using the program XLSTAT 2013. 5.06 software (http://www.xlstat.com/en /). Spatial frequency maps were obtained by using the hg frequencies reported in Figure 2. The frequency data were converted into isofrequency maps using the Surfer software (version 8, Golden Software, Golden, CO), following the kriging algorithm. RESULTS NRY haplogroup composition and frequencies A high level of haplogroup diversity was observed in the sample (Fig. 2) and the Croatian genetic profile corresponds to its geographic position and to the previously reported patterns of Y chromosome diversity in SEE (Battaglia et al., 2009; Kovacˇevic´ et al., 2014; Marjanovic´ et al., 2005; Pericˇic´ et al., 2005; Regueiro et al., 2012). Some 36 different subhaplogroups were present in the American Journal of Human Biology
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sample, and are shown in detail in Figure 2 and Supporting Information Tables 5 and 6. The most common hgs were I, R, E, J, and G, which encompass 96% of the total sample and only three subclades – I2a1b-M423 (33.2%), R1a1a1b1a*-M558 (19.3%), and E1b1b1a2a-V13 (8.75%) harbored > 60% of the total paternal genetic variation in Croatia. When compared to a larger population dataset comprising 4459 samples from other European populations (Balanovsky et al., 2008; Battaglia et al., 2009; Karanachak et al., 2013; King et al., 2011; Kushniarevich et al., 2013; Regueiro et al., 2012), it was observed in the PCA plot (Fig. 3) that the formed clusters clearly
reflected the European geographic architecture, and that most of Croatian samples fit into the wider SEE region. The first principal component encompassed 20% of the observed variance and sharply distinguished Northern and Central European populations from the others, based on the frequency of R1a individuals, while the second one accounted for about 23% of the total variance and was a clear dividing point between Southeastern and Southern Europe and other European populations, based on the increased frequency of I2a2M423. It was observed from spatial gradient maps (Figs. 4–6 and Supporting Information Table 7) that haplogroups I2a1b-M423 and E1b1b1a1b-V13 both exhibited high densities within SEE populations. For example, I2a1b-M423 had a frequency peak in present-day Bosnia and Herzegovina and in Southern Croatia, and E1b1b1a1b-V13 peaked in southern parts of the Balkan Penninsula, namely in Greece and Albania, as suggested in previous studies (Battaglia et al., 2009; Marjanovic´ et al., 2005; Rootsi et al., 2004; Underhill et al., 2007). R1a1a1b1a*M558 reaches its frequency peak in the area of today’s Belarus and Ukraine and decreases to the south, but more prominently to the west of Europe, as noted in Underhill et al. (2015) study. Nevertheless, its percentage in northern Croatia and Central European populations was still considerable. NRY haplotype diversity
Visual representation (PCA plot) of major (sub)haplogroup frequencies in European populations and Croatian samples. Green diamond: North and Eastern Europe, Orange diamond: Central Europe, Purple diamond: Southern Europe, Red diamond: Southeastern Europe, Yellow diamond: Western Europe. Abbreviations: CRO, Croatia; BOS, Bosnia and Herzegovinia; SRB, Serbia; HUN, Hungaria; UKR, Ukraine; POL, Poland; SLO, Slovenia; CZE, Czech Republic; BLRUS, Belarus; RUS, Russia; BULG, Bulgaria; MAC-G, Macedonian Greeks; ALB, Albania; MAC-A, Macedonian Albanians; GRE, Greece; ITAL, Italy; FRA, France. Fig. 3.
Phylogenetic relationships within the three dominant Croatian NRY were evaluated by analyzing eight STR loci on 126 samples and compared to over 500 samples from different European populations (Supporting Information Figs. 1–3, Supporting Information Table 8). The diversity of I2a1b-M423 haplotypes was high and mirrored by the widespread distribution of haplotype clusters, most of which originated in south/southeastern European
Fig. 4. Spatial gradient map illustrating clines in frequency for I2a1b-M423. Comparative population data has been taken from Battaglia et al. (2009), Regueiro et al. (2012), Underhill et al. (2007), and Varzari et al. (2013).
