Ancient DNA at the edge of the world: Continental immigration and the persistence of Neolithic male lineages in Bronze Age Orkney.

Orkney was a major cultural center during the Neolithic, 3800 to 2500 BC. Farming flourished, permanent stone settlements and chambered tombs were constructed, and long-range contacts were sustained. From ∼3200 BC, the number, density, and extravagance of settlements increased, and new ceremonial monuments and ceramic styles, possibly originating in Orkney, spread across Britain and Ireland. By ∼2800 BC, this phenomenon was waning, although Neolithic traditions persisted to at least 2500 BC. Unlike elsewhere in Britain, there is little material evidence to suggest a Beaker presence, suggesting that Orkney may have developed along an insular trajectory during the second millennium BC. We tested this by comparing new genomic evidence from 22 Bronze Age and 3 Iron Age burials in northwest Orkney with Neolithic burials from across the archipelago. We identified signals of inward migration on a scale unsuspected from the archaeological record: As elsewhere in Bronze Age Britain, much of the population displayed significant genome-wide ancestry deriving ultimately from the Pontic-Caspian Steppe. However, uniquely in northern and central Europe, most of the male lineages were inherited from the local Neolithic. This suggests that some male descendants of Neolithic Orkney may have remained distinct well into the Bronze Age, although there are signs that this had dwindled by the Iron Age. Furthermore, although the majority of mitochondrial DNA lineages evidently arrived afresh with the Bronze Age, we also find evidence for continuity in the female line of descent from Mesolithic Britain into the Bronze Age and even to the present day.

Helena's Many Daughters: More Mitogenome Diversity behind the Most Common West Eurasian mtDNA Control Region Haplotype in an Extended Italian Population Sample.

The high number of matching haplotypes of the most common mitochondrial (mt)DNA lineages are considered to be the greatest limitation for forensic applications. This study investigates the potential to solve this constraint by massively parallel sequencing a large number of mitogenomes that share the most common West Eurasian mtDNA control region (CR) haplotype motif (263G 315.1C 16519C). We augmented a pilot study on 29 to a total of 216 Italian mitogenomes that represents the largest set of the most common CR haplotype compiled from a single country. The extended population sample confirmed and extended the huge coding region diversity behind the most common CR motif. Complete mitogenome sequencing allowed for the detection of 163 distinct haplotypes, raising the power of discrimination from 0 (CR) to 99.6% (mitogenome). The mtDNAs were clustered into 61 named clades of haplogroup H and did not reveal phylogeographic trends within Italy. Rapid individualization approaches for investigative purposes are limited to the most frequent H clades of the dataset, viz. H1, H3, and H7.

The Paleo-Indian Entry into South America According to Mitogenomes.

Recent and compelling archaeological evidence attests to human presence ∼14.5 ka at multiple sites in South America and a very early exploitation of extreme high-altitude Andean environments. Considering that, according to genetic evidence, human entry into North America from Beringia most likely occurred ∼16 ka, these archeological findings would imply an extremely rapid spread along the double continent. To shed light on this issue from a genetic perspective, we first completely sequenced 217 novel modern mitogenomes of Native American ancestry from the northwestern area of South America (Ecuador and Peru); we then evaluated them phylogenetically together with other available mitogenomes (430 samples, both modern and ancient) from the same geographic area and, finally, with all closely related mitogenomes from the entire double continent. We detected a large number (N = 48) of novel subhaplogroups, often branching into further subclades, belonging to two classes: those that arose in South America early after its peopling and those that instead originated in North or Central America and reached South America with the first settlers. Coalescence age estimates for these subhaplogroups provide time boundaries indicating that early Paleo-Indians probably moved from North America to the area corresponding to modern Ecuador and Peru over the short time frame of ∼1.5 ka comprised between 16.0 and 14.6 ka.

Mitogenome Diversity in Sardinians: A Genetic Window onto an Island's Past.

Sardinians are "outliers" in the European genetic landscape and, according to paleogenomic nuclear data, the closest to early European Neolithic farmers. To learn more about their genetic ancestry, we analyzed 3,491 modern and 21 ancient mitogenomes from Sardinia. We observed that 78.4% of modern mitogenomes cluster into 89 haplogroups that most likely arose in situ. For each Sardinian-specific haplogroup (SSH), we also identified the upstream node in the phylogeny, from which non-Sardinian mitogenomes radiate. This provided minimum and maximum time estimates for the presence of each SSH on the island. In agreement with demographic evidence, almost all SSHs coalesce in the post-Nuragic, Nuragic and Neolithic-Copper Age periods. For some rare SSHs, however, we could not dismiss the possibility that they might have been on the island prior to the Neolithic, a scenario that would be in agreement with archeological evidence of a Mesolithic occupation of Sardinia.

