Analysis of the R1b-DF27 haplogroup shows that a large fraction of Iberian Y-chromosome lineages originated recently in situ

1 SCIENtIFIC RePoRTS | 7: 7341 | DOI:10.1038/s41598-017-07710-x www.nature.com/scientificreports Analysis of the R1b-DF27 haplogroup shows that a large fraction of Iberian Y-chromosome lineages originated recently in situ Neus Solé-Morata 1, Patricia Villaescusa 2, Carla García-Fernández1, Neus Font-Porterias1, María José Illescas2, Laura Valverde2, Francesca Tassi3, Silvia Ghirotto3, Claude Férec4,5,6,7, Karen Rouault4,5, Susana Jiménez-Moreno8, Begoña Martínez-Jarreta9, Maria Fátima Pinheiro10, María T. Zarrabeitia11, Ángel Carracedo12,13, Marian M. de Pancorbo2 & Francesc Calafell 1 Haplogroup R1b-M269 comprises most Western European Y chromosomes; of its main branches, R1b-DF27 is by far the least known, and it appears to be highly prevalent only in Iberia. We have genotyped 1072 R1b-DF27 chromosomes for six additional SNPs and 17 Y-STRs in population samples from Spain, Portugal and France in order to further characterize this lineage and, in particular, to ascertain the time and place where it originated, as well as its subsequent dynamics. We found that R1b-DF27 is present in frequencies ~40% in Iberian populations and up to 70% in Basques, but it drops quickly to 6–20% in France. Overall, the age of R1b-DF27 is estimated at ~4,200 years ago, at the transition between the Neolithic and the Bronze Age, when the Y chromosome landscape of W Europe was thoroughly remodeled. In spite of its high frequency in Basques, Y-STR internal diversity of R1b-DF27 is lower there, and results in more recent age estimates; NE Iberia is the most likely place of origin of DF27. Subhaplogroup frequencies within R1b-DF27 are geographically structured, and show domains that are reminiscent of the pre-Roman Celtic/Iberian division, or of the medieval Christian kingdoms. Although it contains ~1% of the genome length in a human male cell, the lack of recombination along most of the Y chromosome makes constructing phylogenies for genetic variation relatively easy. Coupled with a robust geographic differentiation, this trait has provided a comprehensive phylogeography of Y chromosome haplotypes (usually referred to as haplogroups), that has been thoroughly characterized. Thus, the origin, dispersal, and geographic spread of many haplogroups are known. Moreover, both the genotyping of fast-mutating short tandem repeats (STRs) in the non-recombining region of the Y chromosome (NRY), and the recent availability of ascertainment-bias-free whole sequences of the NRY have reliably added a temporal scale to the deployment of the Y-chromosome diversity. One of the most salient features of the recent evolutionary history of human Y chromosomes is that it seems to have happened in bursts, with haplogroups rising to high frequency in the wake 1Institut de Biologia Evolutiva (CSIC-UPF), Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Catalonia, Spain. 2BIOMICs Research Group, Lascaray Research Center, University of the Basque Country UPV/EHU, Vitoria-Gasteiz, Spain. 3Dipartimento di Scienze della Vita e Biotecnologie, Università di Ferrara, Ferrara, Italy. 4Inserm, UMR 1078, Brest, France. 5Laboratoire de Génétique Moléculaire, CHRU Brest, Hôpital Morvan, Brest, France. 6Université de Bretagne Occidentale, Brest, France. 7Etablissement Français du Sang-Bretagne, Brest, France. 8Forensic and Legal Medicine Area, Department of Pathology and Surgery, University Miguel Hernández, Elche, Spain. 9Department of Forensic Medicine, University of Zaragoza, Zaragoza, Spain. 10Forensic Genetics Department, National Institute of Legal Medicine and Forensic Sciences, Porto, Portugal. 11Unit of Legal Medicine, University of Cantabria, Santander, Spain. 12Genomic Medicine Group, CIBERERUniversity of Santiago de Compostela, Galician Foundation of Genomic Medicine (SERGAS), Santiago de Compostela, Spain. 13Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia. Correspondence and requests for materials should be addressed to F.C. (email: [email protected]) Received: 6 April 2017 Accepted: 28 June 2017 Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 SCIENtIFIC RePoRTS | 7: 7341 | DOI:10.1038/s41598-017-07710-x of major lifestyle shifts and technological innovations such as the advent of the Neolithic or the recently acknowledged demographic upheaval caused by the Bronze Age in Europe1, 2. The most frequent Y-chromosome haplogroup in W Europe is R1b-M269, with frequencies ranging from 41% (Germany) to 83% (Ireland)3. Precisely, the higher frequency of this haplogroup in W Europe rather than in E Europe or W Asia led previous authors to believe it had a post-glacial Palaeolithic origin4, 5; however, a larger STR variance in SE European and W Asian R1b-M269 chromosomes and direct TMRCA dating pointed to R1b-M269 having surfed the Neolithic wave of advance3, 6, the evidence for which other authors did not find conclusive7. Finally, direct dating from NRY sequence variation puts the origin of R1b-M269 in the Early Bronze Age, ~4500 years ago (ya)1, 8, consistent with the growing ancient DNA record, where a surge in R1b-M269 is indeed seen at that time2, 9. Note, though, that R1b-M415, a branch ancestral to R1b-M269, was found as early as 14,000 ya in Italy10 and 7,000 ya in Spain2. Moreover, lack of structure of STR variation within R1b-M26911, 12 points also to an explosive growth. The most important branches of R1b-M269 are R1b-U106, particularly frequent in the Low Countries and NW Germany3, 13, and R1b-S116 (also known as R1b-P312), which is common throughout W Europe3. The latter trifurcates in turn into U152 (frequent in N Italy and Switzerland13), L21 (also known as M529, abundant in the British Isles7), and DF27 (Fig.1; Supplementary Figure1). DF27 was first discovered by citizen scientists14 and, although among the burgeoning amateur genetic genealogy it is known to be frequent in Iberian populations and their overseas offshoots, few academic publications have been devoted to it. It was found in the 1000 Genome Project populations at a frequency of 49% in Iberians, 6% in Tuscans, 7% in British, and it was absent elsewhere except for admixed populations in the Americas: Colombia (40%), Puerto Rico (36%), Mexico (10%), Perú (8%), African-Americans (4%) and Afro-Caribbeans (2%)14, 15. It was first genotyped specifically in a few Iberian populations, Brittany and Ireland as part of a study on R1b-S11616, which indeed confirmed that R1b-DF27 is present at frequencies >40% in Spain and Portugal. Subsequently, 12 SNPs within DF27 were genotyped in four N Spanish populations17, confirming its high frequency and hinting at some substructure within the Iberian Peninsula. As for its presence elsewhere, the frequency of R1b-S116 (xL21, U152) can be used as an upper bound for the frequency of R1b-DF27. R1b-S116 (x L21, U152) was found at frequencies 0–10% in Germany3, 18, 7% in the Netherlands3, 8–12% in Flanders19, 6–12% in Switzerland3, and 1–12% in Italy3, 20. Here, by extending population sampling to cover France as well as by improving coverage in the Iberian Peninsula, we aim to i) further characterize the spatial distribution of R1b-DF27, ii) estimate its time of origin, and iii) explicitly model its expansion in relation both to its phylogenetic context, and to the demographic events that thoroughly reshaped the genetic diversity of W Europe around 4500 ya. Results Over one thousand individuals carrying DF27 were typed for six additional SNPs (Table1, Fig.1) and 17 Y-STRs. DF27 itself was found at frequencies 0.3–0.5 in Iberia (with a mean of 0.42), with the notable exception of native Basques, where it reached 0.74 (for this and all subsequent frequency values, see Fig.2 and Supplementary Table1). In France, it dropped to a range of 0.06–0.20 and a mean of 0.11. Elsewhere, it was 0.15 in Britain (but <0.01 in Ireland) and 0.08 in Tuscany. Most (50–100%, with a proportion that dropped from East to West) DF27 Y chromosomes were also derived for Z195; thus, the highest