Genetic studies have identified substantial non-African admixture in the Horn of Africa (HOA). In the most recent genomic studies, this non-African ancestry has been attributed to admixture with Middle Eastern populations during the last few thousand years. However, mitochondrial and Y chromosome data are suggestive of earlier episodes of admixture. To investigate this further, we generated new genome-wide SNP data for a Yemeni population sample and merged these new data with published genome-wide genetic data from the HOA and a broad selection of surrounding populations. We used multidimensional scaling and ADMIXTURE methods in an exploratory data analysis to develop hypotheses on admixture and population structure in HOA populations. These analyses suggested that there might be distinct, differentiated African and non-African ancestries in the HOA. After partitioning the SNP data into African and non-African origin chromosome segments, we found support for a distinct African (Ethiopic) ancestry and a distinct non-African (Ethio-Somali) ancestry in HOA populations. The African Ethiopic ancestry is tightly restricted to HOA populations and likely represents an autochthonous HOA population. The non-African ancestry in the HOA, which is primarily attributed to a novel Ethio-Somali inferred ancestry component, is significantly differentiated from all neighboring non-African ancestries in North Africa, the Levant, and Arabia. The Ethio-Somali ancestry is found in all admixed HOA ethnic groups, shows little inter-individual variance within these ethnic groups, is estimated to have diverged from all other non-African ancestries by at least 23 ka, and does not carry the unique Arabian lactase persistence allele that arose about 4 ka. Taking into account published mitochondrial, Y chromosome, paleoclimate, and archaeological data, we find that the time of the Ethio-Somali back-to-Africa migration is most likely pre-agricultural.

The Horn of Africa (HOA) occupies a central place in our understanding of modern human origins. This region is the location of the earliest known modern human fossils, a possible source for the out-of-Africa migration, and one of the most genetically and linguistically diverse regions of the world. Numerous genetic studies over the last decades have identified substantial non-African ancestry in populations in this region. Because there is archaeological, historical, and linguistic evidence for contact with non-African populations beginning about 3,000 years ago, it has often been assumed that the non-African ancestry in HOA populations dates to this time. In this work, we find that the genetic composition of non-African ancestry in the HOA is distinct from the genetic composition of current populations in North Africa and the Middle East. With these data, we demonstrate that most non-African ancestry in the HOA cannot be the result of admixture within the last few thousand years, and that the majority of admixture probably occurred prior to the advent of agriculture. These results contribute to a growing body of work showing that prehistoric hunter-gatherer populations were much more dynamic than usually assumed.

Funding: This work was supported by NSF BCS-0518530 to CJM and by research funds provided by the School of Natural and Social Sciences of Lehman College to RLR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In order to investigate the discrepancy among the archaeological, historical, mitochondrial, Y chromosome, and genome-wide data for recent vs. more ancient evidence of admixture in the HOA, we generated new genome-wide SNP data for a Yemeni sample and analyzed these new data with publicly available data [16] , [43] , [48] – [51] . Our objectives were to verify the presence of admixture in the HOA, determine the affinities of any HOA non-African ancestry, and evaluate the number of distinct admixture episodes and their timing.

Y chromosome data are also suggestive of at least two episodes of non-African migration into the HOA prior to 3 ka. First, HOA populations carry E-M78 Y chromosomes at high frequencies [40] , [41] . E-M78 originated in northeastern Africa around 19 ka with a descendant lineage (E-V32) unique to the HOA that arrived by at least 6 ka [41] . Because northern African populations in this timeframe are inferred to have substantial non-African ancestry [42] , [43] , the expansion south of E-M78 could have introduced non-African ancestry into the HOA prior to 6 ka. Second, some HOA populations carry moderate to high frequencies of T-M70 (previously K2-M70) Y chromosomes [44] – [46] . The T haplogroup originated in the area of the Levant approximately 21 ka and the T-M70 sub-haplogroup was present in northeast Africa by at least 14 ka, possibly arriving in the HOA as early as 5 ka [44] , [45] , [47] .