A genetic chronology for the Indian Subcontinent points to heavily sex-biased dispersals.

BACKGROUND: India is a patchwork of tribal and non-tribal populations that speak many different languages from various language families. Indo-European, spoken across northern and central India, and also in Pakistan and Bangladesh, has been frequently connected to the so-called "Indo-Aryan invasions" from Central Asia ~3.5 ka and the establishment of the caste system, but the extent of immigration at this time remains extremely controversial. South India, on the other hand, is dominated by Dravidian languages. India displays a high level of endogamy due to its strict social boundaries, and high genetic drift as a result of long-term isolation which, together with a very complex history, makes the genetic study of Indian populations challenging. RESULTS: We have combined a detailed, high-resolution mitogenome analysis with summaries of autosomal data and Y-chromosome lineages to establish a settlement chronology for the Indian Subcontinent. Maternal lineages document the earliest settlement ~55-65 ka (thousand years ago), and major population shifts in the later Pleistocene that explain previous dating discrepancies and neutrality violation. Whilst current genome-wide analyses conflate all dispersals from Southwest and Central Asia, we were able to tease out from the mitogenome data distinct dispersal episodes dating from between the Last Glacial Maximum to the Bronze Age. Moreover, we found an extremely marked sex bias by comparing the different genetic systems. CONCLUSIONS: Maternal lineages primarily reflect earlier, pre-Holocene processes, and paternal lineages predominantly episodes within the last 10 ka. In particular, genetic influx from Central Asia in the Bronze Age was strongly male-driven, consistent with the patriarchal, patrilocal and patrilineal social structure attributed to the inferred pastoralist early Indo-European society. This was part of a much wider process of Indo-European expansion, with an ultimate source in the Pontic-Caspian region, which carried closely related Y-chromosome lineages, a smaller fraction of autosomal genome-wide variation and an even smaller fraction of mitogenomes across a vast swathe of Eurasia between 5 and 3.5 ka.

Erratum to: Quantifying the legacy of the Chinese Neolithic on the maternal genetic heritage of Taiwan and Island Southeast Asia.

In the original article, one of the co-authors' (Ken Khong Eng) given name has been published incorrectly. The correct given name should be Ken Khong. The original article has been corrected.

Quantifying the legacy of the Chinese Neolithic on the maternal genetic heritage of Taiwan and Island Southeast Asia.

There has been a long-standing debate concerning the extent to which the spread of Neolithic ceramics and Malay-Polynesian languages in Island Southeast Asia (ISEA) were coupled to an agriculturally driven demic dispersal out of Taiwan 4000 years ago (4 ka). We previously addressed this question using founder analysis of mitochondrial DNA (mtDNA) control-region sequences to identify major lineage clusters most likely to have dispersed from Taiwan into ISEA, proposing that the dispersal had a relatively minor impact on the extant genetic structure of ISEA, and that the role of agriculture in the expansion of the Austronesian languages was therefore likely to have been correspondingly minor. Here we test these conclusions by sequencing whole mtDNAs from across Taiwan and ISEA, using their higher chronological precision to resolve the overall proportion that participated in the "out-of-Taiwan" mid-Holocene dispersal as opposed to earlier, postglacial expansions in the Early Holocene. We show that, in total, about 20% of mtDNA lineages in the modern ISEA pool result from the "out-of-Taiwan" dispersal, with most of the remainder signifying earlier processes, mainly due to sea-level rises after the Last Glacial Maximum. Notably, we show that every one of these founder clusters previously entered Taiwan from China, 6-7 ka, where rice-farming originated, and remained distinct from the indigenous Taiwanese population until after the subsequent dispersal into ISEA.

Reconciling migration models to the Americas with the variation of North American native mitogenomes.

