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Genotypes of predomestic horses match phenotypes
painted in Paleolithic works of cave art
Melanie Pruvost
a,b
, Rebecca Bellone
c
, Norbert Benecke
b
, Edson Sandoval-Castellanos
d
, Michael Cieslak
a
,
Tatyana Kuznetsova
e
, Arturo Morales-Muñiz
f
, Terry O
Connor
g
, Monika Reissmann
h
, Michael Hofreiter
i,1
,
’
and Arne Ludwig
a,1
a
Research Group of Evolutionary Genetics, The Leibniz Institute for Zoo and Wildlife Research, 10252 Berlin, Germany;
b
Department of Natural Sciences,
German Archaeological Institute, 14195 Berlin, Germany;
c
Department of Biology, University of Tampa, Tampa, FL 33606;
d
Laboratorio de Genética Ecológica
y Evolución, Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autonoma de Mexico, Mexico City, 04510, Mexico;
e
Department
of Palaeontology, Faculty of Geology, Moscow State University, Moscow 119899, Russia;
f
Laboratory of Archaeozoology, Universidad Autonoma Madrid,
28049 Madrid, Spain;
g
Department of Archaeology, and
i
Department of Biology, University of York, Heslington, York YO10 5DD, United Kingdom; and
h
Department of Crop and Animal Sciences, Humboldt University, 10115 Berlin, Germany
Edited* by Richard G. Klein, Stanford University, Stanford, CA, and approved October 5, 2011 (received for review June 6, 2011)
and Ariège regions. Although it can be concluded that naturalistic
depictions of animals in cave art constitute a restricted phenom-
enon, with more than 80% of the examples being found in two of
the regions mentioned above (Ariège and Périgord in France and
the Cantabrian coast in Spain), four important sites (i.e., Ignatieva
and Kapova in Russia, Cuciulat in Romania, and Badanj in
Bosnia) are located outside Western Europe (Fig. 1) (8).
Chronologically, most of the evidence dates to the Magdalenian
period (16
Archaeologists often argue whether Paleolithic works of art, cave
paintings in particular, constitute re
ections of the natural en-
vironment of humans at the time. They also debate the extent to
which these paintings actually contain creative artistic expres-
sion, re
ect the phenotypic variation of the surrounding envi-
ronment, or focus on rare phenotypes. The famous paintings
“
depicting spotted horses
on the walls of a cave in Pech-Merle, France, date back
The Dappled Horses of Pech-Merle,
”
25,000
y, but the coat pattern portrayed in these paintings is remarkably
similar to a pattern known as
∼
–
11 kyBP) although the earliest testimonies go back to
the Aurignacian of Chauvet Cave in France (i.e., 31 kyBP) (9,
10). Post-Paleolithic art, shifting to more abstract and stylized
forms, is of much less relevance for the discussion about possible
naturalistic animal depictions (4, 5).
Where animal species can be condently identied, horses are
depicted at the majority of these sites. With more than 1,250
documented depictions (
in modern horses. We
have genotyped nine coat-color loci in 31 predomestic horses
from Siberia, Eastern and Western Europe, and the Iberian Pen-
insula. Eighteen horses had bay coat color, seven were black, and
six shared an allele associated with the leopard complex spotting
(
“
leopard
”
), representing the only spotted phenotype that has been dis-
covered in wild, predomestic horses thus far.
LP
was detected in
four Pleistocene and two Copper Age samples from Western and
Eastern Europe, respectively. In contrast, this phenotype was ab-
sent from predomestic Siberian horses. Thus, all horse color phe-
notypes that seem to be distinguishable in cave paintings have
now been found to exist in prehistoric horse populations, sug-
gesting that cave paintings of this species represent remarkably
realistic depictions of the animals shown. This
nding lends sup-
port to hypotheses arguing that cave paintings might have con-
tained less of a symbolic or transcendental connotation than
often assumed.
