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LETTER

doi:10.1038/nature14558

An early modern human from Romania with a recent

Neanderthal ancestor

Qiaomei Fu

1,2,3

, Mateja Hajdinjak

, Oana Teodora Moldovan

, Silviu Constantin

, Swapan Mallick

2,6,7

, Pontus Skoglund

Nick Patterson

, Nadin Rohland

, Iosif Lazaridis

, Birgit Nickel

, Bence Viola

3,7,8

, Kay Pru

fer

, Matthias Meyer

, Janet Kelso

David Reich

2,6,9

& Svante Pa

Neanderthals are thought to have disappeared in Europe approxi-

mately 39,000–41,000 years ago but they have contributed 1–3% of

the DNA of present-day people in Eurasia

. Here we analyse DNA

from a 37,000–42,000-year-old

modern human from Pes

̧tera cu

Oase, Romania. Although the specimen contains small amounts of

human DNA, we use an enrichment strategy to isolate sites that are

informative about its relationship to Neanderthals and present-

day humans. We find that on the order of 6–9% of the genome of

the Oase individual is derived from Neanderthals, more than any

other modern human sequenced to date. Three chromosomal seg-

ments of Neanderthal ancestry are over 50 centimorgans in size,

indicating that this individual had a Neanderthal ancestor as

recently as four to six generations back. However, the Oase indi-

vidual does not share more alleles with later Europeans than with

East Asians, suggesting that the Oase population did not contrib-

ute substantially to later humans in Europe.

Between 45,000 and 35,000 years ago, anatomically modern

humans spread across Europe, while the Neanderthals, present since

before 300,000 years ago, disappeared. How this process occurred

has long been debated

1,3–5

. Comparisons between the Neanderthal

genome and the genomes of present-day humans have shown that

Neanderthals contributed approximately 1–3% of the genomes of all

people living today outside sub-Saharan Africa

6,7

suggesting that

human populations ancestral to all non-Africans mixed with

Neanderthals. The size of segments of Neanderthal ancestry in

present-day humans suggests that this occurred between 37,000

and 86,000 years ago

. However, where and how often this occurred

is not understood. For example, Neanderthals share more alleles

with East Asians and Native Americans than with Europeans, which

may reflect additional interbreeding in the ancestors of eastern non-

Africans

9–12

. Surprisingly, analyses of present-day genomes have not

yielded any evidence that Neanderthals mixed with modern humans

in Europe, despite the fact that Neanderthals were numerous

there and cultural interactions between the two groups have been

proposed

13,14

More direct insight into the interactions between modern and

archaic humans can be obtained by studying genomes from modern

humans who lived at a time when they could have met Neanderthals.

Recent analyses of genomes from a

43,000–47,000-year-old modern

human from western Siberia

and a

36,000–39,000-year-old mod-

ern human from eastern Europe

showed that Neanderthal gene flow

into modern humans occurred before these individuals lived. The

Siberian individual’s genome contained some segments of

Neanderthal ancestry as large as 6 million base pairs (bp), suggesting

that some Neanderthal gene flow could have occurred a few thousand

years before his death

We report genome-wide data from a modern human mandible,

Oase 1, found in 2002 in the Pes

̧tera cu Oase, Romania. The age of

this specimen has been estimated to be

37,000–42,000 years by direct

radiocarbon dating

2,17,18

. Oase 1 is therefore one of the earliest modern

humans in Europe. Its morphology is generally modern but some

aspects are consistent with Neanderthal ancestry

19–21

. Subsequent

excavations uncovered a cranium from another, probably contempor-

aneous individual, Oase 2, which also carries morphological traits that

could reflect admixture with Neanderthals

17,19

We prepared two DNA extracts from 25 mg and 10 mg of bone

powder removed from the inferior right ramus of Oase 1. We treated

an aliquot of each of these extracts with

Escherichia coli

uracil-DNA

glycosylase (UDG), an enzyme that removes uracils from the interior

parts of DNA molecules, but leaves a proportion of uracils at the ends

of the molecules unaffected. Uracil residues occur in DNA molecules

as a result of deamination of cytosine residues, and are particularly

prevalent at the ends of ancient DNA molecules

9,22

. Among the DNA

fragments sequenced from these two extracts, 0.18% and 0.06%,

respectively, could be mapped to the human reference genome. We

prepared three additional DNA libraries from the extract containing

0.18% human-like molecules, but omitted the UDG treatment to

increase the number of molecules in which terminal C-to-T substitu-

tions could be seen and used to identify putatively ancient fragments.