In this study we evaluated migration models to the Americas by using the information contained in native mitochondrial genomes (mitogenomes) from North America. Molecular and phylogeographic analyses of B2a mitogenomes, which are absent in Eskimo-Aleut and northern Na-Dene speakers, revealed that this haplogroup arose in North America ∼11-13 ka from one of the founder Paleo-Indian B2 mitogenomes. In contrast, haplogroup A2a, which is typical of Eskimo-Aleuts and Na-Dene, but also present in the easternmost Siberian groups, originated only 4-7 ka in Alaska, led to the first Paleo-Eskimo settlement of northern Canada and Greenland, and contributed to the formation of the Na-Dene gene pool. However, mitogenomes also show that Amerindians from northern North America, without any distinction between Na-Dene and non-Na-Dene, were heavily affected by an additional and distinctive Beringian genetic input. In conclusion, most mtDNA variation (along the double-continent) stems from the first wave from Beringia, which followed the Pacific coastal route. This was accompanied or followed by a second inland migratory event, marked by haplogroups X2a and C4c, which affected all Amerindian groups of Northern North America. Much later, the ancestral A2a carriers spread from Alaska, undertaking both a westward migration to Asia and an eastward expansion into the circumpolar regions of Canada. Thus, the first American founders left the greatest genetic mark but the original maternal makeup of North American Natives was subsequently reshaped by additional streams of gene flow and local population dynamics, making a three-wave view too simplistic.

Whole mitochondrial genomes unveil the impact of domestication on goat matrilineal variability.

BACKGROUND: The current extensive use of the domestic goat (Capra hircus) is the result of its medium size and high adaptability as multiple breeds. The extent to which its genetic variability was influenced by early domestication practices is largely unknown. A common standard by which to analyze maternally-inherited variability of livestock species is through complete sequencing of the entire mitogenome (mitochondrial DNA, mtDNA). RESULTS: We present the first extensive survey of goat mitogenomic variability based on 84 complete sequences selected from an initial collection of 758 samples that represent 60 different breeds of C. hircus, as well as its wild sister species, bezoar (Capra aegagrus) from Iran. Our phylogenetic analyses dated the most recent common ancestor of C. hircus to ~460,000 years (ka) ago and identified five distinctive domestic haplogroups (A, B1, C1a, D1 and G). More than 90 % of goats examined were in haplogroup A. These domestic lineages are predominantly nested within C. aegagrus branches, diverged concomitantly at the interface between the Epipaleolithic and early Neolithic periods, and underwent a dramatic expansion starting from ~12-10 ka ago. CONCLUSIONS: Domestic goat mitogenomes descended from a small number of founding haplotypes that underwent domestication after surviving the last glacial maximum in the Near Eastern refuges. All modern haplotypes A probably descended from a single (or at most a few closely related) female C. aegagrus. Zooarchaelogical data indicate that domestication first occurred in Southeastern Anatolia. Goats accompanying the first Neolithic migration waves into the Mediterranean were already characterized by two ancestral A and C variants. The ancient separation of the C branch (~130 ka ago) suggests a genetically distinct population that could have been involved in a second event of domestication. The novel diagnostic mutational motifs defined here, which distinguish wild and domestic haplogroups, could be used to understand phylogenetic relationships among modern breeds and ancient remains and to evaluate whether selection differentially affected mitochondrial genome variants during the development of economically important breeds.

Mitochondrial DNA signals of late glacial recolonization of Europe from near eastern refugia.

Human populations, along with those of many other species, are thought to have contracted into a number of refuge areas at the height of the last Ice Age. European populations are believed to be, to a large extent, the descendants of the inhabitants of these refugia, and some extant mtDNA lineages can be traced to refugia in Franco-Cantabria (haplogroups H1, H3, V, and U5b1), the Italian Peninsula (U5b3), and the East European Plain (U4 and U5a). Parts of the Near East, such as the Levant, were also continuously inhabited throughout the Last Glacial Maximum, but unlike western and eastern Europe, no archaeological or genetic evidence for Late Glacial expansions into Europe from the Near East has hitherto been discovered. Here we report, on the basis of an enlarged whole-genome mitochondrial database, that a substantial, perhaps predominant, signal from mitochondrial haplogroups J and T, previously thought to have spread primarily from the Near East into Europe with the Neolithic population, may in fact reflect dispersals during the Late Glacial period, ∼19-12 thousand years (ka) ago.

A novel in-frame 18-bp microdeletion in MT-CYB causes a multisystem disorder with prominent exercise intolerance.

