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Post by Admin on Mar 29, 2021 21:06:19 GMT
Fig. 2. Admixture among ancient groups based on genome sequence data. (A) Admixture graph with four migration edges for the individual with the highest sequencing coverage for each geographic site, region, or time period. To improve clarity, branch lengths are not drawn to scale and represent only the hierarchical clustering (see SI Appendix, Fig. S10 for the topology drawn to scale). (B) Genetic affinity of the different early farmer individuals (f4-statistic) to a Central European Mesolithic individual (Loschbour, highest coverage Mesolithic genome), as a function of the dating of the early farmer individuals. A central European LBK individual is used as a baseline early Neolithic farmer (highest coverage early Neolithic individual, but the choice of reference individual does not affect the qualitative result) (SI Appendix, section S11). The Bronze Age, Chalcolithic, and Scandinavian farmers show greater levels of admixture with HG groups than the temporally older Neolithic Central European farmers (Pearson correlation R2 = 0.69, P = 0.001). To further investigate the relationship between the El Portalón farmers and modern-day individuals, we inferred admixture fractions (22) among a large set of modern-day individuals from Eurasia and North Africa (Fig. 3A and Datasets S1 and S5). All modern-day Iberian groups displayed ancestry from early farmers and hunter–gatherers and also showed admixture from North Africa (Fig. 3A, yellow component) (23) and the Caucasus/Central Asia (Fig. 3A, dark purple component), potentially related to the observed migration during the Bronze Age (24, 25) or the later Roman Empire ruling of Iberia. Basques (including French Basques) were an exception; they display ancestry from early farmers and hunter–gatherers, similar to other modern-day Iberian groups, but little or no admixture from North Africa and the Caucasus/Central Asia (1, 23) (Fig. 3A and SI Appendix, section S10). Interestingly, among all European groups, Basques and Sardinians displayed strong genetic affinity to the El Portalón farmers (Fig. 3B and SI Appendix, Fig. S8). However, all other early farmers were closer to Sardinians (SI Appendix, Figs. S11 and S12), and Basques were closer to El Portalón individuals (or equally close for Gok2) compared with all other early farmers (SI Appendix, Fig. S13). To further test the scenario of Basques being the genetically most similar group to the El Portalón farmers, we computed D-statistics for different population topologies. All topologies where Basques were an outgroup to the highest coverage El Portalón individual (ATP2) and another modern-day Spanish population—D(Mbuti, Basques; other Spanish, ATP2)—were rejected [36 tests, false discovery rate (FDR) < 0.01] (SI Appendix, section S11) whereas all topologies using Basques as an ingroup with ATP2, and another Spanish population as an outgroup, were consistent with the data. Test results for the other ATP individuals showed qualitatively similar patterns (SI Appendix, section S11 and Datasets S6 and S7). Our data suggest that modern-day Basques traced their genetic ancestry to early Iberian farmers. Fig. 3. Population structure of ancient and modern-day individuals. (A) Admixture fractions among modern-day individuals from Eurasia and North Africa together with 16 ancient individuals. Only ancient and modern-day individuals from Southwestern Europe are shown (see Dataset S1 for the complete plot with all individuals). Admixture components are labeled based on the populations/geographic regions in which they are modal. (B) Genetic similarity (measured using outgroup f3 statistics) between Southwestern European groups and four El Portalón individuals. Error bars show ±1 SE. Higher values represent greater genetic similarity between the El Portalón farmers and the modern-day Southwestern populations in the legend. This high similarity of modern Basques to El Portalón individuals was surprising because Basques have been posited as a remnant isolated population with a close relationship to the Mesolithic inhabitants of the region, based on classical genetic markers (26) and mtDNA haplogroup data (8), although the level of continuity has been unclear (12, 13). The Basque language (Euskara) is a linguistic isolate, with no proven relationships with any languages now spoken in Europe or elsewhere (11), and it has commonly been concluded that the Basque language is a relict of the ancient, preagricultural linguistic diversity of Europe, with roots as far back as the Paleolithic (SI