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Fig. 5. Spatial gradient map illustrating clines in frequency for R1a1a1b1a*-M558. Comparative population data has been taken from Underhill et al. (2015).
Fig. 6. Spatial gradient map illustrating clines in frequency for E1b1b1a1b-V13. Comparative population data has been taken from Battaglia et al. (2009), Cruciani et al. (2007), and Regueiro et al. (2012).
populations. Two dominant star-like clusters were evident with a major portion of south/southeastern European samples placed within them, indicating a rapid expansion from a common founder. Unlike for hg I2a2, the prevailing diversity of R1a1a1b1a*-M558 was among the Northern, Eastern and Central European populations. One major cluster was evident, with only a minor presence of South/ Southeastern Europeans. Its star-like form, relative
shortness of the branches and observed frequency pattern in Europe were consistent with a model of recent hg R1a diversification followed by range expansions and subsequent population growth across Europe. The dominant portion of the E1b1b1a2a-V13 haplotype diversity occurred among southern and Southeastern European countries, in line with the previous findings that indicated Southeastern/Southern Europe as the geographic source American Journal of Human Biology
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of this particular E-subclade (Battaglia et al., 2009; Semino et al., 2004). One dominant cluster was evident, with most of its representatives belonging to Balkan populations and Croatia. It is important to stress that the proposed old age of the I2a1b-M423 and R1a1a1b1a*-M558 lineages obtained in previous studies (Battaglia et al., 2009; Pericˇic´ et al., 2005; Rootsi et al., 2004; Underhill et al., 2007, 2015) has been based on STR analysis (8 and 10 loci, respectively) and recent studies clearly indicate that the STR-based age calculations tend to yield overestimated dates (Batini et al., 2015; Hallast et al., 2015; Karmin et al., 2015). For example, the expansion time estimates of R1a lineages in Europe have been dated much later, about 5 kya, based on 70 complete NRY sequences collected worldwide (Karmin et al., 2015). Some 56 different haplotypes were detected in the I2a1b-M423 clade, congruent with its high haplotype diversity (0.999 6 0.003) and a scattered distribution in the phylogenetic network. The distribution of 44 different haplotypes within the R1a1a1b1a*-M558 lineage, also illustrated in the network projection, similarly correlated with the high diversity observed within this haplogroup (0.995 6 0.005). The most haplotype sharing and slightly lower diversity values were evident within E1b1b1a1b V13 lineage (0.981 6 2.580), suggesting a somewhat reduced degree of gene flow within the Balkan populations for this clade (Table 1). Genetic distances obtained by the pairwise difference method using eight STR loci (Supporting Information Table 9) were visualized in form of multidimensional scaling plots for three major hgs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, and DYS439) (Supporting Information Figs. 4–6). As expected, the obtained genetic distances between Croatia and other European populations for hg I2a1b-M423 place Croatia close to its geographic neighboring countries. The observed peak frequency of this lineage in Bosnia (Marjanovic´ et al., 2005) could be responsible for it’s slightly outlying position in the plot. Slovenia is the only SEE country in the plot that doesn’t show affinity to this region, which coincides with previous findings based on mtDNA analyses ( Sarac et al., 2014). As for the R1a1a1b1a*-M558 lineage, most SEE populations (Croatia, Bosnia, Serbia, Montenegro) are positioned relatively close to each other, indicating their genetic, as well as geographic closeness. The only surprising result is the E1b1b1a2-V13 plot, since the Croatian population does not cluster tightly with the rest of the SEE ones. This result could possibly be explained by a maritime influx into Croatia through the Mediterranean Sea, which brought some untypical E1b1b1a1b-V13 haplotypes into the genetic structure of Croatia and caused its alienated position. DISCUSSION Our results indicate that a high level of haplogroup and haplotype diversity exists in the Croatian sample set, suggesting a dynamic gene flow in the SEE region throughout history. Y chromosome haplogroup frequency distributions in the Croatian population correspond to the previously reported results for the Croatian population (Barac´ et al., 2003; Pericˇic´ et al., 2005) and they fit within a Southeast European paternal genetic landscape (Batta American Journal of Human Biology
