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Hybridization between closely related species, and the concomitant transfer or introgression of DNA, is widespread in nature (1, 2). In hom-inin evolution, the sequencing of Neandertals (3) and their sister lineage, Denisovans (4, 5), provided evidence for introgression of these lineages into modern humans. Specifically, ~1-3% of each non-African human genome is estimated to have been inherited from Neandertals (3, 5). Although initial inferences of introgression between Neandertals and humans may not have been robust to alternative explanations, most no-tably archaic population structure (3, 6), subsequent analyses have pro-vided evidence for gene flow (7–9).

We hypothesized that a substantial amount of the Neandertal ge-nome may be recovered from the analysis of contemporary humans de-spite the limited amounts of admixture, as introgressed sequences may vary among individuals (Fig. 1A). Indeed, coalescent simulations for a broad range of admixture models suggest that 35-70% of the Neandertal genome persists in the DNA of present-day humans (figs. S1 and S2) (10). By identifying Neandertal sequence from a large sample of modern humans, we hope to discover surviving lineages that may come from multiple archaic ancestors (Fig. 1A), allowing population level data to be recovered.

To identify surviving Neandertal lineages, we developed a two-stage computational strategy (fig. S3) (10). First, we identify candidate intro-gressed sequences using an extension of a previously developed sum-mary statistic referred to as S* (11), which is sensitive to the signatures of introgression (Fig. 1B) and is calculated without using the Neandertal reference genome. We performed coalescent simulations for a wide vari-ety of demographic scenarios, and found that our implementation of S* can distinguish introgressed from non-introgressed sequences (Fig. 1C and fig. S4). Second, we refine the set of candidate introgressed se-quences using an orthogonal approach by comparing them to the Nean-dertal reference genome and testing whether they match significantly more than expected by chance (10). We estimate that using S* alone, as compared to our two-staged approach, would recover ~30% of Neander-tal lineages at a FDR = 20% (fig. S5) (10).

We applied this framework to whole-genome sequences from 379

Europeans and 286 East Asians, from the 1000 Genomes Project (table S1) (12). Specifically, we calculated S* in 50kb sliding windows (tables S2 to S8) (10), and determined statistical signifi-cance through coalescent simulations using a computationally efficient ap-proach (fig. S6) (10). At an S* thresh-old corresponding to p-value ≤ 0.01, we identified ~40 Gb of candidate intro-gressed sequence. Note, S* p-values are robust to demographic uncertainty (fig. S7). The distribution of Neandertal match p-values for this set of candidate introgressed sequences (Fig. 1D) demonstrates a strong skew toward zero, consistent with the hypothesis that they are strongly enriched for Neander-tal lineages. The distribution of Nean-dertal match p-values for sequences that do not possess significant evidence of introgression, as revealed by S*, is approximately uniform (Fig. 1D) (10), indicating that our statistical approach is able to distinguish between intro-gressed and non-introgressed lineages (fig. S8) (10).

At FDR = 5%, we identified over 15 Gb of introgressed sequence across all individuals, which spans ~20% (600 Mb) of the Neandertal genome (Fig. 1E and table S9). Of the 600 Mb of unique sequence, ~25% (149 Mb) was shared between Euro-peans and East Asians. On average, we found 23 Mb of introgressed sequence per individual (Fig. 1F), with East Asian individuals inheriting 21% more Neandertal sequence than Europeans. Within subpopulations, we found small but statistically significant variation in the amount of introgressed sequence among Europeans (Kruskal-Wallis rank sum test; p-value = 4.2 × 10−12), but not among East Asians (p-value = 0.43).

The average length of introgressed haplotypes was ~57kb (Fig. 2A), and ~26% of all protein-coding genes had one or more exons that over-lapped a Neandertal sequence (Fig. 2B). At a broad scale, the genomic distribution of Neandertal lineages exhibits marked heterogeneity, with particular chromosomal arms, such as 8q and 17q, depleted of Neander-tal sequence (Fig. 2A). These qualitative patterns were confirmed by multiple logistic regression, which showed that chromosomal arm was a significant predictor (p-value < 10−16) of the odds that a 50kb window possessed introgressed sequence (10) (Fig. 2C and figs. S9 and S10). Furthermore, odds ratios were negatively correlated with fixed differ-ences between modern humans and Neandertal (Fig. 2D; Spearman’s ρ = –0.80; p-value < 5.8 × 10−8). A strong depletion of Neandertal lineages spanning ~17 Mb on 7q encompasses the FOXP2 locus (Fig. 2A), a tran-scription factor that plays an important role in human speech and lan-guage (13). The observed negative correlation between odds ratio and divergence remained significant when East Asians and Europeans were analyzed separately (fig. S11) and when explicitly controlling for the presence of Neandertal lineages in modern humans (10) (figs. S12 and S13). These results suggest that sequence divergence between modern humans and Neandertals was a barrier to gene flow in some regions of the genome and associated with deleterious fitness consequences (14).