A novel heteroplasmic mitochondrial DNA (mtDNA) microdeletion affecting the cytochrome b gene (MT-CYB) was identified in an Italian female patient with a multisystem disease characterized by sensorineural deafness, cataracts, retinal pigmentary dystrophy, dysphagia, postural and gait instability, and myopathy with prominent exercise intolerance. The deletion is 18-base pair long and encompasses nucleotide positions 15,649-15,666, causing the loss of six amino acids (Ile-Leu-Ala-Met-Ile-Pro) in the protein, but leaving the remaining of the MT-CYB sequence in frame. The defective complex III function was cotransferred with mutant mtDNA in cybrids, thus unequivocally establishing its pathogenic role. Maternal relatives failed to show detectable levels of the deletion in blood and urinary epithelium, suggesting a de novo mutational event. This is the second report of an in-frame intragenic deletion in MT-CYB, which most likely occurred in early stages of embryonic development, associated with a severe multisystem disorder with prominent exercise intolerance.

Association of the mtDNA m.4171C>A/MT-ND1 mutation with both optic neuropathy and bilateral brainstem lesions.

BACKGROUND: An increasing number of mitochondrial DNA (mtDNA) mutations, mainly in complex I genes, have been associated with variably overlapping phenotypes of Leber's hereditary optic neuropathy (LHON), mitochondrial encephalomyopathy with stroke-like episodes (MELAS) and Leigh syndrome (LS). We here describe the first case in which the m.4171C>A/MT-ND1 mutation, previously reported only in association with LHON, leads also to a Leigh-like phenotype. CASE PRESENTATION: A 16-year-old male suffered subacute visual loss and recurrent vomiting and vertigo associated with bilateral brainstem lesions affecting the vestibular nuclei. His mother and one sister also presented subacute visual loss compatible with LHON. Sequencing of the entire mtDNA revealed the homoplasmic m.4171C>A/MT-ND1 mutation, previously associated with pure LHON, on a haplogroup H background. Three additional non-synonymous homoplasmic transitions affecting ND2 (m.4705T>C/MT-ND2 and m.5263C>T/MT-ND2) and ND6 (m.14180T>C/MT-ND6) subunits, well recognized as polymorphisms in other mtDNA haplogroups but never found on the haplogroup H background, were also present. CONCLUSION: This case widens the phenotypic expression of the rare m.4171C>A/MT-ND1 LHON mutation, which may also lead to Leigh-like brainstem lesions, and indicates that the co-occurrence of other ND non-synonymous variants, found outside of their usual mtDNA backgrounds, may have increased the pathogenic potential of the primary LHON mutation.

IκBε deficiency accelerates disease development in chronic lymphocytic leukemia.

The NFKBIE gene, which encodes the NF-κB inhibitor IκBε, is mutated in 3-7% of patients with chronic lymphocytic leukemia (CLL). The most recurrent alteration is a 4-bp frameshift deletion associated with NF-κB activation in leukemic B cells and poor clinical outcome. To study the functional consequences of NFKBIE gene inactivation, both in vitro and in vivo, we engineered CLL B cells and CLL-prone mice to stably down-regulate NFKBIE expression and investigated its role in controlling NF-κB activity and disease expansion. We found that IκBε loss leads to NF-κB pathway activation and promotes both migration and proliferation of CLL cells in a dose-dependent manner. Importantly, NFKBIE inactivation was sufficient to induce a more rapid expansion of the CLL clone in lymphoid organs and contributed to the development of an aggressive disease with a shortened survival in both xenografts and genetically modified mice. IκBε deficiency was associated with an alteration of the MAPK pathway, also confirmed by RNA-sequencing in NFKBIE-mutated patient samples, and resistance to the BTK inhibitor ibrutinib. In summary, our work underscores the multimodal relevance of the NF-κB pathway in CLL and paves the way to translate these findings into novel therapeutic options.

BTK and PLCG2 remain unmutated in one-third of patients with CLL relapsing on ibrutinib.