Appendix, section S12) (9). Our data, suggesting that Basques trace their genetic ancestry to early Iberian farmers, challenges this assumption. The alternative interpretations of the linguistic history of Europe are, however, unclear. The remaining languages of Western Europe belong to the Indo-European family (27). The origin of the Indo-European language family is itself controversial (28), with most debate polarized between proponents of the steppe hypothesis, that Indo-European was introduced into Europe from the East during the Bronze Age (∼4,500 yBP) (29), and the Anatolian hypothesis, that Indo-European language dispersed from Anatolia during the Neolithic (30, 31). There was genetic turnover associated with Yamnaya and Corded Ware cultures at ∼4,500 yBP, which may thus be associated with a primary or secondary dispersal of Indo-European languages (25). A possible interpretation of the role of Basque in this scenario would be that it is a descendent of the language (or one of the languages) of the early farmers, and some scholars have posited that the Basque language was related to the pre-Roman language of Sardinia (Paleosardo) (32). The two Southern European population isolates of Sardinians and Basques were genetically associated with the early farmers of Europe that drove the Neolithic transition (1⇓–3), and close contacts between Iberia and Sardinia in the Neolithic are also indicated by archaeological finds (33). However, the possibility remains that the Basque language is a retention of the preagricultural linguistic diversity. Conclusions In summary, our ancient genomic sequence data from the El Portalón individuals and our analyses suggest the following model of events: The incoming early farmers, who could have spoken a non–Indo-European language, assimilated resident hunter–gatherers, with this admixed group becoming the ancestors of modern-day Iberian groups. Basques remained relatively isolated (compared with other Iberian groups) with marked continuity since the Neolithic/Chalcolithic period, but not since the Mesolithic (contrary to refs. 8, 9, and 26). Later migration into Iberia, possibly during the long reign of the Roman Empire and the 7th to 13th century period of Moorish rule of the peninsula, led to distinct and additional admixture in all Iberian groups but the Basque population (23).
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Post by Admin on Mar 29, 2021 23:11:17 GMT
Materials and Methods Archaeological Samples. Sixteen bone and teeth human remains, representing sixteen individuals from the Chalcolithic and Bronze Age site of El Portalón (Spain) (14) were sampled for ancient DNA analyses. The samples had been excavated between 2000 and 2012, and C14 dates were obtained for each of them using accelerator mass spectrometry (AMS). See SI Appendix, section S1 for details. Sequencing. DNA was extracted from bones and teeth (34⇓–36); DNA extracts were converted into blunt-end Illumina libraries (37). All samples were prepared in dedicated ancient DNA (aDNA) facilities at the Evolutionary Biology Center in Uppsala, Sweden. The libraries were sequenced on Illumina’s HiSeq platform at the SNP&SEQ Technology Platform SciLife Sequencing Centre in Uppsala. All 16 samples were screened for human DNA, and only individuals with over 1% of human DNA content (n = 8) were used for downstream analysis. See SI Appendix, section S2 and Dataset S8 for details. Next Generation Sequencing Data Processing and Authentication. Paired-end reads were merged, and remaining adapters were trimmed (38). The merged and trimmed reads were subsequently mapped to the human reference genome using BWA (39); potential PCR duplicates with identical start and end coordinates were collapsed into consensus sequences. The sequences showed a deamination pattern toward fragment ends, which are characteristic for ancient DNA (16). Contamination was estimated based on discordant sites in mitochondria and the X chromosome in males (40⇓–42). A detailed description can be found in SI Appendix, section S3. Uniparental Haplogroups. Consensus sequences for the mitochondrial genomes of all samples were called using the samtools package (43). We used haplofind (44) to assign the mitochondrial genome to known mitochondrial haplogroups. Y haplogroups were assigned based on PhylotreeY (45). We excluded all non-SNP sites, transition sites (to avoid deamination damage), and A/T and G/C SNPs (to avoid strand misidentification). See SI Appendix, sections S4 and S5 for details. Modern Reference Data. The ancient samples were merged with the Human Origins genotype data (1, 46), excluding transition