LP
30% of all animal illustrations) rang-
ing from the Early Aurignacian of Chauvet to the Late Magda-
lenian (several post
–
12-kyBP sites in France and Spain) (11), and
from the Iberian Peninsula to the Ural mountains, horses are the
most frequent of the more than 30 mammal species depicted in
European Upper Paleolithic cave art (5, 12). Depictions are
commonly in a caricature form that slightly exaggerates the most
typical
∼
features (13). Although taken as a whole, images
of horses are often quite rudimentary in their execution, some
detailed representations, from both Western Europe and the
Ural mountains, are realistic enough to at least potentially rep-
resent the actual appearance of the animals when alive. In these
cases, attributes of coat color may also have been depicted with
deliberate naturalism, emphasizing colors or patterns that char-
acterized contemporary horses. For example, the brown and
black horses dominant at Lascaux and Chauvet, France, phe-
notypically match the extant coat colors bay and black. However,
the depictions in the cave of Pech-Merle, France, dated to
24.7 kyBP (14), featuring spotted horses in a frieze that includes
hand outlines and abstract patterns of spots, have led pre-
historians to argue for more complex explanations for several
reasons. First, the juxtaposition of elements in this depiction raises
the question of whether the spotted pattern is in some way sym-
bolic or abstract, and second, a spotted coat phenotype is, at least
by many researchers, considered unlikely for Paleolithic horses.
“
horsey
”
|
|
ancient DNA
transient receptor potential cation channel subfamily M1
|
|
single nucleotide polymorphism
leopard complex spotting
Franco-Cantabrian region
P
rehistoric representations of animals have the potential to
provide rst-hand insights into the physical environment that
humans encountered thousands of years ago and the phenotypic
appearance of the animals depicted. However, the motivation
behind, and therefore the degree of realism in, these depictions
is hotly debated and it has yet to be shown to what extent they have
been executed in a naturalistic manner. Neuropsychological ex-
planations include
in which an internally gen-
erated image is perceived in external space (1), whereas others
have argued for shamanistic signi
“
hyperimagery,
”
cance (2) or simply art for
art
’
s sake (3). Some paleontologists argue that cave paintings are
re
ections of the natural environment of humans at the time (4),
but not all researchers agree with this opinion (5).
Exact numbers of Upper Paleolithic sites with animal depic-
tions are uncertain because of ongoing debates regarding the
taxonomic identication of some images and the dating of some
(e.g., ref. 6). However, art of this period has been identied in at
least 40 sites in the Dordogne
–
Périgord region, a similar number
in coastal Cantabria [although Bicho and coauthors (7) argue for
many more sites], and around a dozen sites in each of the Ardèche
Author contributions: M.P., N.B., M.R., and A.L. designed research; M.P., M.C., M.R., and
M.H. performed research; R.B. contributed new reagents/analytic tools; M.P., N.B., E.S.-C.,
T.K., A.M.-M., M.R., and A.L. analyzed data; and R.B., T.O., M.H., and A.L. wrote the paper.
The authors declare no con
ict of interest.
*This Direct Submission article had a prearranged editor.
1
To whom correspondence may be addressed. E-mail: l
udwig@izw-berlin.de
or
msh503@
york.ac.uk
.
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1108982108/-/DCSupplemental
.
www.pnas.org/cgi/doi/10.1073/pnas.1108982108
PNAS Early Edition
|
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assuming a large panmictic population, the 95% con
dence
interval (C.I.) for the actual allele frequency in the ancient horse
population is 0.082
0.418. Therefore, the true allele frequency
was most likely above 0.1. In contrast, we did not detect LP
in
–
ve predomestic Asian samples from the Pleistocene. In
postglacial times (i.e., after 11,700 y ago), our sample set of
predomestic horses is geographically patchy because some peri-
ods are characterized by an absence of horse remains for some
regions. The leopard complex spotted allele was not detected in
the six Iberian remains dating to the Mesolithic, but 2 of 10 of the
predomesticated, postglacial horses from Eastern Europe carried
the allele associated with leopard complex spotting (Table 1).
Fig. 1. Map of key locations of Paleolithic cave art containing horse
paintings. The Franco–Cantabrian region containing most of the Paleolithic
cave paintings is highlighted.