frequencies of Z195 (0.29–0.41) were reached both in the Basque Country and in E Iberia (Catalonia, Valencia), and it becomes as rare in Portugal as it is in France. Conversely, the highest frequencies of R1b-DF27* (xZ195) are found in Native Basques and Western Iberian populations such as Asturias, Portugal and Galicia, which may harbor yet unknown branches of R1b-DF27. In turn, Z195 splits into two branches, namely L176.2 and Z220 (Fig.1). Note that L176 is a recurrent mutation that defines two clades in the Y phylogeny: L176.1 within R1a, and L176.2 under R1b-DF27; throughout this Figure 1. Simplified phylogenetic tree of the R1b-M269 haplogroup. SNPs in italics were not analyzed in this manuscript. www.nature.com/scientificreports/ 3 SCIENtIFIC RePoRTS | 7: 7341 | DOI:10.1038/s41598-017-07710-x manuscript, we will refer exclusively to the latter. L176.2 and Z220 peak, respectively, in E Iberia and the Basque Country. L176.2 is further subdivided into M167 (SRY2627, ref. 21), with the highest frequencies in Catalonia and the lands settled from Catalonia in the 13th century (Valencia, the Balearics). This marker had been typed in a number of Iberian and other European populations4, 18–20, 22–25, and the overall frequency pattern found (Supplementary Figure1) confirms a distribution centred in the eastern half of Iberia, although with higher frequencies (up to 0.16) in the upper Ebro river valley and the Pyrenees. As mentioned above, Z220 is most frequent in the Basque Country (0.28), and a similar pattern is found for its successive nested clades, namely Z278 and M153. For the latter, available additional data22, 23, 25 showed it confined to the Iberian Peninsula, with frequencies 0.06–0.40 among Basque subpopulations, but rarely above 0.01 elsewhere (Supplementary Figure1). The subhaplogroup frequencies were summarized in a PC plot (Fig.3). The first PC separated the Iberian populations (save for the three westernmost samples, namely Portugal, Galicia, and Asturias) from the rest, explained 68.6% of the total variation, and was positively correlated with DF27 and all of its subhaplogroups. On the contrary, PC2 (20.9%) was positively correlated with L176.2 and M167 and most negatively correlated with Z278 and M153, and separated most Eastern Iberian populations from the rest. In order to quantify the structure of subhaplogroup frequencies, we performed AMOVA with several population groupings. Thus, if we compared Iberian populations vs. the rest, the proportion of the variance explained by the differences among these two groups (i.e., FCT) was 12.40% (p < 10−4), while the proportion of the variance found within groups (i.e., FSC) was 3.20% (p < 10−4). If the native Basques were split from the Iberians, then FCT = 13.57% (p < 10−4) and FSC = 1.37% (p < 10−4). Finally, if Eastern Iberians are also split from the rest of Iberians, then FCT = 11.68% (p < 10−4) and FSC = 0.31% (p = 0.0106). In conclusion, the differences among the groups that are apparent in the PCA plot are highly statistically significant. Population abbr. Region lon lat Total N Source N DF27 typed Additional R1b(1) Additional P312 (2) Additional Z195 (3) SNP typing STR typing Alacant ALA Valencia −0.56 38.36 142 (113)16, (29)27 22 0 0 0 this work16, 27 [27], [54] Alsace ALS France 7.75 48.58 80 [39] 6 0 0 0 this work39 [39] Andalucía AND NA −6 37.5 100 [16] 47 0 0 0 this work16 NA Aragón ARA Aragón −0.86 41.63 92 [17] 34 0 0 0 [17] [26] Asturias AST N. Central Spain −5.87 43.34 63 [16] 27 0 0 0 [17] [26] Auvergne AUV France 3.09 45.78 89 [39] 5 0 0 0 this work39 [39] Barcelona BCN Catalonia 2.13 41.4 571 (99)16, (472)27 245 11 8 6 this work16, 27 [26], [27] Brittany BRI NA −4.49 48.39 145 [16] 28 0 0 0 this work16 NA Cantabria CAN N. Central Spain −3.83 43.35 96 [16] 43 0 0 0 [16], [17] [41] Castelló CAS Valencia −0.06 40 49 [27] 22 2 0 1 this work27 [27] Galicia GAL NA −8.55 42.88 70 [16] 28 0 0 0 this work16 NA Girona GIR Catalonia 2.81 41.99 131 [27] 42 2 3 0 this work27 27 Île-de-France IDF France 2.35 48.86 