Archaeological data indicate trade between the HOA and Arabia by at least 8 ka [30] , [31] and genetic analyses of mitochondrial and Y chromosome data suggest much earlier migrations into the HOA. Mitochondrial data are suggestive of as many as three waves of prehistoric non-African migration into the HOA. First, HOA populations carry several unique M1 lineages of the otherwise South and East Asian mitochondrial haplogroup M [13] , [32] – [34] . Many of these HOA M1 lineages have deep roots, diverging from M1 representatives elsewhere 20–30 ka [34] – [36] . Second, representatives of N1a and N2a in the HOA diverged from their most closely related haplotypes in the Middle East and the Caucasus 15–20 ka [37] . Third, in the Eurasian mitochondrial HV1 and R0a lineages there are several sub-haplogroups (HV1a3, HV1b1, R0a2b, R0a2g) that are found in both the HOA and the Arabian Peninsula. Within these shared sub-haplogroup lineages, the HOA and Arabian haplotypes are distinct, suggesting that the migration that brought these lineages into the HOA happened soon after the sub-haplogroups began to diversify at 6–10 ka [38] , [39] .

However, more recent archaeological research shows that non-African influences in the HOA were limited and transient. Of the early first millennium BCE inscriptions in non-African scripts complete enough to identify a language, only a small proportion are written in a non-African (South Arabian) language - the majority are written in indigenous proto-Ge'ez [24] . In the HOA, architecture with non-African (primarily South Arabian) elements is entirely monumental or ritual [25] and ritual items with exclusively non-African elements are rare [26] . There are few to no indications of non-African material culture in everyday objects: the ceramics and lithics found outside of the ritual context are almost entirely indigenous with clear local precedents [24] , [25] , [27] . While earlier scholarship conceived of a South Arabian origin D'MT polity with sovereignty over much of the northern HOA, it is now clear that this polity, if it ever existed at all as an integrated state [24] , was geographically restricted to the regions around Yeha and Aksum in what is now the Tigray region of Ethiopia [25] . Artifacts with non-African features are effectively absent in the material culture (ritual or otherwise) of contemporaneous populations in the Eritrean highlands on the Asmara plateau (the “Ancient Ona”) [25] , [28] , [29] . Prior to the first millennium BC, the archaeology of the HOA is less well studied, but what is available shows no substantial non-African material culture beyond trade relations [25] . Taken all together, the archaeological data could be consistent with limited non-African (primarily South Arabian) migration into the north Ethiopian highlands at the outset of the first millennium BCE, but cannot support large-scale population movements from any foreign population.

Populations in the Horn of Africa (HOA: Ethiopia, Eritrea, Djibouti, and Somalia) have substantial non-African ancestry [11] – [15] . The most recent genomic studies estimate 30–50% non-African ancestry in the Cushitic and Semitic speaking populations of the HOA resulting primarily from admixture around 3 ka [16] , [17] . This timeframe corresponds to the estimated time of origin of the Ethiosemitic languages [18] and there are some carved inscriptions in South Arabian scripts associated with temple ruins and ritual items in South Arabian styles dated to the early first millennium BCE in the north Ethiopian highlands [19] – [23] . These linguistic and archaeological connections have been cited in the recent population genomic studies to support a hypothesis of high levels of non-African migration into the HOA around 3 ka.

The timing and extent of migration and admixture are questions that are central to the entire scope of human evolutionary history from the origin of our species to the present day. The most important event underlying human population structure is the origin of anatomically modern humans in Africa and their subsequent migration around the globe [1] – [3] . Following the initial out-of-Africa migration, the rate of migration between sub-Saharan Africa and the rest of the Old World was low throughout prehistory, but not absent; there is statistically significant evidence for a deep history of intercontinental migration [4] – [7] . Beginning around 11 ka (thousand years ago), the switch to reliance on domesticated plants and animals is associated with major population and language expansions from multiple centers of domestication around the world [8] – [10] . Finally, migration and admixture accelerated during the last few thousand years with increasing international trade, including the trade in slaves and the transplantation and shuffling of populations in the colonial era, culminating in the modern era of high international migration.

Results and Discussion

For these analyses, we generated new genome-wide SNP data using the Illumina 370K array from 61 Yemenis, chosen to represent all geographic regions of the country. These new data were merged with published data from the HOA [16], the Middle East [48], North Africa [43], Qatar [50], southern Africa [51], west Africa [49], the HapMap3 project [52], and the Human Genome Diversity Project [53]. After reduction to SNPs shared across all source datasets and quality control, the main merged dataset included 2,194 individuals from 81 populations for 16,766 SNPs (Table S1).