Discussion
The results of this study bear directly on debates concerning the
nature of Paleolithic representations of animals, specically
whether these depictions constitute literal representations of
phenotypic variation in the contemporary animal populations or
not. We found evidence for a long-term existence of the leopard
complex spotted phenotype in the European horse population.
So far, LP is the only spotted phenotype that has been found
in both predomestic and domestic horses. In addition to the LP
horses and in striking agreement with Paleolithic cave paintings,
only bay (e.g., Lascaux, France) and black (e.g., Chauvet,
France) genotypes were discovered in predomestic wild horse
remains (21), whereby bay seems to be the most common color
phenotype in predomestic times (18 of 31 typed samples so far)
and is also the most commonly painted phenotype. So far, no
evidence has been found for horses with chestnut, white, diluted,
or other spotted phenotypes in predomestic times (this study and
ref. 21). Only a single chestnut allele of MC1R was discovered
in a sample from Pietrele, Romania (6,300 yBP) (Table 1). It is
likely that dun dilution was present in predomestic horses as it is
for example in modern Przewalski horses. However, because the
dun mutation has not yet been identi
However, the spotted horses depicted at Pech-Merle closely
resemble the leopard complex spotting (LP) seen in some
modern horse breeds. Leopard complex spotting is characterized
by white spotting patterns that range from horses having a few
white spots on the rump to horses that are almost completely
white. The white area of these horses can also have pigmented
oval spots known as
“
leopard spots
”
(15) after which one of the
specic phenotypes (
“
leopard
”
) was named. Today, leopard is a
popular phenotype in several horse breeds, including Knab-
strupper, Appaloosa, and Noriker. Leopard complex spotting is
caused by an incompletely dominant locus (LP) located on horse
chromosome 1 (15, 16). Modier genes are thought to be respon-
sible for the variation in the amount of white patterning observed
(15, 17). In the Appaloosa and Miniature horse breeds, homo-
zygosity for LP has been associated with congenital stationary
night blindness (CSNB). Horses with this disorder have problems
with seeing at low-light conditions and the retinal rod pathway of
vision is disrupted as shown by the diagnostic negative electro-
retinograph (ERG) (18, 19). Therefore, CSNB should have been
under negative selection in wild horses. Recently, a single SNP in
the TRPM1 gene (ECA1:108,249,293C
>
T) was found to be
linked to both LP and CSNB in Appaloosa horses (20).
So far, ancient DNA studies have produced evidence for bay
and black horses only, whereas no evidence for white spotted
phenotypes in predomestic horses has been found (21). Here we
test the possibility that the leopard complex spotting phenotype
was already present in horses and accurately depicted by their
human contemporaries, nearly 25,000 y ago. To investigate
whether LP spotting was present in ancient horses, we genotyped
the associated SNP in predomestic horses from Siberia, Western
and Eastern Europe, and the Iberian Peninsula.
ed, we cannot distinguish
between dun and nondun horses at the moment.
Our previous ancient DNA study of coat coloration in pre-
domestic horses produced evidence that the only phenotypes
present in ancestral, predomestic horse populations were bay
and black (21). Today, bay
–
dun is still found in the Przewalski
horse (Equus ferus przewalskii), which is listed as the last
remaining wild horse by the International Union for Conserva-
tion of Nature (IUCN) and often discussed as a close relative of
domestic horses (22), although its taxonomic status is contro-
versial and there is genetic evidence for admixture between
Przewalski and domesticated horses (e.g., ref. 23). Recently,
studies of both maternal (mtDNA) (24
–
25) and paternal lineages
(Y chromosomal DNA) (26) found that the Przewalski horse
displays DNA haplotypes not present in modern or ancient do-
mestic horses, suggesting that Przewalski horses are not directly
ancestral to modern domestic horses. However, independently of
its taxonomic status, several lines of evidence suggest that the
bay phenotype of the Przewalski horse represents an ancestral
character. Firstly, several wild ass species, which undoubtedly
represent wild equids, also show a bay
Results
The samples investigated for the SNP associated with leopard
complex spotting have previously (21) been genotyped for eight
coat-color SNPs in six genes (MC1R, ASIP, SILV, MATP,
EDNRB, and KIT) including basic coat colors (bay, black, and
chestnut), dilution phenotypes (cream and silver), and white
spottings (tobiano, sabino, and overo) (Table 1). All samples that
had previously been successfully typed could also be typed for
the TRPM1 SNP. We were able to type 31 predomestic samples
for the TRPM1 SNP and identied the LP-associated allele in
6 samples, all of which were heterozygous and thus not affected
by CSNB. The tobiano and sabino alleles were previously iden-
tied in 8 and 3 ancient domestic samples, respectively (21), but
never in predomestic or wild horses. Thus, leopard spotting is the
only spotted phenotype found in predomestic horses to date.
