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Direct evidence for positive selection of skin, hair, and
eye pigmentation in Europeans during the last 5,000 y
Sandra Wilde
a
, Adrian Timpson
b,c
, Karola Kirsanow
a
, Elke Kaiser
d
, Manfred Kayser
e
, Martina Unterländer
a
,
Nina Hollfelder
a,1
, Inna D. Potekhina
f
, Wolfram Schier
d
, Mark G. Thomas
b,2
, and Joachim Burger
a
a
Institute of Anthropology, Johannes Gutenberg University Mainz, 55128 Mainz, Germany;
b
Research Department of Genetics, Evolution and Environment,
University College London, London WC1E 6BT, United Kingdom;
c
Institute of Archaeology, University College London, London WC1H 0PY, United Kingdom;
d
Institute of Prehistoric Archaeology, Freie Universität Berlin, 14195 Berlin, Germany;
e
Department of Forensic Molecular Biology, Erasmus University Medical
Center Rotterdam, 3000 CA, Rotterdam, The Netherlands; and
f
Institute of Archaeology, Academy of Science of the Ukraine, 04210 Kiev-210, Ukraine
Edited by Nina G. Jablonski, The Pennsylvania State University, University Park, Pennsylvania, and accepted by the Editorial Board February 1, 2014 (received
for review September 4, 2013)
Pigmentation is a polygenic trait encompassing some of the most
visible phenotypic variation observed in humans. Here we present
direct estimates of selection acting on functional alleles in three
key genes known to be involved in human pigmentation path-
ways—HERC2,SLC45A2,andTYR—using allele frequency estimates
from Eneolithic, Bronze Age, and modern Eastern European samples
and forward simulations. Neutrality was overwhelmingly rejected
for all alleles studied, with point estimates of selection ranging
from around 2–10% per generation. Our results provide direct ev-
idence that strong selection favoring lighter skin, hair, and eye
pigmentation has been operating in European populations over
the last 5,000 y.
ancient DNA
|
computer simulations
|
natural selection
|
Neolithic/Bronze Age
|
Eastern Europe
Genomic signatures of natural selection in humans are usually
obtained from modern population genetic data and take the
form of patterns of variation outside those expected under
neutrality (1), including strong correlations between allele fre-
quencies and hypothesized ecological drivers of selection (2) and
identifying alleles with unusually recent age estimates for their
frequencies (1). All such indirect approaches have poor sensi-
tivity and temporal resolution, most are confounded by past
demographic processes, and many are insensitive to selection
acting on standing variation (3). With advances in ancient DNA
analysis techniques it is possible to obtain direct estimates of
natural selection over specific time periods by estimating allele
frequency change, permitting changes in selection intensity to be
detected through time and a more detailed understanding of the
forces shaping human evolution. However, to date no such esti-
mates have been made.
Pigmentation is a particularly conspicuous human phenotypic
variation and in the past has been misleadingly used as a proxy
for deep biogeographical origins (4). Dark pigmentation is thought
to be the ancestral state in humans and to have been maintained
by purifying selection in low-latitude, high-UVR regions to protect
against folate photolysis, UV radiation (UVR)-induced DNA
damage, and possibly damage to immunoglobulins (5, 6). Conti-
nental-scale correlations between skin pigmentation and incident
UVR levels strongly indicate positive ecological adaptation (5),
although sexual selection—particularly in relation to eye and hair
coloration (7)—and relaxation of selective constraints (6) may also
have been important.
Melanin, a derivative of tyrosine, is the primary biopolymer
responsible for constitutive animal pigmentation and is found in
two forms in humans, eumelanin (black-brown) and pheomela-
nin (red-yellow). It is synthesized in melanosomes, organelles
located in melanocytes in several tissues including the basal layer
of the epidermis, hair follicles, and the iris. Variation in pig-
mentation depends mainly on differences in the amount and type
of melanin synthesized and the shape and distribution of mela-
nosomes in different tissues (8). The products of several genes
are involved in melanin synthesis and distribution, and known
DNA polymorphism in those genes explains a substantial pro-
portion of human pigmentation variation (9–12). Signatures of
recent natural selection have been detected in some pigmenta-
tion genes, including both shared and regionally specific alleles
associated with lighter skin pigmentation in eastern and western
Eurasia (13, 14), using modern population genetic data (1, 12,
13, 15, 16).