Horn of Africa populations in the regional genetic landscape We first investigated the position and dispersion of HOA populations in the genetic landscape in a multi-dimensional scaling (MDS) analysis of pairwise identity by state (IBS). Consistent with prior analyses of global genome-wide genetic variation [3], [53], [54], the first dimension of the IBS MDS analysis separates sub-Saharan Africans from non-Africans (Figure 1A). The HOA samples are broadly dispersed between the main sub-Saharan Africa cluster and the non-African populations and several sub-clusters of HOA samples are apparent. To see the specific distribution of all of the included HOA samples, we plotted the HOA samples in isolation (Figure 1B). While we include many more African and non-African population samples than prior analysis of these HOA data, our results in the MDS analysis for the HOA samples are not qualitatively different than those of Pagani et al. [16], who showed that the different HOA clusters correspond to linguistic groups: the Gumuz are Nilotic-speaking, the Ari and Wolayta are Omotic-speaking, and the rest speak Cushitic or Semitic languages. The dispersion of HOA samples between the sub-Saharan and non-African clusters is suggestive of admixture between African and non-African ancestors [55], [56]. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Multidimensional scaling analysis shows the great genetic diversity within the Horn of Africa. We plotted the first two dimensions of a multidimensional scaling analysis of pairwise identity by state across all study populations. (A) The HOA populations are broadly scattered between out-of-African populations and the bulk of sub-Saharan African populations along the first dimension. Some clusters of HOA individuals are much closer to the main sub-Saharan African cluster, while others are much closer to North African and Arabian clusters. (B) In this plot, we zoom in on the HOA samples and leave out all other populations. While the region as a whole covers a broad swath of the first MDS dimension, most individual populations are tightly clumped, with groups separated by language. The Nilo-Saharan speaking Gumuz are on the far left, the Omotic speaking Ari are in the center, and the Cushitic and Semitic speaking populations are on the right. https://doi.org/10.1371/journal.pgen.1004393.g001 In order to better understand the genetic structure of HOA populations, we analyzed the SNP data using the model-based, maximum likelihood ancestry estimation procedure implemented in the ADMIXTURE software [57] for K values from 2 to 20 (Figure S1). For this analysis, we excluded SNPs in strong linkage disequilibrium, which reduced the main dataset to 16,420 SNPs. We used the cross-validation method encoded in the ADMIXTURE software in an attempt to estimate the optimal number of inferred ancestral components (K) [58]. This cross validation procedure splits the genotype data into partitions and masks (marks as missing) each partition in turn, predicting the genotypes of the masked sites from the remaining unmasked data. For our data, the cross-validation error is minimized at K = 12, but there is little difference in error from K = 9 to K = 14 (Figure S2). For HOA populations, the ADMIXTURE estimates for K = 9–14 fall into three distinct patterns at K = 9–10, K = 11, and K = 12–14 (Figure S1). Here we focus on the ancestral component estimates for K values of 10, 11, and 12 as representative of these three patterns (Figure 2). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Population structure of Horn of Africa populations in a broad context. ADMIXTURE analysis reveals both well-established and novel ancestry components in HOA populations. We used a cross-validation procedure to estimate the best value for the parameter for the number of assigned ancestral populations (K) and found that values from 9 to 14 had the lowest and similar cross-validation errors (Figure S2). (A) The differences in inferred ancestry from K = 9–14 are most pronounced in the HOA for K = 10–12, where two ancestry components that are largely restricted to the HOA appear (the dark purple and dark green components). (B) Surface interpolation of the geographic distribution of eight inferred ancestry components that are relatively unchanging and common to the ADMIXTURE results from K = 10–12. (C) Individual ancestry estimation for HOA populations (with language groups indicated) and surface plots of the changing distributions of the Nilo-Saharan (light blue) and Arabian (brown) ancestry components for K = 10–12. At K = 11, a new HOA-specific ancestry component that we call Ethiopic appears (dark purple) and at K = 12 a second new ancestry component that we call Ethio-Somali (dark green) appears with its highest frequencies in the HOA. https://doi.org/10.1371/journal.pgen.1004393.g002 There are ten inferred ancestry components (IACs) that are consistent across all three focal K values (Figure 2) and are congruent with published analyses of African and Eurasian population structure [3], [49], [59], [60]. Four IACs are found predominantly in sub-Saharan African populations: (1) one with high frequencies in the Mbuti and Biaka pygmies that is colored pink in the figure; (2) one with high frequencies in Khoesan speaking populations of southern Africa that is colored light purple; (3) one with high frequencies in Niger-Congo speaking populations throughout sub-Saharan Africa that is colored dark blue; and (4) one with high frequencies in Nilo-Saharan speaking populations that is colored light blue. Five IACs are found predominantly in Eurasia: (1) one with high frequencies in Central