Four of 10 of the Western European horses from the Pleisto-
cene had a genotype indicative of the leopard complex pheno-
type (Fig. 2), suggesting that this phenotype was not rare in
Western Europe during the Pleistocene. For our sample size and
dun phenotype; and
secondly, horses of this phenotype are depicted in remarkable
detail in Paleolithic cave paintings (e.g., in Chauvet). Whereas
black or black
–
dun and leopard spotted phenotypes also oc-
curred at measurable frequencies in Pleistocene and Copper Age
wild horses, as shown by both contemporary depictions and our
genotyping results, their absence in modern Przewalski
–
’
s horses
is probably explained by the severe population bottleneck that
they have undergone (27), possibly in combination with the
Asian origin of these horses, where LP seems to have been rarer,
if not entirely absent.
Most modern populations of wild animals display uniform
coloration, whereas domesticated species show a remarkable
variation in coat color (28). Most scientists believe that changes
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www.pnas.org/cgi/doi/10.1073/pnas.1108982108
Pruvost et al.
Table 1. Genotyping results for nine coat-color loci in the 31 ancient DNA samples
Basic
color
Age
Region
Sample
Spotting Dilution ASIP EDNRB
KIT13
KIT16 MATP MC1R SILV9 SILV11 TRPM1
Pleistocene
Siberia
SP1181A Bay
No
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
SP1181B Bay
No
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
SP1181C Bay
No
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
SP1181E Bay
No
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
SP1181F Bay
No
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
Europe
PET6
Bay
Leopard
complex
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
LP/lp
PET5
Bay
Leopard
complex
No
A/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
LP/lp
PET3
Bay
No
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
PET2
Bay
No
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
PET1
Bay
Leopard
complex
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
LP/lp
KG5
Bay
Leopard
complex
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
LP/lp
KG4
Bay
No
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
KG3
Bay
No
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
KG2
Bay
No
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
KG1
Bay
No
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
Mesolithic-Neolithic Iberian Peninsula
44
Bay
No
No
A/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
3
Black
No
No
a/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
31
Bay
No
No
A/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
32
Black
No
No
a/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
34
Black
No
No
a/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
37
Bay
No
No
A/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
Copper Age
Europe
Spa1
Bay
No
No
A/A ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
PIE9
Black
No
No
a/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/e
z/z
z/z
lp/lp
VIT4
Black
No
No
a/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
MAY3
Bay
No
No
A/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
MAY5
Bay
No
No
A/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
MAY6
Bay
No
No
A/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
MAY7
Bay
Leopard
complex
No
A/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
Lp/lp
MAY10
Bay
Leopard
complex
No
A/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
LP/lp
CAS1
Black
No
No
a/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
MOL5
Black
No
No
a/a
ov/ov KM0/KM0 sb1/sb1
C/C
E/E
z/z
z/z
lp/lp
The described phenotype is proposed on the basis of the genotypes at each of the nine loci tested, including basic coat-color (bay, black, or chestnut)
dilution (cream or silver), and spotting pattern (leopard complex, tobiano, sabino, or overo). Gray shading indicates that at least one allele differs from the
proposed wild type of the Przewalski horse.
in coat color and speci
cally an increase in coat-color variability
are a direct consequence of the domestication (28, 29). Previous
work by us supports this notion by demonstrating a comparative
lack of coat-color variation in predomestic horses and an ex-
plosion of color patterns during and following the Iron Age (21).