glia et al., 2009; Kovacˇevic´ et al., 2014; Marjanovic´ et al., 2005; Mirabal et al., 2010; Regueiro et al., 2012). A previous high-resolution analysis of the maternal heritage of SEE (Sarac et al., 2014) based on mitochondrial DNA SNPs is also consistent with the results of the paternal lineage analysis. Three NRY haplogroups that account for the majority of Croatia’s paternal gene pool have been the focus of this study, namely the I2a1b-M423, R1a1a1b1a*M558, and E1b1b1a1b1a-V13 haplogroup, which we view as representing a Balkan, a Central-European and a Neolithic input into the Southeast European genetic landscape. The Balkan I2a1b-M423 haplogroup One of the foci in this study was a I-M438 lineage with a downstream mutation M423—a marker most common in Slovenia, Bosnia and Herzegovina, Croatia and Russia. It is characterized as a European-specific, SEE autochthonous paternal lineage and a genetic signal of the recolonization process of Europe after LGM in the Early Holocene (Rootsi et al., 2004). More recent studies indicate that it most likely spread following the post-Younger Dryas recovery (Karmin et al., 2015, Fig. Suppl. 25). It is now known that the M423 SNP accounts for the majority of the Eastern/Southeastern European hg I chromosomes (>75%) and virtually all I-P37.2 Y individuals in SEE (Battaglia et al., 2009). The results of our study show a considerable frequency of I2a1b-M423 ( >30%) in Croatia and its phylogenetic network also shows high haplotype diversity, mirrored by the widespread distribution of haplotype clusters. The elevated frequency and high diversity of I2a1b-M423 lineages among different SEE populations shows a genetic signature of their common paternal history over a long period of time. The PCA plot clearly shows a SEE cluster comprised of Bosnian and Herzegovinian, Serbian and Croatian samples based predominantly on the I2a1b-M423 component. As shown in the I2a1bM423 spatial gradient map, a clear cline of this clade is evident inside Europe, spreading from the area of Southern Croatia and Bosnia and Herzegovina mostly northward and eastward. Although the initial STR-based time estimate for this clade gave support for a Upper Paleolithic origin (Pericˇic´ et al., 2005), new studies based on whole Y chromosome sequencing suggest a somewhat younger age of this clade, between 5 and 7.5 kya (Batini et al., 2015; Karmin et al., 2015). In addition, the high haplotype diversity of this lineage in Croatia reveals its significant expansion only after the adoption of agriculture by Mesolithic hunter-gatherers in SEE (Battaglia et al., 2009). More evidence of its autochthonous European origin also came recently from European aDNA studies. Lazaridis et al. (2014) sequenced nine ancient genomes (with age estimates of cca 8 kya) and analyzed the complete NRY sequence of five male individuals (one from Luxemburg and four from Sweden). Their results showed all five of them belonged to the I haplogroup. The authors warned that, at present, the limited number of ancient samples for which Y chromosome data are available makes it difficult to assess how statistically surprising it is that this NRY haplogroup occurs in all five of the ancient Mesolithic males but in only a quarter of present-day males from that geographic area. They appear to argue that the haplogroup I today is found in a wider European area at a
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much lower frequency than it occurred around 8 kya. This haplogroup has also been found and described in a Scandinavian Neolithic hunter-gatherer from Sweden (Skoglund et al., 2014), as well as in Neolithic remains from southern France and northern Spain (Lacan et al., 2011a). The fact that a significant portion of investigated Mesolithic males belonged to haplogroup I suggests that this paternal lineage might represent a major pre-Neolithic European clade, and the results obtained in this study support this hypothesis.
2014). Such recent episodes could be the proposed IndoEuropean migrations from the Pontic steppe associated with the Yamnaya and Corded Ware cultures in Late Neolithic/Bronze Age and massive Slavic migrations in a more recent history (3rd – 6th century), together with smaller ones that most probably occurred repeatedly since Paleolithic times and are associated with specific historical events (Haak et al., 2015; Pericˇic´ et al., 2005; Regueiro et al., 2012).