and South Asian populations that is colored red; (2) one with high frequencies in European populations that is colored light orange; (3) one with its highest frequencies in southern Europe, the Middle East, and Central Asia that is colored dark orange; (4) one with its highest frequencies in Arabian populations that is colored brown; and (5) one with its highest frequencies in Central Asian populations of known East Asian ancestry that is colored grey. The tenth shared IAC is colored light green and predominates in North African populations. This “Maghrebi” IAC has been recovered in previous studies of North African populations and is hypothesized to represent a late Pleistocene migration of non-African ancestors back into Africa [43], [61]. From K = 10 to K = 12, the changes in ADMIXTURE results occur primarily in the HOA, where two new IACs appear at high frequencies (Figure 2). At K = 10 the African ancestry of HOA populations is dominated by the Nilo-Saharan IAC and the non-African ancestry is mostly split between Arabian and Maghrebi IACs. At K = 11 a new African IAC, colored dark purple in the figure, which we refer to as “Ethiopic”, replaces much of the previously Nilo-Saharan attributed ancestry. The Ethiopic IAC reaches its highest frequencies in the Omotic speaking Ari and Wolayta populations, and is present at moderate frequencies in Semitic and Cushitic speaking populations. Pagani et al. [16] previously reported the presence of an equivalent Ethiopia-specific IAC (colored yellow in their Figure 1C). At K = 12 a second new IAC replaces almost all of the Maghrebi and much of the Arabian attributed non-African ancestry. This IAC is colored dark green on the figure and is referenced here as “Ethio-Somali”. This Ethio-Somali IAC is found at its highest frequencies in Cushitic speaking Somali populations and at high frequencies in neighboring Cushitic and Semitic speaking Afar, Amhara, Oromo, and Tygray populations. This IAC was not identified in the source study for the HOA SNP data [16], but Tishkoff and colleagues [59], in an analysis of an independent autosomal microsatellite dataset, did recover an equivalent IAC (calling it “Cushitic”). While this Ethio-Somali IAC is found primarily in Africa, it has clear non-African affinities (Text S1). Confident determination of the appropriate K value in an ADMIXTURE-like analysis in most human population genomic studies is problematic because the information required to set K a priori is unknown. In fact, there is no true K value in most cases because the simultaneous diversification model fit by ADMIXTURE is a poor reflection of human population history. Therefore, rather than take the ADMIXTURE IACs for one of K = 10,11,12 at face value, we used these estimates as hypotheses about the genetic structure of HOA populations and then evaluated these hypotheses in separate analysis. First, for all focal K values, the ADMIXTURE analysis suggests that many HOA populations have admixture between African and non-African ancestors in their history. To test this, we conducted three formal tests for admixture: the f 3 -statistic test, the D-statistic test, and a weighted LD test [62], [63]. We found that eight HOA populations (Afar, Amhara, Ari Cultivator, Oromo, Ethiopian Somali, Somali, Tygray, and Wolayta) had statistically significant signals of admixture with non-African populations for all three tests (Tables S2, S3, S4). With this strong support for a history of admixture between African and non-African ancestral populations, the differences among ADMIXTURE IACs across K = 10–12 suggest the following hypotheses for the African ancestry in the HOA: (K = 10) The HOA African ancestry is very similar to that found in neighboring Nilo-Saharan speaking populations. (K = 11,12) There is a distinct, differentiated African ancestry in HOA populations (the Ethiopic IAC). And the following hypotheses for the non-African ancestry in the HOA: (K = 10,11) HOA populations experienced admixture with one or more non-African populations carrying high levels of the Arabian and Maghrebi IACs along with small amounts of the Eurasian IAC. (K = 12) There is a distinct non-African ancestry in the HOA that constitutes most of the non-African ancestry (the Ethio-Somali IAC). And the following hypotheses for the non-African ancestry in the HOA: To evaluate these ADMIXTURE-derived hypotheses, we used the CHROMOPAINTER software [64] to partition the chromosomes of HOA and neighboring populations into segments of African and non-African origin. We then sampled from the painted segments to create composite African and non-African ancestry chromosomes. To ensure that the African and non-African ancestry analyses would be directly comparable, we retained only those SNPs where samples could be generated from both the African and non-African painted segments across all populations, resulting in a dataset that includes 4,340 SNPs (the “4K partitioned” dataset). This dataset includes African and non-African partitioned samples from admixed HOA populations (Afar, Amhara, Ari [Blacksmith and Cultivator combined], Oromo, Somali [Ethiopian Somali and Somali combined], and Tygray) and from admixed Middle Eastern and North African (MENA) samples (Egypt, Mozabite, Palestinian, Yemen) as well as from relatively non-admixed African (Anuak, Gumuz, South Sudanese) and non-African (Bedouin, Druze, Saudi Arabia) populations. Further information on the population selection and ancestry painting methods is detailed in Materials and Methods. We used this 4K partitioned dataset to evaluate hypotheses of gene flow and population structure arising from the ADMIXTURE results.