sites and sites showing indels. Most of the ancient samples have sequencing depths too low to confidently call diploid genotypes. Therefore, we randomly sampled one allele per individual and SNP site. Only reads and bases with a minimum mapping and base quality of 30 were considered. To increase power for the comparison of sequenced ancient individuals, we repeated the same procedure for 1.9 million transversion SNPs, which were polymorphic in Yorubans of the 1000 Genomes Project phase 3 data (21). See SI Appendix, section S7 for a detailed description. Population Genetic Analysis. PCAs of ancient individuals and modern European populations from the Human Origins dataset were conducted using EIGENSOFT (47). Ancient individuals were projected onto the PC1-PC2 space using Procrustes analysis (48). We calculated D-statistics (46) to check for consistency of the data with different tree topologies and f3 and f4 statistics (46, 49) to estimate affinities among populations. See SI Appendix, section S8 for more details. TreeMix (20) was used to infer a bifurcating population tree plus admixture events for the highest coverage individuals from each of the ancient European groups using Denisovans (50) as an outgroup and restricting the analysis to transversion SNPs that were polymorphic in Yorubans (21). Correction for low sample sizes was turned off (-noss), and SEs were estimated using blocks of 500 SNPs. We show the admixture graph for m = 4 in Fig. 2A, and a detailed description of the methods and results for other migration events can be found in SI Appendix, section S9. Model-based clustering of the ancient individuals together with Eurasian and North African populations from the Human Origins dataset (1) was conducted with ADMIXTURE (22). The genotype data were pruned for linkage disequilibrium, and we tested different numbers of clusters from K = 2 to K = 15. The results of 50 iterations per number of clusters were combined using CLUMPP (51) and plotted with distruct (52). We chose to display K = 10 in the main paper (Fig. 3A) because it is the lowest value of K to show a clear distinction between hunter–gatherer, early farmer, North African, and Near Eastern components. The plot for all Ks is shown in Dataset S1, and more details can be found in SI Appendix, section S10. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1509851112/-/DCSupplemental.
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Post by Admin on Mar 30, 2021 0:51:22 GMT
S1. The El Portalón site stratigraphic and cultural sequence The El Portalón cave is an archaeological site in the Sierra de Atapuerca (Burgos, Spain; Fig. S1), a region well known for its Early and Middle Pleistocene sites [1,2], but more recent excavations have shown that it also presents a rich and varied archaeological record [3–5]. Previous studies have been successful in obtaining ancient DNA from faunal remains in the El Portalón cave [6,7].
The El Portalón cave spans the Late Pleistocene to the Holocene and it is during this later period that the cave became one of the main entrances to the Cueva Mayor-Cueva del Silo karst (Fig. S1). Recent excavations at the El Portalón have revealed a stratigraphic sequence starting in the Late Pleistocene and showing evidence of human occupation throughout the Holocene. [3] reported detailed radiocarbon dates for the entire stratigraphy ranging from 30,000 BP to 1,000 BP. Two major sedimentary phases are observed in the El Portalón archaeological record, the lower phase comprises sediments from the Late Pleistocene and has a significant paleontological record and sparse Upper Palaeolithic human artifacts. The upper phase corresponds to the Holocene and is characterized by dark archaeological sediments with abundant archaeological artifacts. The Holocene phase includes Mesolithic, Neolithic, Chalcolithic, Bronze Age, Iron Age, Roman and Medieval periods of human occupation. The palaeoenvironmental data obtained from the speleothem record [8], are of great importance because of the scarcity of this type of information regarding the Upper Palaeolithic, Mesolithic and Neolithic cultural periods in the interior of the Iberian Peninsula. In addition to its vast cultural/technological and faunal remains, a series of burials have been identified in the Chalcolithic period. These burials are often accompanied with grave goods, such as pottery and small animal bones in anatomical position, most commonly sheep. The archaeological content combined with direct radiocarbon dating of the human samples analysed suggest a pre-Bell Beaker Chalcolithic chronocultural assignation for the burials.