We next leveraged the catalog of introgressed sequences in East Asians and Europeans to refine admixture models and infer parameters of gene flow between modern humans and Neandertals (figs. S14 and S15). Specifically, using an ABC framework (10), we statistically tested a model with a single pulse of introgression into the common ancestor of

Resurrecting Surviving Neandertal Lineages from Modern Human Genomes Benjamin Vernot and Joshua M. Akey*

Department of Genome Sciences, University of Washington, Seattle, WA, USA.

*Corresponding author. E-mail: akeyj@uw.edu

Anatomically modern humans overlapped and mated with Neandertals such that non-African humans inherit ~1-3% of their genomes from Neandertal ancestors. We identified Neandertal lineages that persist in the DNA of modern humans, in whole-genome sequences from 379 European and 286 East Asian individuals, recovering over 15 Gb of introgressed sequence that spans ~20% of the Neandertal genome (FDR = 5%). Analyses of surviving archaic lineages suggests that there were fitness costs to hybridization, admixture occurred both before and subsequent to divergence of non-African modern humans, and Neandertals were a source of adaptive variation for loci involved in skin phenotypes. Our results provide a new avenue for paleogenomics studies, allowing substantial amounts of population-level DNA sequence information to be obtained from extinct groups even in the absence of fossilized remains.

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Europeans and East Asians (3) as well as a second model with gene flow both in the common ancestor and a second, smaller pulse into East Asians shortly after the two populations split (Fig. 3A). Consistent with recent inferences (5, 9), observed patterns of introgression were incom-patible with a one pulse model (Fig. 3B), suggesting that gene flow be-tween Neandertals and humans occurred multiple times. Although we varied many parameters of each model (10) (fig. S14), only the ratio of ancestral effective population size between East Asians and Europeans (Ne

ASN/NeEUR) and the relative amount of introgression between the sec-

ond and first pulse (m2/m1) had appreciable effects on model fit (Fig. 3B). We estimate that Ne

ASN/NeEUR is 1.29 (95% CI of 1.15-1.57), and

that East Asians received an additional 20.2% (95% CI of 13.4%-27.1%) more Neandertal sequence in the second pulse (10). We note that addi-tional unexplored models may provide a better fit to the data, and refin-ing demographic models of hominin evolution is an important area of future work.

The collection of surviving Neandertal lineages that we identified al-lows us to search for signatures of adaptive introgression (15, 16). First, we used introgressed variants that exhibit large allele frequency differ-ences between Europeans and East Asians (FST > 0.40; p-value < 0.001 by simulation) (10), to identify four significantly differentiated regions (Fig. 4 and table S10) (10). Introgressed haplotypes in two of these re-gions span genes that play important roles in the integumentary system: BNC2 on chromosome 9 and POU2F3 on chromosome 11. BNC2 en-codes a zinc finger protein expressed in keratinocytes and other tissues (17), and has been associated with skin pigmentation levels in Europeans (18). The adaptive haplotype has a frequency of ~70% in Europeans and is completely absent in East Asians (Fig. 2B). POU2F3 is a homeobox transcription factor expressed in the epidermis and mediates keratinocyte proliferation and differentiation (19, 20). The adaptive haplotype in East Asians has a frequency of ~66% and is found at less than 1% frequency in Europeans (Fig. 2B). No coding introgressed variants were found in BNC2 or POU2F3, although several highly differentiated introgressed variants were located in functional non-coding elements (21) (Fig. 2B), suggesting that modern humans acquired adaptive regulatory sequences at these loci. We also searched for shared signatures of adaptive intro-gression between East Asians and Europeans, identifying six distinct regions that have introgressed haplotype frequencies greater than 40% in both populations (Fig. 4 and table S11; p-value < 10−4 by simulation) (10). One of these regions lies in the type II cluster of keratin genes on 12q13 (table S11), further suggesting that Neandertals provided modern humans with adaptive variation for skin phenotypes. In total, eight of the ten candidate introgressed regions overlap protein-coding genes (Fig. 4).