Patients with chronic lymphocytic leukemia (CLL) progressing on ibrutinib constitute an unmet need. Though Bruton tyrosine kinase (BTK) and PLCG2 mutations are associated with ibrutinib resistance, their frequency and relevance to progression are not fully understood. In this multicenter retrospective observational study, we analyzed 98 patients with CLL on ibrutinib (49 relapsing after an initial response and 49 still responding after ≥1 year of continuous treatment) using a next-generation sequencing (NGS) panel (1% sensitivity) comprising 13 CLL-relevant genes including BTK and PLCG2. BTK hotspot mutations were validated by droplet digital polymerase chain reaction (ddPCR) (0.1% sensitivity). By integrating NGS and ddPCR results, 32 of 49 relapsing cases (65%) carried at least 1 hotspot BTK and/or PLCG2 mutation(s); in 6 of 32, BTK mutations were only detected by ddPCR (variant allele frequency [VAF] 0.1% to 1.2%). BTK/PLCG2 mutations were also identified in 6 of 49 responding patients (12%; 5/6 VAF <10%), of whom 2 progressed later. Among the relapsing patients, the BTK-mutated (BTKmut) group was enriched for EGR2 mutations, whereas BTK-wildtype (BTKwt) cases more frequently displayed BIRC3 and NFKBIE mutations. Using an extended capture-based panel, only BRAF and IKZF3 mutations showed a predominance in relapsing cases, who were enriched for del(8p) (n = 11; 3 BTKwt). Finally, no difference in TP53 mutation burden was observed between BTKmut and BTKwt relapsing cases, and ibrutinib treatment did not favor selection of TP53-aberrant clones. In conclusion, we show that BTK/PLCG2 mutations were absent in a substantial fraction (35%) of a real-world cohort failing ibrutinib, and propose additional mechanisms contributing to resistance.

Mitochondrial haplogroup C4c: a rare lineage entering America through the ice-free corridor?

Recent analyses of mitochondrial genomes from Native Americans have brought the overall number of recognized maternal founding lineages from just four to a current count of 15. However, because of their relative low frequency, almost nothing is known for some of these lineages. This leaves a considerable void in understanding the events that led to the colonization of the Americas following the Last Glacial Maximum (LGM). In this study, we identified and completely sequenced 14 mitochondrial DNAs belonging to one extremely rare Native American lineage known as haplogroup C4c. Its age and geographical distribution raise the possibility that C4c marked the Paleo-Indian group(s) that entered North America from Beringia through the ice-free corridor between the Laurentide and Cordilleran ice sheets. The similarities in ages andgeographical distributions for C4c and the previously analyzed X2a lineage provide support to the scenario of a dual origin for Paleo-Indians. Taking into account that C4c is deeply rooted in the Asian portion of the mtDNA phylogeny and is indubitably of Asian origin, the finding that C4c and X2a are characterized by parallel genetic histories definitively dismisses the controversial hypothesis of an Atlantic glacial entry route into North America.

Two Distinct Clinical Patterns of Ibrutinib-to-Venetoclax Transition in Relapsed Chronic Lymphocytic Leukemia Patients.

Patients with chronic lymphocytic leukemia (CLL) relapsing on ibrutinib are often treated with the Bcl-2 inhibitor venetoclax. However, the transition from one agent to another poses some clinical challenges due to disease flares sometimes occurring right after ibrutinib interruption. Here, we describe three clinical vignettes highlighting two distinct patterns of ibrutinib-to-venetoclax transition. While patients following the favorable pattern transited to venetoclax without experiencing disease flare, the one patient who took the unfavorable path showed rapid disease rebound, with large-cell transformation occurring one week after ibrutinib interruption. A high burden of BTK and PLCG2 mutations was found only in patients with the favorable transition pattern, suggesting that removing BTK inhibition might be particularly harmful if CLL cells are progressing through mechanisms external to the BTK axis.

Biomolecular insights into North African-related ancestry, mobility and diet in eleventh-century Al-Andalus.

Historical records document medieval immigration from North Africa to Iberia to create Islamic al-Andalus. Here, we present a low-coverage genome of an eleventh century CE man buried in an Islamic necropolis in Segorbe, near Valencia, Spain. Uniparental lineages indicate North African ancestry, but at the autosomal level he displays a mosaic of North African and European-like ancestries, distinct from any present-day population. Altogether, the genome-wide evidence, stable isotope results and the age of the burial indicate that his ancestry was ultimately a result of admixture between recently arrived Amazigh people (Berbers) and the population inhabiting the Peninsula prior to the Islamic conquest. We detect differences between our sample and a previously published group of contemporary individuals from Valencia, exemplifying how detailed, small-scale aDNA studies can illuminate fine-grained regional and temporal differences. His genome demonstrates how ancient DNA studies can capture portraits of past genetic variation that have been erased by later demographic shifts-in this case, most likely the seventeenth century CE expulsion of formerly Islamic communities as tolerance dissipated following the Reconquista by the Catholic kingdoms of the north.