In situ European diversification of the R1a1a1b1a*-M558 clade
Several theories about the origin of E1b1b1a1b1a-V13 and its spread have been proposed, and all are, in a certain way, linked to the Neolithic farmers. Early studies suggested that the mutation was brought to the Balkans together with early farming technologies (Semino et al., 2004). Battaglia et al. (2009) proposed an earlier arrival of this lineage in Europe (during the late Mesolithic period), followed by its Neolithic dispersal into Europe with the spread of farming. Most recently, King et al. (2011) suggested that E1b1b1a1b1a-V13 may trace the demographic and socio-cultural impact of Greek colonization. Researchers still debate the fate and interplay between pioneering farmers from the Near East and local Mesolithic societies and search for the clearest signal of their interaction. However, the phylogeography and temporal evidence supports the idea that E1b1b1a1b1a-V13 arose in Southern Europe, with peak frequencies among the Albanians and Greeks (Semino et al., 2004) and with declining patterns towards the north. The lack of any plausible Middle Eastern source of E1b1b1a1b1a-V13 during the Early Neolithic or Bronze Age, together with the low STR variation observed in the Middle East, additionally bolsters this view (Battaglia et al., 2009; King et al., 2008). We support the scenario proposed by Battaglia et al. (2009) and additionally strengthened by two recent studies that the Mediterranean route has had a pronounced role in the spread of farming (Lacan et al., 2011b; Paschoua et al., 2014) and that local South European populations adopted the new technology through their contact with Near Eastern migrants and spread it further into the European area. The high diversity of the E1b1b1a1b1a-V13 obtained in this study supports the cultural diffusion theory that the clade originated in the neighboring area of Southern Europe and spread only after populations adopted farming from Near Eastern pioneers using leapfrog colonization in the Aegean/Adriatic (Richards, 2003; Forenbaher and Miracle, 2005). Lacan et al. (2011b) and Paschoua et al. (2014) also concluded that the Mediterranean route had even greater influence on the peopling of Southern Europe during the Neolithic transition than the Central European one, based on autosomal, mitochondrial and Y chromosome DNA analysis, giving even more importance to this lineage.
The R1a-M420 clade arose in Western Eurasia and today it is most frequently observed in Eastern Europe, Western and Central Asia and southern Siberia. The recent, highly detailed phylogeographic analysis of 2,923 R1a-M420 Y chromosomes revealed two geographically divergent new mutations – the European Z282 and the Asian Z93 SNP, which defined major opposing clines. The Middle East (most probably present-day Iran) has been proposed as the plausible geographic center from which Z282 and Z93 chromosomes spread towards Europe and Asia, respectively (Underhill et al., 2015). This study proposed the M558 marker, which defines a sister clade to the already defined M458 lineage, with high frequencies in central/eastern Europe. Its prevalence drops drastically towards Western Europe, and it occurs at lower but informative frequencies in Balkan populations with known Slavonic heritage. This finding raised the possibility that the wide and rapid spread of the M458 and M558 lineages was associated with an autochthonous Bronze Age Proto-Slavic culture that arose in Central Europe near the Vistula river, the ‘Corded Ware culture.’ The age of R1a1a1b1a*-M558 obtained by Underhill et al. (2015) placed this lineage in the Holocene period (about 9 kya), based on STR data analysis. However, a more recent study conducted by Haak et al. (2015) suggests that the previous time estimates were significantly overestimated and that the Yamnaya culture, a cultural complex that spread across the Pontic Caspian-Ural steppes (beginning around 3,3 kya) brought the R1a clade into Europe in Late Neolithic. Two additional aDNA studies analyzing the Y chromosome showed that R1a individuals are absent or rare in sites dating before 5 kya (Batini et al., 2015; Haak et al., 2008), an observation consistent with the R1a age estimate published by Karmin et al. (2015). In this study, the R1a1a1b*-M458 lineage is not as frequent as its sister clade R1a1a1b1a*-M558, which is five times more common. This finding suggests that certain Central-European migrations left a more pronounced impact on the Croatian genetic landscape, especially the islands where the pronounced influence of genetic drift most probably pushed this particular R1a lineage to higher frequencies. Its high prevalence in our total sample (almost 20%), together with a high haplotype diversity, speaks in favor of several episodes of gene flow from Central Europe that occurred during prehistory and history in SEE, which is also evident in haplotype clusters depicted in the haplogroup’s phylogenetic network. These episodes were, as seems, male-specific, rapid and relatively recent (5 kya and younger), which makes any strong inference about older demographic events questionable (Hellenthal et al.,
