Abstract One of the main findings derived from the analysis of the Neandertal genome was the evidence for admixture between Neandertals and non-African modern humans. An alternative scenario is that the ancestral population of non-Africans was closer to Neandertals than to Africans because of ancient population substructure. Thus, the study of North African populations is crucial for testing both hypotheses. We analyzed a total of 780,000 SNPs in 125 individuals representing seven different North African locations and searched for their ancestral/derived state in comparison to different human populations and Neandertals. We found that North African populations have a significant excess of derived alleles shared with Neandertals, when compared to sub-Saharan Africans. This excess is similar to that found in non-African humans, a fact that can be interpreted as a sign of Neandertal admixture. Furthermore, the Neandertal's genetic signal is higher in populations with a local, pre-Neolithic North African ancestry. Therefore, the detected ancient admixture is not due to recent Near Eastern or European migrations. Sub-Saharan populations are the only ones not affected by the admixture event with Neandertals.

Citation: Sánchez-Quinto F, Botigué LR, Civit S, Arenas C, Ávila-Arcos MC, Bustamante CD, et al. (2012) North African Populations Carry the Signature of Admixture with Neandertals. PLoS ONE 7(10): e47765. https://doi.org/10.1371/journal.pone.0047765 Editor: David Caramelli, University of Florence, Italy Received: July 17, 2012; Accepted: September 20, 2012; Published: October 17, 2012 Copyright: © Sánchez-Quinto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: FS-Q, SC, CA and CL-F are supported by a grant from the Ministerio de Ciencia e Innovación of Spain (BFU2009-06974) and CGL2010-14944/BOS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Probably the most striking finding derived from the Neandertal genome project [1] was the evidence for admixture between Neandertals and a population of modern humans that left Africa between 80 Kya and 50 Kya subsequently expanding into the rest of the world. The study involved the sequencing and comparison of the Neandertal genome to five modern human genomes: two African (Yoruba and San) and three non-Africans (French, Chinese and Melanesian); all the non-African human genomes shared with Neandertals between 1–4% of their genome, in regions of low recombination placed along ten chromosomes [1]. Additional genomic region introgressions from Neandertals, Denisovans and also putative archaic African hominins have been recently described in Eurasian, Oceanic and even African populations, respectively [2]–[7]. However, an alternative scenario in which the ancestral population of today non-Africans was more closely related to Neandertals than the ancestral population of current Africans due to ancient substructure within the African continent, cannot be totally excluded with the present data [8], although it seems unlikely [9]. In light of this, it is unfortunate that North African individuals have not been included in these admixture analyses, since both the putative African substructure and the admixture are likely to differentially affect North African and sub-Saharan African populations. The importance of North Africa in the emergence of modern Homo sapiens has been traditionally neglected. However, recent archaeological and paleontological evidence increasingly points to this area as a potential source of out-of-Africa migrations [10],[11]. Recent dating of the characteristic North African lithic industry, called Aterian, has provided much older dates than previously assumed, now ranging from 145 Kya to 40 Kya [12],[13]. These Aterian people made personal ornaments with shells, a sign of modern human symbolic behavior [14]. Morphometric analyses of the 80 Kya Dar es-Soltan skull (Morocco) and of Aterian hominin teeth show similarities with early modern humans from Qafzeh and Skhul (Israel) and with the later skull of Pestera cu Oase (Romania) [15],[16]. Recent genetic analysis of North African populations [17] have found that, despite the complex admixture genetic background, there is an autochthonous genomic component which is likely derived from “back-to-Africa” gene flow older than 12,000 years ago (ya) (i.e., prior to the Neolithic migrations). This local population substratum seems to represent a genetic discontinuity with the earliest modern human settlers of North Africa (those with the Aterian industry) given the estimated ancestry is younger than 40,000 years ago [17]. The estimated time of Neandertal admixture with modern human populations is between 37,000–86,000 years ago [18]. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Average ancestry proportions in North African populations estimated by ADMIXTURE for k = 4 different ancestries (the best k value determined by cross-validation error). https://doi.org/10.1371/journal.pone.0047765.t001 The aim of this work was to investigate if this autochthonous North African ancestry bares any traces of the introgression with Neandertals, by applying the f4 ancestry ratio statistic test, previously used to detect Denisovan admixture in Asia [3]. We show that North African populations, like all non-African humans [1], also carry the signature of admixture with Neandertals, and that the real geographical limit for Neandertal admixture is between sub-Saharan groups and the rest. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Results of the ADMIXTURE analysis (k = 4) with North African populations. ADMIXTURE was performed on a set of European, North African, Near Eastern and Sub-Saharan populations in order to account for the different admixture proportions in North Africa. Tunisians and Saharawi are the North African populations with highest proportion of autochthonous component, whereas the rest of the populations have greater amounts of admixture with neighboring populations. https://doi.org/10.1371/journal.pone.0047765.g001