To obtain direct estimates of the strength of natural selection
driving depigmentation we analyzed three polymorphic sites in
ancient and modern samples (Table 1) that had previously been
identified through genome-wide association studies (GWAS) (1,
17) and fine-mapping SNP association (18) as influencing pig-
mentation in modern Europeans: HERC2 (rs12913832 A >G)
(18), SLC45A2 (rs16891982 C >G) (13), and TYR (rs1042602
C>A) (11). The product of the TYR gene, tyrosinase, catalyzes
the first two steps of the melanogenesis pathway (8), and its ab-
sence generates an epistatic mask on downstream pigment-coding
genes, halting melanin production. The TYR SNP rs1042602 is
highly polymorphic in Europeans, and the derived A allele has
been associated with light skin (19) and eye color (20) and the
Significance
Eye, hair, and skin pigmentation are highly variable in humans,
particularly in western Eurasian populations. This diversity may
be explained by population history, the relaxation of selection
pressures, or positive selection. To investigate whether posi-
tive natural selection is responsible for depigmentation within
Europe, we estimated the strength of selection acting on three
genes known to have significant effects on human pigmenta-
tion. In a direct approach, these estimates were made using
ancient DNA from prehistoric Europeans and computer simu-
lations. This allowed us to determine selection coefficients for
a precisely bounded period in the deep past. Our results in-
dicate that strong selection has been operating on pigmenta-
tion-related genes within western Eurasia for the past 5,000 y.
Author contributions: S.W., M.G.T., and J.B. designed research; S.W., A.T., M.U., N.H., and
M.G.T. performed research; A.T. and M.G.T. contributed new reagents/analytic tools; E.K.
and W.S. coordinated the acquisition of the archaeological sample material and provided
background information; I.D.P. provided archaeological sample material and background
information; M.G.T. and J.B. coordinated this study; S.W., A.T., N.H., and M.G.T. analyzed
data; and S.W., A.T. , K.K., E.K., M.K., M.U., N. H., I.D.P., W.S., M.G.T., an d J.B. wrote
the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. N.G.J. is a guest editor invited by the Editorial
Board.
Freely available online through the PNAS open access option.
1
Present address: Department of Ecology and Genetics, Evolutionary Biology, Uppsal a
University, 75236 Uppsala, Sweden.
2
To whom correspondence should be addressed. E-mail: m.thomas@ucl.ac.uk.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1316513111/-/DCSupplemental.
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absence of freckles (11). SLC45A2 (MATP)isinvolvedinthe
distribution and intracellular processing of tyrosinase and other
pigmentation enzymes (21). The derived rs16891982 G allele,
which decreases in frequency along a north–south cline in Europe
(13), is associated with lighter skin, hair, and eye pigmentation in
modern populations (13, 22). The HERC2 SNP rs12913832 A >G
is the main determinant of iris pigmentation (brown/blue) (18, 23)
and is also associated with skin and hair pigmentation and the
propensity to tan (24). It is located within an intron of the Hect
Domain and RCC1-like Domain2 (HERC2) gene 21 kb upstream
from the OCA2 promoter and serves as an enhancer for OCA2
expression (23, 25). OCA2 expression is increased in the mela-
nocytes of carriers of the ancestral A allele, and attenuated in
carriers of the derived G allele (25). OCA2 encodes a melanoso-
mal transmembrane protein, protein P, which is involved in the
trafficking and processing of tyrosinase, the regulation of mela-
nosomal pH, and glutathione metabolism (26). Selection favoring
the SLC45A2 rs16891982 G allele has been estimated to have
begun between 11,000 and 19,000 y ago (14), well after the ex-
pansion of anatomically modern humans out-of-Africa. The age of
the derived TYR rs1042602 allele has been estimated using two
different methods: rho statistics place its emergence around 6,100
y ago, whereas the Bayesian coalescent approach using BEAST
dates the allele to ∼15,600 y ago (27).
However, these estimates were made by assuming that the
selected allele arose at the time selection started and so do not
accommodate the possibility of selection acting on standing
variation (3). Furthermore, such estimates are themselves de-
pendent on estimates of mutation and recombination rates. No
estimates of the strength of selection or when it started to act on
HERC2/OCA2 and TYR are available at present.
Results
Ancient DNA was retrieved from 63 out of 150 Eneolithic (ca.