91 [39] 9 2 0 0 this work39 39 Ireland IRE NA −8 53 146 [16] 1 0 0 0 this work16 NA Lleida LLE Catalonia 0.62 41.62 104 [27] 52 2 0 0 this work27 27 Madrid MAD NA −3.72 40.42 99 [16] 49 0 0 0 this work16 NA Mallorca MAL Mallorca 3 39.63 48 [27] 24 0 2 0 this work27 [27] Midi-Pyrénées MPY France 1.45 43.61 67 [39] 7 1 0 0 this work39 [39] Native Basque NBA Basques −2.47 43.17 229 [16] 169 0 0 0 16, 17 [16] Nord-Pas-de-Calais NPC France 3.04 50.63 68 [39] 8 3 0 0 this work39 [39] Portugal POR NA −8.5 39.5 109 [16] 44 0 0 0 this work16 NA Provence–Alpes-Côte d’Azur PAC France 5.45 43.29 45 [39] 5 3 0 0 this work39 [39] Pyrenees PYR Catalonia 1.12 42.49 46 [27] 24 0 1 0 this work27 [27] Resident Basque RBA N. Central Spain −3.69 42.35 111 [16] 53 0 0 0 [16], [17] [16] Tarragona TAR Catalonia 1.17 41.12 120 [27] 47 0 1 1 this work27 [27] Valencia VAL Valencia −0.4 39.48 79 [27] 31 3 2 1 this work27 [27] Total 2990 1072 29 17 9 Table 1. Populations sampled and their original sources. Region: higher population grouping used in some analyses. (1): Chromosomes predicted to be R1b but without further SNP typing; (2): Chromosomes known to be R1b-P312* (xZ195, U152, L21) but without further typing; (3): Chromosomes known to be R1b-Z195 (xM167, Z220), but not typed for L176. www.nature.com/scientificreports/ 4 SCIENtIFIC RePoRTS | 7: 7341 | DOI:10.1038/s41598-017-07710-x Haplotypes comprising 17 Y-STRs were available for 758 individuals (Table2). AMOVA among this set of populations gave RST = 0.72% (p = 0.00386), while, for the same populations, subhaplogroup frequencies yielded FST = 8.33% (p < 10−4). Thus, Y-STRs seem to capture much less phylogeographic structure than SNPs themselves, as described for R1b-M26911, 12. Still, some Y-STR structure may be present within R1b-DF2712, 17. Since a median-joining tree with 688 different haplotypes is unmanageable, we resorted to principal component analysis (PCA) among haplotypes (Fig.4). The first PC explained 15.1% of the STR variation and correlated mostly with DYS437 (r = 0.865), DYS448 (r = 0.858), and YGATAH4 (r = 0.724), and separated haplotypes that carried the derived allele for Z220 (that is, belonging to R1b-Z220*, R1b-Z278* and R1b-M153) from the rest. PC1 Figure 2. Contour maps of the derived allele frequencies of the SNPs analyzed in this manuscript. Population abbreviations as in Table1. Maps were drawn with SURFER v. 12 (Golden Software, Golden CO, USA). www.nature.com/scientificreports/ 5 SCIENtIFIC RePoRTS | 7: 7341 | DOI:10.1038/s41598-017-07710-x coordinates were highly significantly different by subhaplogroup (p ~ 10−150, ANOVA). The median haplotype for Z220-derived chromosomes was 11-14-18 at YGATAH4-DYS437-DYS448 while it was 12-15-19 for the rest of DF27 chromosomes (other STRs showed the same median allele). PC2 explained 8.6% of the STR variation and correlated with DYS390 (r = 0.647) and DYS456 (r = −0.544), and separated R1b-Z220* from R1b-Z278* chromosomes; overall, R1b-DF27 subhaplogroups had significantly different PC2 coordinates (p = 9.12 × 10−4). The same PC results can also be analyzed by population (Fig.4b): PC1 coordinates are statistically significantly different by population (ANOVA, p = 0.00512), with the French samples having higher, positive values in this Figure 3. Principal component analysis of subhaplogroup frequencies. Population abbreviations as in Table1. Circles: Iberian populations; squares: non-Iberian populations. Population N K Dhap Var sd (Var) All 758 688 0.9996 0.330 0.215 Alacant 57 56 0.9994 0.377 0.400 Alsace 5 5 1 0.260 0.292 Aragón 29 29 1 0.372 0.218 Asturias 26 26 1 0.283 0.274 Auvergne 5 5 1 0.187 0.125 Native Basques 154 122 0.9951 0.282 0.174 Barcelona 184 178 0.9995 0.348 0.226 Cantabria 30 30 1 0.403 0.364 Castelló 23 23 1 0.294 0.213 Girona 33 33 1 0.306 0.183 Île-de-France 8 8 1 0.323 0.204 Lleida 39 39 1 0.314 0.187 Mallorca 21 21 1 0.341 0.280 Midi-Pyrénées 7 7 1 0.308 0.314 Nord-Pas-de-Calais 7 7 1 0.432 0.459 Provence–Alpes-Côte d’Azur 3 3 1 0.156 0.278 Pyrenees 17 17 