S1.1. Early Chalcolithic (Pre-Bell Beaker) funerary context, Bronze Age and disturbed layers from the El Portalón cave The burial phase comprises a tumular stacking of decimetric limestone clasts (Figs. S1 & S2). The tumulus appears to be of an oval shape and its structure corresponds to a progressive accumulation of limestone clasts in an agrading (vertical) and prograding fashion over a basal surface defined by floors that have ceramic "pavements" on which there are numerous circular small fire pits filled with partially combusted charcoal fragments. Associated with the fire pits, are nearly complete small ceramic bowls, along with remains of juvenile individuals of domestic fauna, mainly lambs, in anatomical connection (Fig. S2). Among the limestone clasts and sometimes lying on the floor, are different human remains, defining funerary contexts with some archaeological elements typical of grave goods (Fig. S2).
For example, within the funerary contexts, there is a large amount of archaeological material associated to the human remains. Pottery fragments are the most abundant archaeological remains (50%) and although very fragmented, most of them are simple forms, of which closed, globular and ovoid shapes prevail. Ornamental decoration is simple and homogeneous, the motifs are reduced to just a few themes such as cylindrical perforations, impressions, embossed tablets, simple incised or channelled rope lines or incisions. In general, most of these decorations are located below the rim (Fig. S2). The pottery types and decorations, in particular the embossed tablets, are characteristic of the Early Chalcolithic (Pre-Bell Beaker) phase and common for the Chalcolithic in the Duero Basin [9].
The bone industry collection is composed of nineteen pieces that can be divided into two functional groups: bone and shaft tools and personal adornment objects. Among the group of "tools", awls, a gradine, a bipoint, a rod and a spatula with ochre residue have been identified. Regarding the lithic materials, 260 pieces (3%) associated to the tumulus have been recovered and with the exception of two polished elements, the rest are from the carving industry (Fig. S2). The number of adornment objects associated with the funerary context is very scarce, as only seven remains have been recovered.
Within the tumulus, 3,694 faunal bone remains have been recovered, of which 51.2% (1,892) have been anatomically and taxonomically identified and the other 48.8% (1,802) are of undetermined remains (bone fragments or shards). From this large faunal collection, ovicaprines are the most abundant (Ovis aries and Capra hircus) (41.65%), followed by Bos taurus (11.15%), Sus domesticus (7.82%), Canis familiaris (1.64%) and Equus sp. (0.9%). Hunting activity is indicated by the presence of Cervus elaphus, Capreolus capreolus, Vulpes vulpes, Leporidae indet and a small carnivore. Furthermore, a small percentage of fish and turtle shells have been identified.
We suggest that the El Portalón pre-Bell Beaker Copper Age funerary barrow is the result of repetitive burial activities following a similar pattern over time. Each event appears to have disturbed a previous funerary context. In addition, the collapse of a large part of the roof of the cavity during this period and its later use as a habitat and animal stable seemed to have contributed to the disturbance of some of the funerary structures. Under such events, human bone remains are a common occurrence, and grave goods elements often appear scattered among the limestone blocks of the tumular structure (Fig. S2).
Chalcolithic funerary practices in El Portalón were prolonged over six generations (ca. 150 yr), creating a collective but not simultaneous burial site. The continuous use of this space has altered the primary burials, displacing and scattering the bones; usually only isolated bones or small bone sets remain at different levels of anatomical connection.