6,500–5,000 y ago) and Bronze Age (ca. 5,000–4,000 y ago)
samples from the Pontic–Caspian steppe, mainly from modern-
day Ukraine. We used multiplex-PCR enrichment and next-
generation sequencing to genotype the three pigmentation-
associated SNPs (rs12913832, rs16891982, and rs1042602) and
mtDNA hypervariable region 1 (HVR1) sequences plus 32 mtDNA
coding region SNPs and a 9-bp-indel from these individuals (Tables
S1 and S2). Consensus HVR1 sequences were successfully assem-
bled from 60 individuals. Pigmentation gene data were obtained
from 48 samples. We also genotyped the three pigmentation-
associated SNPs in a sample of 60 modern Ukrainians (28) and
observed an increase in frequency of all derived alleles between
the ancient and modern samples from the same geographic re-
gion (Table 1 and Fig. S1). This implies that the pigmentation of the
prehistoric population is likely to have differed from that of
modern humans living in the same area. Modern frequencies of
the derived alleles within all of Europe and outside of Europe
are provided for comparison (Table 1).
Inferring natural selection based on temporal differences in
allele frequency requires the assumption of population continuity.
To this end we compared the 60 mtDNA HVR1 sequences
obtained from our ancient sample to 246 homologous modern
sequences (29–31) from the same geographic region and found
low genetic differentiation (F
ST
=0.00551; P=0.0663) (32).
Coalescent simulations based on the mtDNA data, accommo-
dating uncertainty in the ancient sample age, failed to reject
population continuity under a wide range of assumed ancestral
population size combinations (Fig. 1).
Conversely, continuity between early central European farm-
ers and modern Europeans has been rejected in a previous study
(33). However, the Eneolithic and Bronze Age sequences pre-
sented here are ∼500–2,000 y younger than the early Neolithic
and belong to lineages identified both in early farmers and late
hunter–gatherers from central Europe (33). A plausible expla-
nation for this is that the prehistoric populations sampled in this
study are a product of admixture between in situ hunter–gatherers
and immigrant early farmers during the centuries after the ar-
rival of farming, and that this admixture was a major process
shaping modern patterns of mtDNA variation (34) and possibly
Table 1. Allele frequencies of three functional SNPs associated with pigmentation in ancient and modern populations
Derived allele frequency
Gene SNP Polymorphism Europe Asia Africa Modern Ukrainian sample Ancient sample
HERC2 rs12913832 regulatory
element (OCA2)
A>G 0.710 [758] 0.002 [572] 0.000 [370] 0.651 (0.546–0.744) [86] 0.160 (0.099–0.247) [94]
SLC45A2 (MATP) rs16891982 Leu374Phe C >G 0.970 [758] 0.007 [572] 0.000 [370] 0.927 (0.849–0.965) [82] 0.432 (0.296–0.578) [44]
TYR rs1042602 Ser192Tyr C >A 0.368 [758] 0.002 [572] 0.000 [370] 0.367 (0.279–0.466) [98] 0.043 (0.018–0.106) [92]
Modern allele frequencies are from 1000 Genomes (http://browser.1000genomes.org) (65). Range in parentheses indicates the equal-tailed 95% confidence
interval calculated as described using the qbeta function in R (66). Numbers in square brackets indicate 2Nindividuals. The African American (ASW) data from
1000 Genomes were excluded because this population is admixed to an unknown extent.
120406080100
12345
NN x103
NUP x103
0.00
0.05
0.10
0.15
Fig. 1. Probabilities of obtaining F
ST
equal to or greater than that observed
(0.00551) between 60 Eneolithic (ca. 6,500–5,000 y ago) and Bronze Age (ca.
5,000–4,000 y ago) samples from the Pontic–Caspian steppe, and a combined
sample of 246 homologous modern sequences from the same geographic
region, across a range of assumed ancestral population size combinations.
Two phases of exponential growth were modeled, the first after the initial
colonization of Europe 45,000 y ago, of assumed effective female pop-
ulation size N
UP
(yaxis), and ending when farming began in the region
considered 7,000 y ago, when the assumed effective female population size
was N
N
(xaxis), and the second leading up to the present, when the assumed
effective female population size is 5,444,812. The initial colonizers of Europe
were sampled from a constant ancestral African population of 5,000 effec-
tive females. Gray shaded areas indicate Pvalues >0.05.