1 0.443 0.328 Resident Basques 49 49 1 0.285 0.188 Tarragona 38 38 1 0.351 0.276 Valencia 23 23 1 0.337 0.152 Table 2. Diversity parameters for STR variation within R1b-DF27 chromosomes. K: number of different haplotypes; Dhap: haplotype diversity; Var: average STR allele repeat size variance; sd: standard deviation across loci of Var. www.nature.com/scientificreports/ 6 SCIENtIFIC RePoRTS | 7: 7341 | DOI:10.1038/s41598-017-07710-x PC, while PC2 is not significant across populations (p = 0.781). These results can be explained by the very low frequency of Z220-derived chromosomes outside of Iberia. Internal diversity and ages of DF27 and its derived subhaplogroups. The average STR variance of DF27 and each subhaplogroup is presented in Suppl. Table2. As expected, internal diversity was higher in the deeper, older branches of the phylogeny. If the same diversity was divided by population, the most salient finding is that native Basques (Table2) have a lower diversity than other populations, which contrasts with the fact that DF27 is notably more frequent in Basques than elsewhere in Iberia (Suppl. Table1). Diversity can also be measured as pairwise differences distributions (Fig.5). The distribution of mean pairwise differences within Z195 sits Figure 4. Principal component analysis of STR haplotypes. (a) Colored by subhaplogroup, (b) colored by population. Larger squares represent subhaplogroup or population centroids. www.nature.com/scientificreports/ 7 SCIENtIFIC RePoRTS | 7: 7341 | DOI:10.1038/s41598-017-07710-x practically on top of that of DF27; L176.2 and Z220 have similar distributions, as M167 and Z278 have as well; finally, M153 shows the lowest pairwise distribution values. This pattern is likely to reflect the respective ages of the haplogroups, which we have estimated by a modified, weighted version of the ρ statistic (see Methods). We estimated an age of 4190 ± 140 ya for the whole of DF27. This figure is remarkably similar both to the estimate (4128 ± 71 ya) that can be produced from whole Y-chromosome sequence variability in the 88 DF27-derived individuals present overall in the 1000 genomes project dataset, and to the age estimated from 201 individuals in our dataset for which 21 non-duplicated Y-STRs from the Powerplex Y23 System were available26 (3880 ± 165). Z195 seems to have appeared almost simultaneously within DF27, since its estimated age is actually older (4570 ± 140 ya). Of the two branches stemming from Z195, L176.2 seems to be slightly younger than Z220 (2960 ± 230 ya vs. 3320 ± 200 ya), although the confidence intervals slightly overlap. M167 is clearly younger, at 2600 ± 250 ya, a similar age to that of Z278 (2740 ± 270 ya). Finally, M153 is estimated to have appeared just 1930 ± 470 ya. Haplogroup ages can also be estimated within each population, although they should be interpreted with caution (see Discussion). For the whole of DF27, (Table3), the highest estimate was in Aragon (4530 ± 700 ya), and the lowest in France (3430 ± 520 ya); it was 3930 ± 310 ya in Basques. Z195 was apparently oldest in Catalonia (4580 ± 240 ya), and with France (3450 ± 269 ya) and the Basques (3260 ± 198 ya) having lower estimates. On the contrary, in the Z220 branch, the oldest estimates appear in North-Central Spain (3720 ± 313 ya for Z220, 3420 ± 349 ya for Z278). The Basques always produce lower estimates, even for M153, which is almost absent elsewhere. R1b-DF27 and demography. We tested the dynamics of R1b-DF27 by means of Approximate Bayesian Computing (ABC). In particular, we compared two simple models: constant population size vs. growth since time Tstart. Both the rejection and the regression method undoubtedly favoured the growth model, with associated posterior probabilities that were never lower than 0.99. The principal component analysis (PCA) of the first 3,000 best simulations from each model (i.e. the 3,000 simulation closest to the observed dataset that are generated by each model) actually shows that the point corresponding to the observed data falls