African ancestry in the HOA The hypothesis that African ancestry in the HOA is not distinct from that found in neighboring Nilo-Saharan speaking populations (hypothesis 1A above) requires a history of homogenizing inter-population migration or relatively recent common origin. In the case of homogenizing gene flow, a correlation between genetic and geographic distance might be expected, with nearby populations more alike than distant populations. We calculated within and between population gene identity (the probability that two randomly drawn alleles are identical by state) for all the populations included in the 4K partitioned dataset. We then used the between population gene identity estimates among the predominantly Nilo-Saharan ancestry Anuak, Gumuz, South Sudanese, and the African ancestry partition of the Amhara, Ari, Oromo, Somali, and Tygray to test for a relationship between genetic and geographic distance. No significant relationship was recovered (Mantel test, r = −0.28, p = 0.185) (Figure 3A). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Tests of gene flow and population structure in the African ancestry of HOA populations. Using the African origin partition of the HOA data identified in a chromosome painting analysis, we evaluated the evidence for gene flow with neighboring populations and for population structure within and between the HOA and neighboring populations. (A) Shared gene identity plotted against distance for the HOA populations and the neighboring Anuak, Gumuz, and South Sudanese. (B) Linguistically structured population tree model within the African ancestry partition of HOA populations with the F ST estimate from this tree model, the goodness-of-fit statistic Λ, and the likelihood ratio test statistic K for the improvement in model fit from the unstructured tree. (C) The linguistically structured population tree model for neighboring Nilo-Saharan language family populations. (D) Structured population tree models for the combined HOA and neighboring populations. https://doi.org/10.1371/journal.pgen.1004393.g003 Since the pattern of genetic variation in the African ancestry of Sudanese and HOA populations is not a good fit to one model of ongoing gene flow, we tested the hypothesis that there is population substructure within and between HOA and Nilo-Saharan populations using AMOVA [65] and hierarchical population tree models [66], [67]. First, within the HOA we used AMOVA to test for differentiation between linguistic groups – the Omotic speaking Ari, the Semitic speaking Amhara and Tygray, and the Cushitic speaking Oromo and Somali – and found a significant difference (Φ GT = 0.013, p<0.001). We also fit the HOA data to two population tree models, one without substructure and one with linguistically defined subgroups (Figure 3B), and found that the tree with the linguistic groups is a significantly better fit to the data (K = 30, df = 1, p = 4.3×10−8). Next, we tested for the presence of linguistically delineated subgroups within the Anuak, Gumuz, and South Sudanese. Most southern Sudanese populations speak languages in the Nilotic branch of the Nilo-Saharan language family and the Anuak language is also a Nilotic language [68]. The Gumuz language is either a highly divergent Nilo-Saharan language or a language isolate [69]. AMOVA reveals a statistically significant difference between these linguistic groups (Φ GT = 0.024, p<0.001) and the population tree with linguistically defined subgroups (Figure 3C) is a significantly better fit to the data than the tree without subgroups (K = 132, df = 1, p≈0). Finally, putting all of the populations together in an AMOVA analysis, we find significant differences between linguistic subgroups at both a macro level (Nilo-Saharan vs Afro-Asiatic) (Φ GT = 0.014, p<0.0001) and a micro level (Nilotic, Gumuz, Omotic, Semitic, Cushitic) (Φ GT = 0.022, p<0.0001). Population tree models with these groupings are a significantly better fit to the data than a tree without subgroups (K = 415 and 264, df = 1, p≈0) (Figure 3D). The tree with the larger subgroups (Nilo-Saharan vs Afro-Asiatic) is a slightly better fit to the data (Λ = 662) than the tree with the smaller subgroups (Λ = 812; smaller Λ values indicate better fit). These results support the hypothesis from ADMIXTURE K≥11 of a distinct African ancestry with a long history in differentiated HOA populations (hypothesis 1B above) over the hypothesis from ADMIXTURE K≤10 that African ancestry in the HOA is not substantially differentiated from that found in neighboring populations (hypothesis 1A). In fact, our results suggest a rather more complicated history for these regional populations. Studies of further population samples from ethnic groups in and near the western and southern edges of the Ethiopian escarpment are sure to be interesting.