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also the variability observed in European hair, eye, and
skin color.
To test whether the observed increases in the three light pig-
mentation-associated alleles can be explained by genetic drift
alone or whether natural selection needs to be invoked, we
performed forward computer simulations of drift plus selection,
accommodating uncertainty in ancient and modern allele fre-
quency, population size, and ancient sample age. We assumed
codominance for both SLC45A2 rs16891982 G and TYR rs1042602
A alleles (22, 35) and that the derived HERC2 rs12913832 G allele
is recessive (36). Using these simulations, neutrality (S=0) was
rejected under all assumed ancestral effective population sizes—
ranging from 10
3
to 10
5
at the time of the ancient sample
(SLC45A2 P <1×10
−5
,TYR P <2×10
−5
,andHERC2 P <1×
10
−5
). The values of selection acting on the SLC45A2 rs16891982
G allele, the TYR rs1042602 A allele, and the HERC2 rs12913832
G allele that best explained the observed derived allele frequency
changes were 0.030, 0.026, and 0.036, respectively (Fig. 2).
Whereas there is strong evidence that the derived HERC2
rs12913832 G allele is recessive (36), it is less clear whether the
SLC45A2 and TYR derived alleles are codominant, recessive, or
dominant (22, 35). Under the assumption that both SLC45A2 and
TYR derived alleles are recessive, the selection values that best
explain the observed changes in frequency are 0.022 and 0.104,
respectively, and under the assumption that they are dominant the
selection values are 0.088 and 0.016, respectively; again, neutrality
was rejected for all three alleles (P<4×10
−5
) under all ancestral
population sizes modeled and all assumptions of dominance/
codominance/recessivity (Fig. S2).
Discussion
Our analysis indicates that positive selection on pigmentation
variants associated with depigmented hair, skin, and eyes was still
ongoing after the time period represented by our archaeological
population, 6,500–4,000 y ago. This finding suggests that either
the selection pressures that initiated the selective sweep during
the Late Pleistocene or early Holocene were still operative or that
a new selective environment had arisen in which depigmentation
was favored for a different reason.
The high selection coefficients estimated for pigmentation
genes HERC2,SLC45A2, and TYR are best understood in the
context of estimates obtained for other recently selected loci.
Using spatially explicit simulation and approximate Bayesian
computation, selection on the LCT -13,910*T allele—which is
strongly associated with lactase persistence in Europeans and
southern Asians—was inferred to fall in the range 0.0259–0.0795
and to have begun around 7,500 y ago in the region between the
Balkans and central Europe (37). However, another simulation-
based study incorporating latitudinal effects on selection resulted
in a lower estimate of S(0.008–0.018) (38). The selective ad-
vantage of the G6PD A−and Med deficiency alleles conferring
resistance to malaria have been estimated at 0.019–0.048 and
0.014–0.049, respectively, in regions where malaria is endemic
(39). These alleles are estimated to have arisen ∼6,357 y ago
(G6PD A−) and 3,330 y ago (G6PD Med) (39). Thus, the esti-
mates of Sfor the three pigmentation genes examined in this
study are comparable to those for the most strongly selected loci
in the human genome.
Although these estimated selection coefficients are high, they
are comparable to previous estimates for genes in the pigmenta-
tion complex. The selective sweeps favoring the SLC45A2 de-
rived allele, as well as the derived alleles of SNPs in SLC24A5
and TYRP1, which are also implicated in the lightening of skin
pigmentation, are estimated to have begun between 11,000 and
19,000 y ago, after the separation of the ancestors of modern
Europeans and East Asians (the ages of the selective sweeps
affecting HERC2 and TYR have not yet been estimated) (14, 40).
Beleza et al. (14) recently estimated the coefficient of selection
at the SLC45A2 locus to be 0.05 under a dominant model of
inheritance and 0.04 under an additive model. Selection favoring
the derived alleles of SNPs in SLC24A5 and TYRP1 was found to
be similarly strong.
Estimating selection coefficients using the ancient DNA-based
simulation approach presented here offers considerable advan-
tages over traditional methods based on allele age and frequency
estimates (1): Selection coefficients are estimated over a defined
period; selection acting on standing variation can be accommo-
dated; and our approach is insensitive to the frequently un-
accounted for uncertainties associated with allele age estimation
using molecular or recombination clocks. This latter advantage
is likely to result in considerable improvements in precision.