This study shows that the fragmented remnants of the Neandertal genome carried in the DNA of modern humans can be robustly identi-fied, allowing in aggregate, substantial amounts of Neandertal sequence to be recovered. In principle, our approach can be used in the absence of an archaic reference sequence, potentially allowing the discovery and characterization of previously unknown hominins that interbred with modern humans (22–24). This “fossil free” paradigm of sequencing archaic genomes holds considerable promise to reveal insights into hom-inin evolution, the population genetics characteristics of archaic hom-inins, how introgression has influenced extant patterns of human genomic diversity, and to narrow the search for genetic changes that endow uniquely human phenotypes.

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Acknowledgments: We thank members of the Akey laboratory, S. Browning, and B. Browning, and J. Duffy for critical feedback related to this work, S. Pääbo for providing access to high-coverage Neandertal sequence data, and L. Jáuregui for help in figure preparation. A description of where sequence data used in our analyses can be found is provided in the supplementary materials. J.M.A. is a paid consultant of Glenview Capital.

Supplementary Materials www.sciencemag.org/cgi/content/full/science.1245938/DC1 Materials and Methods Figs. S1 to S15 Tables S1 to S11 References (25–45)

12 September 2013; accepted 6 December 2013 Published online 29 January 2014 10.1126/science.1245938

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Fig. 1. Recovering Neandertal lineages from the DNA of modern humans. (A) Schematic representation illustrating that low levels of introgression may facilitate the recovery of substantial amounts of archaic sequence. Lines represent DNA from contemporary individuals and colored boxes indicate archaic sequences. Different colored boxes represent sequences inherited from distinct archaic ancestors. (B) Genealogies of loci in Europeans and Africans in the presence of introgression. The expected signature of an introgressed lineage (blue) that our method exploits is high levels of divergence that persists over relatively long haplotype blocks. (C) Receiver operator curve illustrating the performance of S* for detecting introgressed sequence in simulated data (10). The diagonal line represents random predictions. (D) Distribution of p-values testing for an enrichment of Neandertal variants for S* candidate and randomly selected regions. (E) Amount of Neandertal sequence recovered as a function of FDR. The inset Venn diagram shows the amount of sequence overlap between East Asians and Europeans at a FDR = 5%. (F) Violin plots showing the distribution of amount of introgressed sequence identified per individual for East Asian and European populations (population abbreviations described in the supplementary materials).

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Fig. 2. Genomic distribution of surviving Neandertal lineages. (A) Neandertal lineages identified in East Asians (ASN, red) and Europeans (EUR, blue). Gray shading denotes regions that did not pass filtering criteria (10). (B) Visual genotype illustrations of introgressed sequence identified in the BNC2 and POU2F3 genes. Rows are individuals, columns are variant sites, and rectangles are colored according to genotype (red = predicted Neandertal variant that matches allele present in the Neandertal reference genome; blue = predicted Neandertal variant that does not match allele present in the Neandertal reference genome; black = other variants). Introgressed variants that overlap a PhastCons conserved element, DNaseI hypersensitive site (DHS), or putative enhancer elements are shown as boxes (10). (C) Odds of finding an introgressed lineage on each chromosomal arm calculated from a logistic regression model (10). Odds ratios (OR) are expressed using chromosome 1p as the baseline level. Horizontal bars represent 95% confidence intervals. (D) Relationship between the OR and number of fixed differences per Mb between humans and Neandertals.

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Fig. 3. Organization and characteristics of Neandertal sequence in Europeans and East Asians suggests at least two admixture events. (A) Schematic diagrams of the one and two pulse admixture models. Ne

ASN and Ne

EUR denote effective population sizes of the ancestral, East Asian, and European populations, respectively. In the one pulse model, gene flow (m1) between Neandertals and the ancestors of Europeans and East Asians occurs at time TI. In the two pulse model, a second pulse of gene flow (m2) occurs into East Asians shortly after the divergence of Europeans and East Asians at time TS. (B) Values of summary statistics calculated from 2000 simulations under each model (red, blue, and grey points; horizontal and vertical bars denote 95% CIs) show that a single pulse model is incompatible with the observed data (white box, corrected for sample size differences between populations; limits of box denote 95% CI). Simulations that varied Ne

ASN/NeEUR are shown in red and those with

variable m2/m1 are shown in blue (color bars indicate parameter values).

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Fig. 4. Signatures of adaptive introgression. Scatter plot of introgressed haplotype frequency in Europeans and East Asians. Significantly differentiated and common shared haplotypes are shown in magenta and blue, respectively. Protein-coding genes that overlap candidate adaptively introgressed loci are shown.