Because the fraction of endogenous DNA is so small, we used hybrid-

ization to DNA probes to isolate human DNA fragments from the

libraries

. Applying this strategy to the mitochondrial genome

allowed the mitochondrial (mt)DNA from the five libraries to be

sequenced to an average coverage of 803-fold (Supplementary Note 1).

At the 3

ends of the DNA fragments, cytosine residues appeared as

thymine residues relative to the human mtDNA reference in 21% of

fragments, reflecting appreciable levels of cytosine deamination. This

suggests that at least some of the human mtDNA is of ancient origin.

We determined mtDNA consensus sequences in two ways: using all

mtDNA fragments, and using only deaminated fragments that carry

C-to-T substitutions at either end relative to the consensus mtDNA

sequence based on these fragments, an approach known to enrich

for endogenous DNA

9,24–26

. The mtDNA sequence based on all frag-

ments clusters with present-day Europeans (Extended Data Fig. 1)

(Supplementary Note 1). In contrast, the mtDNA sequence based on

deaminated fragments is related to a large group of present-day

Eurasian mtDNAs (haplogroup N) but diverges from these before

they diverged from each other. This Oase 1 mtDNA carries a few

private mutations on the basis of which its age can be estimated to

be 36,330 years before present (14,520–56,450; 95% confidence inter-

val). Using six positions at which the mtDNA sequence differs from at

least 99% of 311 present-day humans, we estimate the contamination

These authors contributed equally to this work.

Key Laboratory of Vertebrate Evolution and Human Origins of Chinese Academy of Sciences, IVPP, CAS, Beijing 100044, China.

Department of Genetics, Harvard Medical School, Boston, Massachusetts

02115, USA.

Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig 04103, Germany.

‘‘Emil Racovi ̧

̆’’ Institute of Speleology, Cluj Branch, 400006 Cluj,

Romania.

‘‘Emil Racovi ̧

̆’’ Institute of Speleology, Department of Geospeleology and Paleontology, 010986 Bucharest 12, Romania.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts

02142, USA.

Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Leipzig 04103, Germany.

Department of Anthropology, University of Toronto, Toronto, Ontario, M5S

2S2, Canada.

Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, 02115, USA.

00 MONTH 2015 | VOL 000 | NATURE | 1

2015

Page 2

among all mtDNA fragments to be 67% (95% confidence interval

65–69%). When we restrict to mtDNA fragments that carry terminal

C-to-T substitutions, the contamination estimate is 4% (95% confid-

ence interval of 2–9%) (Supplementary Note 1).

To isolate nuclear DNA from Oase 1, we used three sets of oligo-

nucleotide probes that cover about two million sites that are single

nucleotide polymorphisms (SNPs) in present-day humans and cap-

tured DNA molecules from the five libraries. Of the SNPs targeted,

51% (

1,038,619) were covered by at least one DNA fragment, and

13% (

271,326) were covered by at least one fragment with a

terminal C-to-T substitution. To estimate nuclear DNA contamina-

tion, we tested whether Oase 1 DNA fragments with or without evid-

ence of deamination share more alleles with present-day Europeans or

with East Asians. We found that Europeans share significantly fewer

alleles with Oase 1 fragments that are deaminated than with Oase 1

fragments that are not, consistent with European contamination of

17–30% (Supplementary Note 1). On the basis of these findings and

those from mtDNA, we restricted all subsequent analyses to DNA

fragments that carry terminal C-to-T substitutions. After doing this,

we found that we captured targeted SNPs from the X and Y chromo-

somes at a similar rate, indicating that Oase 1 carried both an X and a

Y chromosome and thus that he was male. The Y chromosome alleles

belong to the F haplogroup, which is carried by most males in Eurasia

today (Supplementary Note 2).

To determine the relationship of the Oase 1 individual to present-

day populations, we first tested whether he shared more alleles with

particular present-day individuals from different populations using

-statistics, which provides a robust estimate of admixture almost

regardless of how SNPs for analysis are chosen

. We find that

Oase 1 shared more alleles with present-day East Asians and Native

Americans than with present-day Europeans, counter to what might

naively be expected for an ancient individual from Europe (Fig. 1)

(5.2

6.4; Extended Data Table 1). However, it has been sug-

gested that Europeans after the introduction of agriculture derive a

part of their ancestry from a ‘basal Eurasian’ population that separated

from the initial settlers of Europe and Asia before they split from

each other

. Therefore, we replaced present-day Europeans with

Palaeolithic and Mesolithic European individuals in these analyses.