However, our approach does require the assumption of pop-
ulation continuity and will not provide direct estimates of when
a selective sweep began.
Although the strength of the selection coefficients in a certain
time window can be estimated with improved precision using our
ancient DNA-based simulation approach, the actual nature of
the selection pressure remains unknown. However, temporal and
geographical information from the prehistoric skeletal pop-
ulation under study can help in formulating reasonable hy-
potheses. Geographic variation in many functional skin pigmentation
gene polymorphisms (13), and lighter skin pigmentation more
generally, correlate strongly with distance from the equator in
long-established populations, suggesting that selective pressure
also occurred along a latitudinal gradient. The samples in our
study were from between 42°N and 54°N, a latitudinal belt in which
3.0 3.5 4.0 4.5 5.0
0
2
4
6
8
10
Log10
founding N
e
Selection (%)
A
3.0 3.5 4.0 4.5 5.0
Log10 founding Ne
Selection (%)
B
3.0 3.5 4.0 4.5 5.0
0
2
4
6
8
10
Log10 founding Ne
Selection (%)
C
0.2
0.4
0.6
0.8
1.0
10
8
6
4
2
0
Fig. 2. Two-tailed empirical Pvalues for obtaining the observed (A)SLC45A2 G allele, (B)TYR A allele, and (C)HERC2 G allele frequency increase. Pvalues
were obtained by forward simulation of drift and natural selection across a range of assumed ancestral population sizes and selection coefficients, assuming
exponential growth to a modern N
e
of 4,845,710. The SLC45A2 rs16891982 G allele and the TYR rs1042602 A allele were assumed to be codominant. The
HERC2 rs12913832 G allele was assumed to be recessive (values less than 0.01 are shaded gray).
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yearly average UVR is insufficient for vitamin D3 photosynthesis
in highly melanized skin (4, 41). Constraints on the ability to
photosynthesize vitamin D3 imposed by low incident UVR in-
tensity may have provided significant selective pressure favoring
lighter pigmentation populations in high-latitude regions such as
the northern Pontic steppe belt. The need to admit UVB radiation
to catalyze the synthesis of vitamin D3, together with the decreased
danger of folate photolysis at higher latitudes, may account for
the observed skin depigmentation from prehistoric to modern
times in this region (5).
Dietary change during the Neolithization process may have
reinforced selection pressure favoring depigmented skin. The
individuals analyzed in this study lived ∼500–2,000 y after the
arrival of farming in the region north of the Black Sea (42, 43).
In many parts of Europe, the Mesolithic–Neolithic transition is
associated with a switch from a vitamin D-rich aquatic or game-
based hunter–gatherer diet (44) to a vitamin D-poor agricul-
turalist diet. In low-UV regimes such as the one prevailing in our
study region, it is difficult to meet vitamin D requirements
without the consumption of significant quantities of oily fish or
animal liver (45, 46). The vitamin D recommended dietary al-
lowance of 800–1,000 IU for adults requires the daily con-
sumption of the equivalent of 100 g of wild salmon (the dietary
input with the greatest measured vitamin D concentration).
Isotopic evidence suggests that the populations sampled in our
study continued to access aquatic resources, primarily river fish,
in the Neolithic, Eneolithic, and Bronze Age, although there was
considerable heterogeneity in fish consumption within the study
region (47–50). However, any diminution in fish consumption
may have been sufficient to generate additional selective pres-
sure favoring depigmentation at this low-incident-UVR latitude.
Although ecological and environmental factors may be suffi-
cient to explain the observed change in European skin pigmen-
tation, these explanations are unlikely to hold for eye and hair
color. The geographic distribution of iris and hair pigmentation
variation does not conform as well to a latitudinal cline model,
with much of the observed phenotypic variation restricted to
Europe and closely related neighboring populations (51, 52).
The blue iris phenotype characteristic of the HERC2 rs12913832
G allele, for example, is almost completely restricted to western
Eurasia and some adjacent regions, its descendant populations,
and populations containing European admixture (51, 52). It is
possible that depigmented irises or the various human hair color
morphs in Europeans are by-products of selection on skin pig-
mentation. There is evidence for gene–gene interaction within
the polygenic system governing complex pigmentation traits;
interactions between HERC2,OCA2, and MC1R, in particular,
have been found to have a statistically significant effect on hair,
iris, and skin color (36). There is also evidence for epistatic
interactions between components of the melanin synthesis
pathway in other mammalian model systems, including interac-
tions between the products of ASIP,MC1R,andTYR (53).