Prevalence, incidence and types of mild anemia in the elderly: the "Health and Anemia" population-based study.

BACKGROUND: Hemoglobin concentrations slightly below the lower limit of normal are a common laboratory finding in the elderly, but scant evidence is available on the actual occurrence of mild anemia despite its potential effect on health. The objectives of this study were to estimate the prevalence and incidence of mild grade anemia and to assess the frequency of anemia types in the elderly. DESIGN AND METHODS: This was a prospective, population-based study in all residents 65 years or older in Biella, Italy. RESULTS: Blood test results were available for analysis from 8,744 elderly. Hemoglobin concentration decreased and mild anemia increased steadily with increasing age. Mild anemia (defined as a hemoglobin concentration of 10.0-11.9 g/dL in women and 10.0-12.9 g/dL in men) affected 11.8% of the elderly included in the analysis, while the estimated prevalence in the entire population was 11.1%. Before hemoglobin determination, most mildly anemic individuals perceived themselves as non-anemic. Chronic disease anemia, thalassemia trait, and renal insufficiency were the most frequent types of mild anemia. The underlying cause of mild anemia remained unexplained in 26.4% of the cases, almost one third of which might be accounted for by myelodysplastic syndromes. In a random sample of non-anemic elderly at baseline (n=529), after about 2 years, the annual incidence rate of mild anemia was 22.5 per 1000 person-years and increased with increasing age. CONCLUSIONS: The prevalence and incidence of mild anemia increase with age and mild anemia affects more than one out of ten elderly individuals. Unexplained anemia is common and may be due to myelodysplastic syndromes in some cases.

Association of mild anemia with hospitalization and mortality in the elderly: the Health and Anemia population-based study.

BACKGROUND: Mild anemia is a frequent laboratory finding in the elderly usually disregarded in everyday practice as an innocent bystander. The aim of the present population-based study was to prospectively investigate the association of mild grade anemia with hospitalization and mortality. DESIGN AND METHODS: A prospective population-based study of all 65 to 84 year old residents in Biella, Italy was performed between 2003 and 2007. Data from a total of 7,536 elderly with blood tests were available to estimate mortality; full health information available to evaluate health-related outcomes was available for 4,501 of these elderly subjects. Mild grade anemia was defined as a hemoglobin concentration between 10.0 and 11.9 g/dL in women and between 10.0 and 12.9 g/dL in men. RESULTS: The risk of hospitalization in the 3 years following recruitment was higher among the mildly anemic elderly subjects than among subjects who were not anemic (adjusted hazard ratio: 1.32; 95% confidence interval: 1.09-1.60). Mortality risk in the following 3.5 years was also higher among the mildly anemic elderly (adjusted hazard ratio: 1.86; 95% confidence interval: 1.34-2.53). Similar results were found when slightly elevating the lower limit of normal hemoglobin concentration to 12.2 g/dL in women and to 13.2 g/dL in men. The risk of mortality was significantly increased in mild anemia of chronic disease but not in that due to beta-thalassemia minor. CONCLUSIONS: After controlling for many potential confounders, mild grade anemia was found to be prospectively associated with clinically relevant outcomes such as increased risk of hospitalization and all-cause mortality. Whether raising hemoglobin concentrations can reduce the risks associated with mild anemia should be tested in controlled clinical trials.

The genomic history of the Iberian Peninsula over the past 8000 years.

We assembled genome-wide data from 271 ancient Iberians, of whom 176 are from the largely unsampled period after 2000 BCE, thereby providing a high-resolution time transect of the Iberian Peninsula. We document high genetic substructure between northwestern and southeastern hunter-gatherers before the spread of farming. We reveal sporadic contacts between Iberia and North Africa by ~2500 BCE and, by ~2000 BCE, the replacement of 40% of Iberia's ancestry and nearly 100% of its Y-chromosomes by people with Steppe ancestry. We show that, in the Iron Age, Steppe ancestry had spread not only into Indo-European-speaking regions but also into non-Indo-European-speaking ones, and we reveal that present-day Basques are best described as a typical Iron Age population without the admixture events that later affected the rest of Iberia. Additionally, we document how, beginning at least in the Roman period, the ancestry of the peninsula was transformed by gene flow from North Africa and the eastern Mediterranean.