Results and Discussion We ran ADMIXTURE for k equal 2 to 7 and obtained CV errors, and determined that the best k (the one with lowest cross-validation error) is k = 4. Results (Figure 1) are coincident with those previously published [17] and show that North Morocco, Libya and Egypt carry high proportions of European and Near Eastern ancestral components, whereas Tunisian Berbers and Saharawi are those populations with highest autochthonous North African component. Particularly, ten Tunisian individuals have more than 99% of their genome assigned to North African ancestry and therefore have been analyzed separately (subsequently referred to as N-TUN) from the overall Tunisian population. In the PCA analysis (Figure 2) Eurasian populations are the closest to Neandertals among modern humans, which is in agreement with previous studies [1]. Sub-Saharan Africans are, as expected, more distant to Neandertal, whereas North African individuals are placed between these two groups. North African individuals with the highest Sub-Saharan African component (as detected by ADMIXTURE) are distant from Neandertal and closer to Sub-Saharan populations. It is interesting to notice that the North African populations closer to Neandertals are populations with a large known European or Near Eastern admixture, but also the Tunisians that have an almost complete autochthonous North African genetic component. The results of the f4 ancestry ratio test (Table 2 and Table S1) show that North African populations vary in the percentage of Neandertal inferred admixture, primarily depending on the amount of European or Near Eastern ancestry they present (Table 1). Populations like North Morocco and Egypt, with the highest European and Near Eastern component (∼40%), have also the highest amount of Neandertal ancestry (∼60–70%) (Figure 3). On the contrary, South Morocco that exhibits the highest Sub-Saharan component (∼60%), shows the lowest Neandertal signal (20%). Interestingly, the analysis of the Tunisian and N-TUN populations shows a higher Neandertal ancestry component than any other North African population and at least the same (or even higher) as other Eurasian populations (100–138%) (Figure 3). Some results of the estimated ancestry in Table 2 are higher than 100%. Because the amount of Neandertal admixture provided by this statistic is in relation to the fraction found in another population, populations with more than 100% values, have more than the observed Neandertal admixture levels found in the “source population” used for comparison (i.e CEU). On the other hand, a negative f4 ancestry ratio value such as that one observed for the Luyha in Table 2 could have several explanations. One possibility is that it reflects an artifact of ascertainment bias on SNP arrays. Ascertainment bias is likely to affect the joint information from Europeans and East Asians, since SNP arrays are most commonly designed based on information from these populations. On the other hand it could also reflect a more complex demographic history (i.e population structure between the populations being compared) than previously assumed. Subsequently, we aimed to compare the results revealed by ADMIXTURE and by the f4 ancestry ratio statistic in an attempt to corroborate that the signal of Neandertal admixture revealed in North African populations is not caused by Eurasian admixture. For this purpose, we performed a Pearson correlation test between the ancestry proportions estimated with ADMIXTURE and the proportions of Neandertal admixture estimated by the f4 ancestry ratio test. Specifically, we tested the correlation between a) both European and Near Eastern components and Neandertal admixture and b) European, Near Eastern and North African admixture components and Neandertal admixture. If signals of gene flow from Neandertals were due exclusively to the European and the Near Eastern components, we would expect that the correlation should significantly decrease in test b), when the North African component is included. On the contrary, the Pearson correlation test reaches significance only when the North African component is included, which is maintained even when Tunisians are removed from the analysis (Table 3). Overall, the correlation analysis and the f4 ancestry ratio statistic show that the North African component actually contributes to the signal of gene flow from Neandertals. Given that the North African autochthonous ancestry seems to be 12,000–40,000 years old [17], our hypothesis is that this ancestral population was descendant from the populations that first interbreed with Neandertals about ∼37,000–86,000 years ago [18] somewhere in the Middle East. Nonetheless further analyses in populations around the contact areas are needed to confirm this hypothesis. A previous study [26] observed that the similarity to Neandertals increases with distance from Africa and suggested this could be explained by SNP ascertainment bias plus a strong genetic drift in East Asian populations. Nonetheless more complex, population-biased, ascertainment schemes might have additional effects (i.e bottlenecks), but these are not expected to significantly increase the rate of false positives in admixture tests [31]. The Tunisian population has been reported to be a genetic isolate [17] so it is plausible that part of the signal detected is actually due to genetic drift. However, this should not affect the other North African groups in our study. Finally, given that SNP arrays are based on common alleles and probably the relevant admixture information is encoded within the rare and very rare alleles, the potential bias, if anything, will underestimate ancient hominid admixture signals, as shown in previous studies [2],[3]. With the current data, however, it is not possible to discard the ancient African substructure hypothesis [8]. Although ours and some previous results [9] tend to favor the admixture hypothesis as the most plausible one, we think that a complete clarification of this issue can only be achieved with a Neandertal high coverage genome, such as this recently achieved for Denisova [32]. This, and sequencing data of North African populations, especially those with a high autochthonous component, may help elucidate more precisely the interbreeding process with Neandertals. In any case, our results show that Neandertal genomic traces do not mark a division between African and non-Africans but rather a division between Sub-Saharan Africans and the rest of modern human groups, including those from North Africa.

Supporting Information Table S1. Stability of the Neandertal admixture estimates. We present each population's estimate ancestry, the standard error in the estimate, and the Z score for different combinations of Sub-Saharan and non-African populations. O (Out-group), BP (Benchmark population, i.e. population which didn't experience any introgression from Neandertals) and SP (Source population i.e. populations in which the amount of introgression from Neandertal is known). https://doi.org/10.1371/journal.pone.0047765.s001 (DOC)

Acknowledgments We are grateful to Brenna M. Henn and Pontus Skoglund for helpful comments on some aspects of the manuscript and the ancestry ratio test, respectively.

Author Contributions Conceived and designed the experiments: DC CDB CL-F. Performed the experiments: FS-Q LR-B SC CA MCA-A. Analyzed the data: FS-Q LR-B SC CA MCA-A. Contributed reagents/materials/analysis tools: DC CDB CL-F. Wrote the paper: DC CL-F.