Additionally, many pigmentation genes, including TYR,HERC2,
and SLC45A2 have pleiotropic effects on skin, hair, and eye
color (11, 36).
Given that intraspecific pigmentation variability in other taxa,
particularly avians, has been attributed to signaling and other
factors associated with mate choice (54) it is possible that depig-
mented irises and the various hair colors observed in Europeans
arose through sexual selection (7). Frequency-dependent sexual
selection in favor of rare variants has been observed in vertebrates
(55, 56), and such selection favoring rare pigmentation morphs
could have driven alleles associated with lighter hair and eye
colors to higher frequency. Once lighter hair and eye pigmenta-
tion phenotypes reached appreciable frequencies in European
populations, these novel traits may have continued to be preferred
as indicators of group membership, facilitating assortative mating.
Assortative mating based on coloration is common in vertebrates
(57), and skin pigmentation has been observed as a criterion for
endogamy in modern human populations (58, 59). In addition,
there is some evidence that lighter iris colors, because of their
recessive mode of inheritance, may be preferred by males in
assortative mating regimes to improve paternity confidence (60).
Consistent with positive assortative mating, an exact test of Hardy–
Weinberg equilibrium reveals an excess of HERC2 rs12913832
homozygotes in both the modern (P=0.0543) and ancient (P=
0.0084) East European samples genotyped here (Table S3), despite
the relatively small sample sizes.
The observed excess of HERC2 rs12913832 homozygotes in
the ancient sample might be explained by population stratifica-
tion in a temporally heterogeneous population sample. Although
we do not observe any chronological or spatial patterning of the
pigmentation markers in our prehistoric sample, we cannot exclude
population stratification in the absence of additional neutral SNPs.
However, we note that neither the TYR nor the SLC45A2 SNPs
investigated here, nor three additional SNPs investigated in the same
ancient and modern samples, showed any significant observable
excess of homozygotes (Table S3), suggesting that the excess
of HERC2 rs12913832 homozygotes is less likely to be due to
population stratification.
In sum, a combination of selective pressures associated with
living in northern latitudes, the adoption of an agriculturalist
diet, and assortative mating may sufficiently explain the observed
change from a darker phenotype during the Eneolithic/Early
Bronze age to a generally lighter one in modern Eastern Euro-
peans, although other selective factors cannot be discounted.
The selection coefficients inferred directly from serially sampled
data at these pigmentation loci range from 2 to 10% and are
among the strongest signals of recent selection in humans.
Methods
Ancient DNA Extraction, Amplification, and Sequencing. Sample information,
detailed descriptions of DNA extraction, amplification, and sequencing methods
as well as validation of the ancient DNA data are provided in Supporting In-
formation owing to the extensive nature of the experimental setup.
Skeletal material from 150 Eneolithic and Bronze Age individuals from the
west and north Pontic region were available for ancient DNA analyses. From
all but one sample DNA was extracted twice independently using 0.5–1.0 g
bone powder; 403 bp of the hypervariable region 1 [nucleotide positions
(np) 16,011–16,413] were amplified using seven overlapping primer pairs
(Table S4). They were integrated in a triple multiplex setup that included 32
clade-determining coding region SNPs and a 9-bp-indel (Table S5), as well as
used in single-locus PCRs. rs12913832, rs16891982, and rs1042602 were
amplified in a multiplex PCR together with 18 other nuclear loci (Table S4).
Sequencing of the PCR products was primarily done by 454 sequencing by
GATC Biotech. Before that, samples were pooled according to a protocol
modified after Meyer et al. (61). Raw data (454) were sorted by barcode and
primer sequences of the multiplex PCRs and then analyzed with SeqMan
ProTM (DNASTAR Lasergene 8, 9, and 10). For authentication purposes mi-
tochondrial haplotypes are based on at least three, and SNP genotypes on at
least four, independent amplification products from two extracts. In cases
where the authentication scheme could not be fulfilled using the available
454 data, those loci were additionally amplified in single-locus PCRs, fol-
lowed by direct sequencing after Sanger.