We then find that the Oase 1 individual shares equally many alleles

with these early Europeans as with present-day East Asians and Native

Americans (Fig. 1) (

1.5 inExtended Data Table 1). Restricting this

analysis to transversion polymorphisms, which are not susceptible to

errors induced by cytosine deamination, does not influence this result

(Extended Data Table 2 and Supplementary Note 3). This suggests that

the Oase 1 individual belonged to a population that did not contribute

much, or not at all, to later Europeans. This contrasts, for example, with

the

36,000–39,000-year-old Kostenki 14 individual from western

Russia, who was more closely related to later Europeans than to East

Asians (1.9

13.7; Extended Data Table 1)

To assess whether the ancestors of the Oase 1 individual mixed with

Neanderthals, we tested whether the Altai Neanderthal genome shares

more alleles with the Oase 1 genome than with sub-Saharan Africans.

We find this to be the case (

7.7; Supplementary Note 4). We then

asked whether the amount of Neanderthal ancestry in the Oase 1

genome is similar to that in present-day non-Africans. Surprisingly,

the Neanderthal genome shares more alleles with the Oase 1 individual

than it does with any present-day people in Eurasia that we tested,

indicating that he carries more Neanderthal-like DNA than present-

day people (5.0

8.2; Extended Data Table 3). We also observe

more Neanderthal-like alleles in the Oase 1 individual when we com-

pare him to four early modern humans: an 8,000-year-old individual

from Luxembourg, and three individuals from Russia who vary in age

between 24,000 and 45,000 years (3.6

6.8; Extended Data

Table 3). Thus, the Oase 1 individual appears to have carried more

Neanderthal-like DNA than any other modern human analysed to

date. This observation cannot be explained by residual present-day

human contamination among the DNA fragments that carry terminal

C-to-T substitutions, because all modern humans studied to date carry

less Neanderthal ancestry than the Oase 1 genome, and thus contam-

ination would lower, rather than increase, the apparent Neanderthal

ancestry.

We estimated the proportion of Neanderthal DNA in the Oase 1

genome using three different statistics

7,29

(Supplementary Note 4).

Although the results differ, they all yield point estimates between

6.0% and 9.4% (Table 1). For one of the statistics, none of the 90%

confidence intervals for Neanderthal ancestry in the other modern

-score

Asian and

Native American

Asian

Native

American

–2

–4

–6

European

Western Eurasians before a

riculture

Loschbour

Kostenki 14

Ust’-Ishim

Loschbour

Kostenki 14

Ust’-Ishim

Figure 1

Allele sharing between the Oase 1 individual and other genomes.

Each point indicates the extent to which the Oase 1 genome shares alleles with

one or other of a pair of genomes from different populations indicated above

and below (see Extended Data Table 1 for numbers).

-scores with an absolute

value greater than 2 indicate an excess of allele sharing (grey).

Table 1

Estimated fraction of the Oase 1 genome that derives from Neanderthals

Statistic 1

(

Denisova

Altai

;

Mbuti

)

(

Denisova

Altai

;

Mbuti

Mezmaiskaya

)

Statistic 2

{

(

Mbuti

Chimp

;

Denisova

)

(

Mbuti

Chimp

;

Dinka

Denisova

)

Statistic 3

(

Mbuti

;

Denisova

Chimp

)

(

Altai

Mbuti

;

Denisova

Chimp

)

Sample

Proportion

s.e.m.

90% CI

Proportion

s.e.m.

90% CI

Proportion

s.e.m.

90% CI

Oase 1

8.1%

2.0%

4.8–11.3%

9.4%

1.1%

7.5–11.3%

6.0%

2.0%

2.8–9.3%

Ust’-Ishim

3.6%

0.9%

2.2–5.0%

5.5%

0.7%

4.3–6.6%

0.4%

1.2%

0.0–2.5%

Kostenki 14

3.8%

1.0%

2.1–5.5%

2.9%

0.8%

1.6–4.2%

1.7%

1.3%

0.0–3.9%

MA1

1.2%

1.1%

0.0–3.0%

3.5%

0.8%

2.2–4.8%

2.3%

1.3%

0.1–4.5%

Loschbour

1.3%

0.9%

0.0–2.8%

3.9%

0.7%

2.7–5.1%

0.5%

1.2%

0.0–2.6%

La Bran

3.1%

1.0%

1.4–4.7%

1.9%

0.7%

0.7–3.1%

1.4%

1.2%

0.0–3.4%

Stuttgart

3.0%

0.9%

1.5–4.4%

2.5%

0.7%

1.3–3.7%

0.4%

1.2%

0.0–2.4%

Han

2.2%

0.9%

0.6–3.7%

2.2%

0.8%

1.0–3.5%

1.0%

1.2%

0.0–3.1%

Dai

2.6%

0.9%

1.1–4.0%

1.0%

0.8%

0.0–2.3%

0.7%

1.2%

0.0–2.6%

French

3.0%

0.9%

1.6–4.5%

3.0%

0.7%

1.8–4.2%

0.2%

1.2%

0.0–2.2%

CI, confidence interval; s.e.m., standard error of the mean; negative values are truncated to 0%.