The Mesolithic E1b1b1a1b1a-V13 foragers
CONCLUSION The genetic structure of modern SEE has been shaped by the complexity of human movements through the Paleolithic, Neolithic, the Bronze Ages and the most recent history of the last two millennia (involving the overlapping of different cultural and demic expansions). Molecular genetic analyses of the modern Croatian paternal genetic pool performed in this study confirm the American Journal of Human Biology
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extraordinary heterogeneity and complexity of this population and suggest a dynamic gene flow in the Croatian population and SEE throughout history. Our NRY analyses point to three distinct genetic signals traceable in the Croatian paternal genetic landscape - the dominant Balkan genetic heritage (embodied in hg I2a1b-M423), along with a Central European input from Slavicspeaking populations (R1a1a1b*-M558) and a recognizable, but moderate Neolithic influx (E1b1b1a2-V13). However, concerning the timing of the proposed lineages, recent studies show that most paternal lineages were greatly affected by more recent and rapid demographic changes that occurred in the last 5,000 years, making it difficult to make inferences about older demographic events. Also, one has to bear in mind that some of the haplotypes present in our sample have most probably been brought into the SEE gene pool at different time periods than the ones indicated in this study, some of them even very recently, and that evolutionary processes involve whole populations and not only certain haplogroups. Nonetheless, this study offers the most complete picture of Croatian paternal genetic diversity to date and offers additional insights into the specific genetic and possible historic events that shaped the current genetic landscape of Croatia and the wider SEE region. Subsequent whole Y chromosome sequencing and the advances in the aDNA research combined with historical, archeological, linguistic, and paleoclimatic perspectives will then further improve our understanding of the population movements in Croatia and SEE, as well as the underlying demographic and historic processes. Author Contributions All authors contributed to the work presented in this paper. J. S. analyzed data and wrote the paper, T. S., D. H A. and N. N. analyzed data, N. V., M. M., B. G., M. K., B. N., A. G. and S. M. collected samples, S. R. and P. R. supervised the project and wrote the paper. ACKNOWLEDGMENTS This research is a part of the project Population structure of Croatia – anthropogenetic approach Laboratory analyses were carried out at the Department of Evolutionary Biology, University of Tartu and Estonian Biocentre, Tartu, Estonia. REFERENCES Balanovsky O, Rootsi S, Pshenichnov A, Kivisild T, Churnosov M, Evseeva I, Pocheshkhova E, Boldyreva M, Yankovsky N, Balanovska E, Villems R. 2008. Two sources of the Russian patrilineal heritage in their Eurasian context. Am J Hum Genet 82: 236–250. Bandelt HJ, Forster P, R € ohl A. 1999. Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol 16: 37–48. Barac´ L, Pericˇic´ M, Klaric´ IM, Rootsi S, Janic´ijevic´ B, Kivisild T, Parik J, Rudan I, Villems R, Rudan P. 2003. Y chromosomal heritage of Croatian population and its island isolates. Eur J Hum Genet 11: 535–542. Batini C, Hallast P, Zadik D, Delser PM, Benazzo A, Ghirotto S, ArroyoPardo E, Cavalleri GL, de Knijff P, Dupuy BM, Eriksen HA, King TE, L opez de Munain A, L opez-Parra AM, Loutradis A, Milasin J, Novelletto A, P amjav H, Sa jantila A, Tolun A, Winney B, Jobling MA. 2 015. Largescale recent expansion of European patrilineages shown by population resequencing. Nat Commun 6: 7152. Battaglia V, Fornarino S, Al-Zahery N, Olivieri A, Pala M, Myres NM, King RJ, Rootsi S, Marjanovic´ D, Primorac D, Had ziselimovic´ R, Vidovic´ S, Drobnicˇ K, Durmishi N, Torroni A, Santachiara-Benerecetti AS, Underhill PA, Semino O. 2009. Y-chromosomal evidence of the cultural
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