Non-African ancestry in the HOA The ADMIXTURE-derived hypothesis that non-African ancestry in the HOA derives from admixture with a population or populations with high levels of the Arabian and Maghrebi IACs and some of the Eurasian IAC (hypothesis 2A above) suggests that HOA populations should have higher levels of shared gene identity with populations with higher proportions of those ancestries. To evaluate this prediction, we examined the relationship between shared gene identity and the ADMIXTURE-estimated proportion of the Arabian, Eurasian, and Maghrebi IACs in MENA population samples for each of the non-African ancestry partitions of the admixed HOA populations using varying intercepts linear models. Only the Maghrebi IAC analysis shows the expected relationship: shared gene identity between HOA and MENA populations increases as the proportion of Maghrebi ancestry increases (Figure 4A). Contrary to expectations, shared gene identity decreases between HOA populations and MENA populations as the proportion of the Arabian IAC (Figure 4B) and the Eurasian IAC (Figure 4C) increases. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. Relationship between non-African ADMIXTURE ancestry components and shared gene identity between HOA and MENA populations. ADMIXTURE results for K = 10,11 suggest that the non-African ancestry in HOA populations is indistinguishable from the “Maghrebi,” “Arabian,” and “Eurasian” ancestry components found in MENA populations. If this is a correct inference, then the shared gene identity of HOA populations should be higher with MENA populations with higher proportions of these ancestries. (A) There is significant positive relationship between shared gene identity and the proportion of Maghrebi ancestry in MENA populations. While there is variation across HOA populations in the overall shared gene identity (different intercepts for each population), the magnitude of the relationship is consistent (adding varying slopes did not significantly improve the model fit). This relationship holds whether or not the Mozabite (a high Maghrebi ancestry outlier) are included in the model. However, contrary to expectations, both the Arabian (B) and Eurasian (C) ancestry components showed a reduction in shared gene identity as the representation of these ancestry components in MENA populations increased. https://doi.org/10.1371/journal.pgen.1004393.g004 Next, we looked for evidence for extended inter-population gene flow in the correlation of geographic distance and shared gene identity. We found no relationship between geographic and genetic distance within either HOA or MENA populations. We then examined this relationship for HOA populations to North African (Egypt, Mozabite), Levantine (Bedouin, Druze, Palestinian), and Arabian (Saudi Arabia, Yemen) populations (Figure S3). For North Africa and Arabia, we calculated both straight-line distances and distances involving a waypoint through Egypt. The only group for which there is a clear gradient of genetic similarity decreasing with geographic distance is for the straight-line distances with Arabian populations (Mantel test, r = −0.74, p = 0.0033) (Figure 5A). This relationship between genetic and geographic distance between HOA and Arabian populations might support a hypothesis of long-term equilibrium gene flow among these populations in an isolation-by-distance model. However, if this hypothesis were true, we would expect the highest levels of pairwise gene identity to be between HOA and Arabian populations, but this is not the case. The highest levels of shared gene identity are between HOA populations and the Levantine Palestinian and the North African Mozabite population samples (Figure 5B). Thus, it is more likely that the genetic-geographic HOA-Arabia distance gradient reflects secondary admixture of Arabian migrants into HOA populations already carrying substantial non-African ancestry or already admixed HOA populations sending migrants into Arabian populations. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. Tests of gene flow and population structure in the non-African ancestry of HOA populations. Using the non-African origin partition of the HOA data identified in a chromosome painting analysis, we evaluated the evidence for gene flow with MENA populations and for population structure within and between the HOA and MENA populations. (A) The only clear and statistically significant pattern of decreasing gene identity with geographic distance was between HOA populations and the Yemen and Saudi Arabia populations on the Arabian peninsula as evaluated by a Mantel test. This relationship only held for “as the crow flies” geographic distances; the relationship disappears using a waypoint through Egypt (Figure S3). (B) Shared gene identity between the non-African ancestry partition of HOA populations and MENA populations presented in increasing order. (C) Structured population tree model within the non-African ancestry partition of HOA populations with the F ST estimate from this tree model, the goodness-of-fit statistic Λ, and the likelihood ratio test statistic K for the improvement in model fit from the unstructured tree. (D) Structured population tree model within the non-African ancestry partition of MENA populations. (E) Structured population tree models for the non-African ancestry partitions of both HOA and MENA populations. Both are significantly better fits to the data than the unstructured tree and the regional structure (HOA vs MENA) is a slightly better fit to the data as measured by the goodness-of-fit Λ statistic. https://doi.org/10.1371/journal.pgen.1004393.g005 While these results suggest some history of gene flow between HOA populations and MENA populations, there is no simple pattern that emerges. In order to better understand the partitioning of genetic variation among these populations, we tested for population substructure within and between HOA and MENA populations using AMOVA and hierarchical population tree models. First, within the HOA, we tested for linguistically defined substructure between Cushitic, Semitic, and Omotic speaking populations, but found no significant differentiation (Φ GT = 0.010, p = 0.066). We then used the ADMIXTURE results to inform subgroup formation. At K = 12, the Amhara, Tygray, Oromo, and Afar all have similar proportions of