2 | NATURE | VOL 000 | 00 MONTH 2015

RESEARCH

LETTER

2015

Page 3

human samples overlap with the confidence interval in Oase 1. When

we restrict analysis to transversion SNPs, the point estimates of

Neanderthal ancestry are even higher (range of 8.4% to 11.3%)

(Extended Data Table 4).

To study the spatial distribution of Neanderthal DNA across the

Oase 1 genome, we designed capture probes for around 1.7 million

nucleotide positions at which nearly all individuals in a sub-Saharan

African population carry one allele whereas Neanderthal genomes

carry a different allele. We used these probes to isolate DNA fragments

from the Oase 1 individual. A total of 78,055 sites were covered by

deaminated DNA fragments from the Oase 1 individual and were also

covered by DNA fragments sequenced from the

36,000–39,000-

year-old Kostenki 14 individual from western Russia

, the

43,000–47,000-year-old individual from Ust’-Ishim in Siberia

and three present-day human genomes from China, France and

Sudan (Supplementary Note 5). Because the Dinka from Sudan are

thought to have little or no Neanderthal ancestry

, we subtracted the

number of alleles that match the Neanderthals in the Dinka individual

(485) from the number in the other genomes to estimate the number of

alleles attributable to Neanderthal ancestry. The resulting numbers of

putative Neanderthal alleles are 3,746 in the Oase 1 individual, 1,586

and 1,121 in the Ust’-Ishim and Kostenki 14 individuals, respectively,

and 1,322 and 1,033 in the Chinese and the European individuals

(Extended Data Table 5). Thus, the Neanderthal contribution to the

Oase 1 genome appears to be between 2.3- and 3.6-fold larger than to

the other genomes analysed. Assuming that the Neanderthal contri-

bution to the European individual is 2% (ref. 7), this suggests that 7.3%

of the Oase 1 genome is of Neanderthal origin. When the numbers of

alleles matching the Neanderthal genome are compared per chrom-

osome (Extended Data Table 5), the highest numbers are always

observed for the Oase 1 genome, except in the case of chromosome

21, in which the Ust’-Ishim individual carries a large segment of likely

Neanderthal ancestry.

We plotted the positions of Neanderthal-like alleles across the

Oase 1 genome (Fig. 2). We detect three segments that are over

50 centimorgans (cM) in size, suggesting that the Neanderthal contri-

bution to the Oase 1 individual occurred so recently in his family tree

that chromosomal segments of Neanderthal origin had little time to

break up due to recombination. To estimate the date of the most recent

Neanderthal contribution to the Oase 1 genome, we studied the size

spans of seven segments of the genome that appeared to be recently

derived from Neanderthals. Their genetic lengths suggest that the

Oase 1 individual had a Neanderthal ancestor as a fourth-, fifth- or

sixth-degree relative (Supplementary Note 5). This would predict that an

average of 1.6% to 6.3% of the Oase 1 genome derived from this recent

Neanderthal ancestor. Visual inspection of the Oase 1 genome sug-

gests that in addition to these seven segments, other smaller segments

also carry Neanderthal-like alleles (Fig. 2). When we remove the seven

longest segments, the estimate of Neanderthal ancestry in Oase 1 drops

from 7.3% to 4.8%, which is still around twice the 2.0–2.9% estimated

for the French, Han, Kostenki and Ust’-Ishim individuals in this

remaining part of the genome. This additional Neanderthal ancestry

100

150

200

100

150

050100

050

050100

100

150

100

150

050100

050

100

150

200

250

050

050100

050

100

150

100

150

100

150

200

250

Figure 2

Spatial distribution of alleles matching Neanderthals in modern

humans.

Coloured vertical lines indicate alleles shared with Neanderthals and

no colour indicates alleles shared with the great majority of West Africans.

D, Dinka; F, French; H, Han; K, Kostenki 14; O, Oase 1; U, Ust’-Ishim. The

seven grey bars indicate segments of putative recent Neanderthal ancestry. This

analysis is based on 78,055 sites. Numbers refer to chromosomes.

00 MONTH 2015 | VOL 000 | NATURE | 3

LETTER

RESEARCH

2015

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Citations96
References66
Linked Data13
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