In this tumular funerary structure, we have discerned human remains from a minimum of eight individuals (three adults and five subadults). In just one case, we have found a practically intact burial and the skeleton of the individual with grave goods (Fig. S2). The rest are isolated and partially articulated bones found in secondary position. The well-preserved burial, provides significant information regarding the potential characteristics of the funeral rites conducted during the Chalcolithic period at this cave (Fig. S2). This burial belongs to a complete subadult individual found in primary position accompanied by grave goods.
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Post by Admin on Mar 30, 2021 2:47:07 GMT
According to [10], this individual corresponds to a male child of an estimated height of 101 cm and due to the state of tooth eruption, he must have died at around 6-7 years of age. Macroscopic analysis and computerized axial tomography reveal a set of lesions both on the cranium and on the long bones, indicating that this individual likely suffered from rickets and/or scurvy at different stages of his life. The etiology of the two metabolic diseases could be attributed to an abnormal diet [10].
The boy´s burial was accompanied by different grave goods; for example, his feet, legs, pelvic area and head were covered by pieces of large fragmented ceramic (Fig. S2). On top of the body and the ceramic, a covering of green clay was deposited. Comprising part of the ensemble and also covered by the same clay, a nearly whole calf skeleton (Bos taurus) was found in anatomical connection. There are substantial amounts of pottery fragments (more than 200), including all vessel parts (rims, bodies, bases) among the grave goods associated with this burial. These fragments have allowed for the reconstruction of several vessels, among which a truncated cone-shaped morphology is notable (Fig. S2).
In addition to the pottery, several tools were found as part of the boy´s grave goods, among these, the most notable ones were a pedunculated arrowhead, a medial fragment of bitruncated white flint flake with marks from wear on both blades, a simple honey-colored flint flake of color with retouching on one of its blades, a quartzite un-retouched spall and a tubular awl on the left distal tibia of the ovicaprine with beveling on the far end, showing cut marks and abrasion prints.
The presence of other burials in the excavated area of the tumular structure is reflected by the existence of sets of human bones (in some cases in anatomical connection). The minimum number of individuals represented by the bones associated to the funerary tumulus (211) is eight: three adults and five subadults including the aforementioned complete child (6-7 years of age). This makes a total of 8 individuals that have been found within the funerary tumulus. Note that while we report aDNA recovery from 8 individuals only 5 come from the tumulus, the others come from the Bronze Age level (n=1) and the clandestine excavation (n=2), detailed below.
Furthermore, 43 additional human bone fragments have been recovered in Middle Bronze Age levels at the space known as the Salón del Coro or Galería Principal, which is also part of the El Portalón site itself.
Finally, in the 2001 field season we identified an area of clandestine excavation in the central part of Portalón carried out by unknown individuals [3]. From 2001 to 2006 we excavated this disturbed area in which we founded significant archaeological and paleontological materials out of their original context, including 91 human bones.
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Post by Admin on Mar 30, 2021 20:22:36 GMT
S4. Mitochondrial analysis The mitochondrial genome coverage varied between 3.6x and 341x among the 8 individuals. The number of SNPs that supports each called haplotype varied between 41 and 50 and they are reported as deviations from the Reconstructed Sapiens Reference Sequence (RSRS) [34] (Table S4). Six of the consensus sequences lacked sequence data at between one and seven of the hg defining position for the called haplotype. A few additional mutations that can be found elsewhere in the mtDNA phylogeny were observed in the sequences.
These were mainly transitions at positions with low coverage, and even though a few of these may be true mutations, most can probably be attributed to post-mortem deaminations. Note that polymorphisms reported at np 195, 4769, 8860, 14766 and 16519 are derived in relation to RSRS but considered ancestral in relation to rCRS [35] All eight individuals from Atapuerca displayed unique haplotypes (Figure 1B, Table S2, Table S4). The most abundant haplogroup, U5, was found in three temporally non-overlapping individuals. Two belonged to subtypes of U5b (U5b3 and U5b1b) and one belonged to U5a (U5a1c). Note that the U5b1b individual (ATP9) have the T6189C back-mutation but display the ancestral state for G12618A. Two individuals belonged to H3. They were dated to within the same time-frame but were not maternally related as one of them carried a T to C transition at np 12957 classifying it to H3c. The remaining three individuals belonged to the haplogroups J, K and X (J1c1b1, K1a2b and X2c).