non-African ancestries that differ from that seen in the Ari and Somali (Figure 2). This observation suggests a geographical structuring between the Amhara, Tygray, Oromo, and Afar in the Ethiopian highlands, the Somali in eastern Ethiopia and the Somalia lowlands, and the Ari in the southwestern Ethiopian Rift. AMOVA of these three population groups reveals significant between group differentiation (Φ GT = 0.017, p<0.0001). In addition, the population tree with these geographic subgroups (Figure 5C) is a significantly better fit to the data than the tree without subgroups (K = 126, df = 1, p≈0). Within MENA populations, linguistic subgroups cannot be defined, so we tested several historic/geographic groupings. Between population differentiation was maximized in the AMOVA analysis with three subgroups: the northwest African Mozabite; the ethnic and religious isolate Druze; and the populations with histories entwined with the development and expansion of Islam - the Egyptians, Palestinians, Bedouin, Saudi Arabians, and Yemeni. For this set of subgroups, between population differentiation was statistically significant (Φ GT = 0.011, p<0.0001) and the population tree with these subgroups (Figure 5D) is a significantly better fit to the data than the tree without subgroups (K = 67, df = 1, p = 3.3×10−16). Finally, putting all of the populations together in an AMOVA analysis, we find significant differences between HOA and MENA subgroups at both a macro level (HOA vs MENA) (Φ GT = 0.014, p<0.0001) and a micro level (all of the individual subgroups identified above) (Φ GT = 0.016, p<0.0001). Population tree models for both the simple (HOA vs MENA) and more complex (all individual regional subpopulations) groups are a significantly better fit to the data than a tree without subgroups (Figure 5E). As measured by the goodness-of-fit statistic Λ of Long and Kittles [67], the simple HOA vs MENA structure is the better first level structure fit to the data than the more complex structure with six different subgroups. Even though there is strong evidence for admixture between HOA and MENA populations, there is also clearly detectable substructure both within and between the HOA and the Middle East and North Africa. If the majority of the non-African ancestry in the HOA had entered during the last few thousand years (hypothesis 2A above), then population groups should be less differentiated within the HOA then within MENA samples. This expectation is reinforced by the smaller geographic area of sampling within the HOA when compared to the geographic spread of the MENA samples. However, what we observe is that population groups within the HOA are more differentiated (Φ GT = 0.017; F ST = 0.105) than population groups across the MENA region (Φ GT = 0.011; F ST = 0.098). All together, these results offer greater support to the hypothesis from ADMIXTURE K≥12 that there is a distinct non-African ancestry in the HOA that is well-differentiated from the non-African ancestry in neighboring Middle Eastern and North African populations (hypothesis 2B).

Non-genetic evidence for the timing of the Ethio-Somali back-to-Africa migration Agriculture was established in the HOA by at least 7 ka [14], [80], which suggests that local population densities were likely to have been relatively high from that time forwards. An external migration that occurred recently leading to 30–60% total genome-wide representation into pre-existing agricultural populations (Table S5) would require large or sustained population movements, which is not supported by either the historical or archaeological record [4], [14]. The Ethio-Somali ancestry is more likely to have arrived during an earlier hunter-gatherer phase, when a smaller migration could make a significant contribution. As a point of reference, the slave trade into North Africa and the Middle East of over 11 million sub-Saharan Africans over the last 1,400 years [81] has led to a maximum of 30% total African ancestry in these populations. Paleoclimate data offer some information on time ranges when human migration back-to-Africa would be most likely to succeed. During arid periods in North Africa and the Middle East, most plausible routes into Africa experienced desertification, reducing the likelihood of successful migration. In our time frame of interest, there have been two major peaks of aridity in the region, the Last Glacial Maximum (LGM: ∼21.5 ka) and the Younger Dryas (YD: ∼12.5 ka), during which successful human migrations would not have been likely [82]–[86]. Since the end of the YD there have been fluctuations of arid and wet phases, but no arid periods as extreme or long lasting as these earlier two intervals [82]. Thus, if the Ethio-Somali ancestors diverged from all other non-African populations by 23 ka and were present in the HOA before the advent of HOA agriculture at around 7 ka [80], then there are three possible window of migration: post-YD, between the YD and the LGM, and pre-LGM. Because agriculturalist populations were expanding rapidly in the Middle East beginning about 12 ka and early agriculture in the HOA has an independent origin [80], the earlier YD-LGM and pre-LGM windows are favored. There is abundant archaeological material in the HOA dating to between 5 and 30 ka, but most of the published literature is descriptions of surface surveys or test excavations [87], [88]. More extensive investigations have focused on patterns of resource utilization [89]–[92], a key archaeological research goal, but less helpful for identifying cultural or biological affinities of early HOA populations. Contemporary HOA populations have occasionally been included in craniometric or dental studies of the biological affinities of ancient North Africans and Egyptians [93]–[95], but very little comparative analysis is available for the few prehistoric HOA skeletal collections [89], [96]. One possible indication of ancient Ethio-Somali admixture might be found in studies of Late Pleistocene Nubians (∼12 ka) from the Nile River Valley, who have been variously interpreted as sharing affinities with contemporaneous North African Iberomaurusians [97] and with sub-Saharan Africans [98]. Admixture of Ethio-Somali ancestors with African-origin populations in this region might explain these divergent interpretations of this Late Pleistocene Nubian population.