in the middle of the results obtained simulating growth, thus confirming that the growth model is also able to generate the observed variation (Suppl. Fig.2). We then estimated the demographic parameters associated with the growth model. The median value for Tstart has been estimated at 103 generations (Table4), with a 95% highest probability density (HPD) range of 50–287 generations; effective population size increased from 131 (95% HPD: 100–370) to 72,811 (95% HPD: 52,522–95,334). Considering patrilineal generation times of 30–35 years27, our results indicate that R1b-DF27 started its expansion ~3,000–3,500 ya, shortly after its TMRCA. As a reference, we applied the same analysis to the whole of R1b-S116, as well as to other common haplogroups such as G2a, I2, and J2a. Interestingly, all four haplogroups showed clear evidence of an expansion (p > 0.99 in all cases), all of them starting at the same time, ~50 generations ago (Table4), and with similar estimated initial and final populations. Thus, these four haplogroups point to a common population expansion, even though I2 (TMRCA, weighted ρ, 7,800 ya) and J2a (TMRCA, 5,500 ya) are older than R1b-DF27. It is worth noting that the expansion of these haplogroups happened after the TMRCA of R1b-DF27. Figure 5. Cumulative distributions of the number of pairwise absolute differences in repeat size among individuals, by subhaplogroup. www.nature.com/scientificreports/ 8 SCIENtIFIC RePoRTS | 7: 7341 | DOI:10.1038/s41598-017-07710-x Discussion We have characterized the geographical distribution and phylogenetic structure of haplogroup R1b-DF27 in W. Europe, particularly in Iberia, where it reaches its highest frequencies (40–70%). The age of this haplogroup appears clear: with independent samples (our samples vs. the 1000 genome project dataset) and independent methods (variation in 15 STRs vs. whole Y-chromosome sequences), the age of R1b-DF27 is firmly grounded Haplogroup ages N age 95% CI DF27 total 758 4194 (4055, 4333) Aragón 29 4527 (3831, 5223) Basques 154 3931 (3617, 4245) Catalonia 311 4367 (4163, 4571) France 35 3428 (2911, 3945) Mallorca 21 4091 (3360, 4822) N. Central Spain 105 3614 (3293, 3935) Valencia 103 4525 (4166, 4884) Z195 total 510 4569 (4398, 4740) Aragón 18 4177 (3293, 5061) Basques 83 3257 (2869, 3645) Catalonia 246 4584 (4349, 4819) France 34 3450 (2923, 3977) Mallorca 14 4028 (3081, 4975) N. Central Spain 44 3917 (3407, 4427) Valencia 71 4541 (4114, 4968) L176.2 total 189 2964 (2737, 3191) Aragón 10 2759 (1626, 3892) Basques 14 3059 (2222, 3896) Catalonia 108 2931 (2651, 3211) France 15 3016 (2273, 3759) Mallorca 7 1465 (440, 2490) N. Central Spain 8 2365 (1465, 3265) Valencia 27 2933 (2370, 3496) M167 total 137 2602 (2351, 2853) Aragón 4 3458 (1919, 4997) Basques 7 1221 (217, 2225) Catalonia 81 2597 (2289, 2905) France 12 2812 (2010, 3614) Mallorca 6 1626 (466, 2786) N. Central Spain 6 2050 (1082, 3018) Valencia 21 2404 (1818, 2990) Z220 total 267 3318 (3114, 3522) Aragón 7 1956 (1080, 2832) Basques 62 2693 (2291, 3095) Catalonia 123 3380 (3094, 3666) Mallorca 7 3198 (2079, 4317) N. Central Spain 27 3718 (3105, 4331) Valencia 40 3683 (3166, 4200) Z278 total 130 2745 (2475, 3015) Aragón 3 1716 (464, 2968) Basques 46 2298 (1877, 2719) Catalonia 41 2425 (1994, 2856) N. Central Spain 20 3417 (2733, 4101) Valencia 20 3084 (2369, 3799) M153 total 34 1926 (1454, 2398) Basques 15 1582 (933, 2231) Catalonia 9 1202 (438, 1966) Valencia 8 2504 (1393, 3615) Table 3. Haplogroup ages estimated from STR variation with the weighted ρ method. 