Population Genetic Analyses. Continuity between Neolithic and present-day
populations in the geographic region encompassing Bulgaria, Romania,
Ukraine, and the southwest of the Russian Federation was tested by calcu-
lating the F
ST
between ancient and modern observed mtDNA HVR1 samples
(29–32) and comparing this with F
ST
s between ancient and modern DNA
samples generated by coalescent simulation (33). These simulated DNA
samples were generated using the program Fastsimcoal (62) under the null
model of a single continuous population, using plausible population pa-
rameter ranges, and serial ancient and modern samples that replicated the
observed sample numbers and dates. Details of the continuity test are pro-
vided in Supporting Information.
To test whether changes in HERC2 rs12913832 G, TYR rs1042602 A, or
SLC45A2 rs16891982 G allele frequencies (Fig. S1) can be explained by
genetic drift, or whether natural selection needs to be invoked, and to
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estimate the strength of natural selection where appropriate, we used a
forward simulation approach. In each forward simulation we first drew the
ancestral allele frequency estimate from a random Beta (n
p
+1, n
q
+1) dis-
tribution, where n
p
and n
q
were the number of ancestral and derived alleles
observed in our ancient sample, respectively, to reflect uncertainty in an-
cestral allele frequencies. Then drift and natural selection were forward-
simulated by binomial sampling across generations and using a standard
selection equation (63), respectively. We assumed codominance for both
SLC45A2 rs16891982 G and TYR rs1042602 A alleles (22, 35) and that the
derived HERC2 rs12913832 G allele is recessive (36) (Fig. 2). However, al-
though there is strong evidence that the derived HERC2 rs12913832 G allele
is recessive (36), it is less clear whether the SLC45A2 and TYR derived alleles
are codominant, recessive, or dominant (22, 35). For this reason we also
performed the same simulations assuming both dominance and recessivity
for the SLC45A2 and TYR derived alleles (Fig. S2). Exponential population
growth was modeled from a range of values of N
e
at the time of the ancient
sample (50 equally spaced log
10
values between 1,000 and 100,000) to
a modern N
e
of 4,845,710 (1/10 of the census population size of Ukraine in
2001, the year that the modern Ukrainian sample was collected; http://en.
wikipedia.org/wiki/Demographics_of_Ukraine). The number of generations
forward-simulated was drawn at random from a pool of 600,000 date esti-
mates for the ancient samples reported here, generated by pooling each set
of 10,000 date estimates for all 60 ancient samples. In the final generation of
each forward simulation, simulated modern sample allele frequencies were
picked from a random binomial with Nequal to the modern sample size
(HERC2 n =86, SLC45A2 n =82, and TYR n =98). Forward simulations were
repeated 100,000 times for each combination of the 50 assumed N
e
values at
the time the ancient sample and 50 selection coefficient (S) values, starting
at zero. Finally, the simulated distribution of modern derived allele fre-
quencies was compared with those observed using the equation 1 −2×
j0.5 –Pj, where P is the proportion of simulated modern allelefrequencies that
are greater than that observed. This yielded a two-tailed empirical Pvalue for
the observed allele frequency increase for each combination of the de-
mographic and natural selection model parameters (64) (Fig. 2 and Fig. S2).
ACKNOWLEDGMENTS. We thank all colleagues who contributed their ar-
chaeological knowledge and samples to this project, notably D. Agre,
St. Alexandrov, V. Bubulici, A. N. Gei, A. A. Khokhlov, I. N. Klyuchneva,
A. Kozak, N. Neradenko, A. V. Nikolova, V. P. Petrenko, Yu. Ya. Rassamakin,
V. A. Romashko, S. N. Sanzharov, E. N. Sava, N. N.Shishlina, and D. Ya. Teslenko.
We also thank Tom Gilbert and colleagues for giving us a hands-on introduc-
tion to bar coding and 454 sequencing workflow, and providing us with pro-
tocols. Thanks to Benjamin Rieger for his “sort3”perl script. The radiocarbon
dates were provided by the Research Laboratory for Archaeology and
the History of Art (University of Oxford), and financially supported by the
Excellence Cluster 264 Topoi, Berlin. The authors acknowledge the use of the
UCL Legion High Performance Computing Facility and associated support serv-
ices in the completion of this work. The project was funded by German Federal
Ministry of Education and Research Grant 01UA0809A.
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