Relationship to the North African back-to-Africa migration Like the Ethio-Somali, the Maghrebi IAC in North African populations derives from a early back-to-Africa migration [34], [43], [61], [99]–[102]. Studies of North African populations reveal a complex layered history of admixture in North Africa, with an inferred pre-Last Glacial Maximum settlement of North Africa by a non-African population followed by gene flow from European, Middle Eastern, and sub-Saharan African populations dating from the end of the LGM to the recent past [43], [103]–[105]. A single prehistoric migration of both the Maghrebi and the Ethio-Somali back into Africa is the most parsimonious hypothesis. That is, a common ancestral population migrated into northeast Africa through the Sinai and then split into two, with one branch continuing west across North Africa and the other heading south into the HOA. For the Ethio-Somali, the lowest F ST value from the ADMIXTURE estimated ancestral allele frequencies is with the Maghrebi (Text S1), which is consistent with a common origin hypothesis. In contrast, the Maghrebi component has lower F ST values with Arabian, European, and Eurasian ancestral populations than with the Ethio-Somali, which suggests that the Maghrebi diverged most recently from those populations, and might indicate separate back-to-Africa migrations for the Ethio-Somali and the Maghrebi. Unfortunately, the F ST estimates alone are not robust enough to distinguish between single or separate back-to-Africa migrations. While the F ST estimates for the ancestral populations are, in theory, free of confounding admixture, they derive from a simplified model of population history that is known to be inaccurate (simultaneous divergence) and are all assumed to be in Hardy-Weinberg equilibrium [57], [106]. As a result, fine-scale differences in pairwise F ST among ancestral populations should be interpreted with care. Mitochondrial M1 and U6 lineages – sub-clades of mitochondrial haplogroups that are otherwise found only in Eurasian populations – are found both in North Africa and the HOA [34]. U6 has its highest frequencies and diversity in Northwest Africa and M1 has its highest frequencies and diversity in the HOA. The differing representation of deeply diverging M1 and U6 mitochondrial lineages in North Africa and the HOA shows that these regions have exchanged few female migrants since approximately 20 ka [36]. While these mitochondrial data further support our hypothesis that most of the non-African ancestry in the HOA has an ancient origin, we still cannot distinguish between single or separate migrations of the Maghrebi and Ethio-Somali back-to-Africa. If we could identify the geographical origins of both M1 and U6 and if these lineages originated in the same area, then a common migration hypothesis would seem more likely. The geographical origin of a mitochondrial clade is usually inferred from the presence of diverse early branching lineages within a region. To date, no region has been identified with a diversity of early branching lineages of either M1 or U6. Given the exclusively Eurasian distribution of the larger M and U haplogroups, it is generally inferred that M1 and U6 originated outside of Africa [34], [35], [100] but since all other early branches of M1 and U6 appear to have gone extinct, it is not possible to specify their location of origin. Most recently, Pennarun and colleagues [36] found that sub-lineages within U6 began diversifying in North Africa about 10,000 years before M1 sub-lineages began diversifying in the HOA (∼30 ka vs. ∼20 ka). This difference in coalescence times might be taken as evidence for separate migrations, but could also be explained by smaller population sizes in the HOA ancestors between 30 and 20 ka following a common migration.