95% CI: 95% confidence interval. www.nature.com/scientificreports/ 9 SCIENtIFIC RePoRTS | 7: 7341 | DOI:10.1038/s41598-017-07710-x around 4000–4500 ya, which coincides with the population upheaval in W. Europe at the transition between the Neolithic and the Bronze Age2, 9. Before this period, R1b-M269 was rare in the ancient DNA record, and during it the current frequencies were rapidly reached2, 9, 10. It is also one of the haplogroups (along with its daughter clades, R1b-U106 and R1b-S116) with a sequence structure that shows signs of a population explosion or burst1. STR diversity in our dataset is much more compatible with population growth than with stationarity, as shown by the ABC results, but, contrary to other haplogroups such as the whole of R1b-S116, G2a, I2 or J2a, the start of this growth is closer to the TMRCA of the haplogroup. Although the median time for the start of the expansion is older in R1b-DF27 than in other haplogroups, and could suggest the action of a different demographic process, all HPD intervals broadly overlap, and thus, a common demographic history may have affected the whole of the Y chromosome diversity in Iberia. The HPD intervals encompass a broad timeframe, and could reflect the post-Neolithic population expansions from the Bronze Age to the Roman Empire28. While when R1b-DF27 appeared seems clear, where it originated may be more difficult to pinpoint. If we extrapolated directly from haplogroup frequencies, then R1b-DF27 would have originated in the Basque Country; however, for R1b-DF27 and most of its subhaplogroups, internal diversity measures and age estimates are lower in Basques than in any other population. Then, the high frequencies of R1b-DF27 among Basques could be better explained by drift rather than by a local origin (except for the case of M153; see below), which could also have decreased the internal diversity of R1b-DF27 among Basques. An origin of R1b-DF27 outside the Iberian Peninsula could also be contemplated, and could mirror the external origin of R1b-M269, even if it reaches there its highest frequencies. However, the search for an external origin would be limited to France and Great Britain; R1b-DF27 seems to be rare or absent elsewhere: Y-STR data are available only for France, and point to a lower diversity and more recent ages than in Iberia (Table3). Unlike in Basques, drift in a traditionally closed population seems an unlikely explanation for this pattern, and therefore, it does not seem probable that R1b-DF27 originated in France. Then, a local origin in Iberia seems the most plausible hypothesis. Within Iberia, Aragon shows the highest diversity and age estimates for R1b-DF27, Z195, and the L176.2 branch, although, given the small sample size, any conclusion should be taken cautiously. On the contrary, Z220 and Z278 are estimated to be older in North Central Spain (N Castile, Cantabria and Asturias). Finally, M153 is almost restricted to the Basque Country: it is rarely present at frequencies >1% elsewhere in Spain (although see the cases of Alacant, Andalusia and Madrid, Suppl. Table1), and it was found at higher frequencies (10–17%) in several Basque regions25; a local origin seems plausible, but, given the scarcity of M153 chromosomes outside of the Basque Country, the diversity and age values cannot be compared. Within its range, R1b-DF27 shows same geographical differentiation: Western Iberia (particularly, Asturias and Portugal), with low frequencies of R1b-Z195 derived chromosomes and relatively high values of R1b-DF27* (xZ195); North Central Spain is characterized by relatively high frequencies of the Z220 branch compared to the L176.2 branch; the latter is more abundant in Eastern Iberia. Taken together, these observations seem to match R1b-DF27 Mean Median Mode 95% HPD-LowB 95% HPD-UppB Tstart 128 103 75 50 287 Na 162 131 100 100 350 Nc 72685 72812 73624 52523 95334 GSM 0.047 0.046 0.044 0.004 0.089 R1b-S116 Tstart 50 50 50 50 52 Na 182 134 110 100 370 Nc 99844 99854 99860 99690 100000 GSM 0.239 0.240 0.246 0.094 0.380 G2a Tstart 82 61 51 50 175 Na 145 122 100 100 340 Nc 10448 7984 5865 1549 25267 GSM 0.179 0.177 0.179 0.023 0.325 I2 Tstart 82 67 58 50 136 Na 178 140 100 100 370 Nc 40622 37366 28135 9162 81946 GSM 0.036 0.027 0 0 0.099 J2a Tstart 55 52 51 50 61 Na 154 129 100 100 350 Nc 94264 95421 96953 85633 100000 GSM 0.022 0.017 0.000 0.000 0.069 Table 4. ABC results for R1b-DF27 and reference haplogroups. GSM: Generalized Stepwise Mutation; HPD: Highest Probability Density.