The mitochondrial lineages of the ATP individuals show a heterogeneous ancestry and can be traced back both to hunter-gatherer (HG) and subsequent farmer contexts (Dataset S2 (external file), Figure S4). The most frequent haplogroup in ATP, U5, is commonly found in HG groups in Iberia and across Europe and Scandinavia [19,20,36–43]. U5 subhaplogroups are also found in Neolithic farmer populations in Europe although at lower frequencies [36,42,44–48]. The remaining four haplogroups found in ATP, H, J, K and X, are present in other farmer populations from the Neolithic and onwards [20,38,48–50]. In southern Europe (e.g. Spain, Portugal and Italy), however, haplogroup H is also frequent in Paleolithic and Mesolithic HG populations [26,36,42].
Even though some haplogroups (U5b and H) are shared between ATP and HGs from Mesolithic Iberia (southern hunter-gatherers SHG), the general haplogroup composition between the groups differ [19,36,43] (Dataset S2, Figure S4), similar to the differences between other farmer and HG populations in Europe[19,20,37–40,48]. None of the previously investigated Neolithic farmer populations from Iberia have similar haplogroup distribution as ATP (Dataset S2, Figure S4). These farmer groups also differ from each other [42,51]. Analysis of haplogroup frequency data have for example shown that early Neolithic north-eastern Iberian populations cluster with early- and middle Neolithic populations from central Europe while other Neolithic Iberian populations (from Basque Country and Navarre, NBQ [42] and Portugal, NPO [36]) share a closer affinity to HG populations [48]. NBQ is the population that share the largest number of haplogroups with ATP (X, H, J, U5b and K [42] although the frequencies differ and NBQ also display additional haplogroups (U, T, HV and I). The Chalcolithic individuals from El Mirador (MIR), a cave located in the same mountain system as ATP (Sierra de Atapuerca), present a somewhat different haplogroup distribution than ATP. MIR clusters with early Neolithic Iberians and early and middle Neolithic central European populations [52]. They lack the U5 subhaplogroups found in ATP and instead display T2 and U3 [52]. RFLP data from another Chalcolithic population from the Basque Country show the same main haplogroups as found in ATP and MIR (38% H, 17% U, 13% J, 21% K and 9% T+X, note that these frequencies are not added to Dataset S2 or to Figure S4 as they are not generated from sequence data), although the lower resolution of the data cannot specify which population (ATP or MIR) that it is most similar to [53]. It has further been suggested that the mt-haplogroup composition of Basque populations differs between Chalcolithic [53] and historical times (600-700 AD) [54] with increasing frequencies of H anv V haplotypes and with increasing similarities to present-day western European populations.
The picture of the ancient farmers in Iberia remains unresolved and the limited level of information retrieved from mitochondrial DNA has not been able to go beyond the above described observations.
Present-day European populations are genetically quite homogenous in terms of mitochondrial haplogroup distributions and it is mainly haplogroup frequency differences that separate different populations. It is therefore not a straightforward process to assess the potential connections between ATP and specific present-day populations. We note that the most abundant lineages in the ATP individuals are found in higher frequencies in some Basque-speaking populations (U5b; [55], and H3; [55,56]) than in other European populations. Further, several haplotypes have been suggested to be autochthonous to present-day Basque populations (see e.g. [57–60]). Two of these are J1c1 [61], a lineage ancestral to the J2c1b1 haplotype in ATP7, and H3c2a [57], a lineage that derives from the H3 and H3c haplotypes found in ATP17 and ATP12-1420 .
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