Although our results presented here may, at
ulations compared with their modern counterparts (30
32), and
it is likely that this increased variability extended to color phe-
notypes as well. However, the overall picture still supports the
notion that arti
–
cial selection was the driving force behind the
rapid increase of coat-color variation in domestic animals and
resulting in their remarkable modern variability.
Recently it was discovered that homozygosity for the leopard
spotted SNP typed in this study is associated with congenital
stationary night blindness in leopard spotted horses (18
rst glance, seem to
contradict this pattern, the general picture of increased pheno-
typic variability in early domestic horses compared with their
wild ancestors holds up, also in light of our recent results. In-
cluding the results in the current study, we have so far found 3
coat-color phenotypes in predomestic horses and 11 in early
domestic horses. Predomestic horses inhabited, in vast numbers,
large areas of Eurasia, and some extant species that still occupy a
similarly large area, such as gray wolves, are also found in dif-
ferent color morphs. It is therefore not entirely surprising that
not all wild horses shared the bay
–
dun or black
–
dun phenotypes.
Moreover, previous studies suggested that morphological
–
20),
which should have caused strong purifying selection against ho-
mozygote LP individuals in predomestic times. Nevertheless, we
found several occurrences of the LP allele in Pleistocene and
Copper Age samples. Although we can only speculate about
potential processes that resulted in the Pleistocene frequency of
the leopard phenotype, such as selective advantage due to
camouage in the snowy Pleistocene environment, sexual selec-
tion, or simply genetic drift, the reason why it did not disappear
due to the CSNB after it had been established seems to be less
and
genetic
—
variability was much larger in Pleistocene animal pop-
—
Pruvost et al.
PNAS Early Edition
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Fig. 2. Horse phenotypes found in Paleolithic artwork from caves in Lascaux (bay) (photo from N. Aujoulat from the Ministère de la Culture et de la Com-
munication, France. The animal corresponds to the second horse from the “Panel of the Chinese horses.”); Chauvet (black) [The picture is showing a panel of
horses (detail L., about 1.10 m). The photo (slide no. 12) is used with permission from the French Ministry of Culture and Communication, Regional Direction for
Cultural Affairs, Rhône-Alpes region, Regional Department of Archeology], and Pech-Merle (
“
leopard
”
spotted) (photo from P. Cabrol ©, Centre de Préhistoire
du Pech Merle. The picture shows the panel of the dappled horses
—“
Le panneau des Chevaux ponctués
”
, Cabrerets, Lot France), all France, and their genetic
s horse (genotype: A
A
/
E
E
/
C
C
/C
C
CH
ch
/CH
ch
D
D
/
LP
lp
/LP
lp
Z
z
/Z
z
); black
counterparts in modern horses. (Left to Right) Bay
–
dun Przewalski
’
−
−
−
–
dun Konik with
winter coat (genotype: A
a
/A
a
E
E
/
C
C
/C
C
CH
ch
/CH
ch
D
D
/
LP
lp
/LP
lp
Z
z
/Z
z
); black
−
−
–
dun Konik with summer coat (same genotype); and leopard complex spotted
Knabstrupper (genotype: A
a
/A
a
E
E
/
C
C
/C
C
CH
ch
/CH
ch
D
d
/D
d
LP
LP
/LP
lp
Z
z
/Z
z
).
−
obscure. Considering that deleterious alleles may stay for a long
time in a population at low frequency despite purifying selection,
the fact that the frequency of the LP allele was comparatively
low in the Holocene and the Copper Age samples could indicate
that its low frequency protected the allele from being purged
from the population.
Our results suggest that, at least for wild horses, Paleolithic
cave paintings, including the remarkable depictions of spotted
horses, were closely rooted in the real-life appearance of the
animals. Therefore, any interpretation of those depictions from a
symbolic or transcendental standpoint will necessarily need to
draw upon data other than the coat pattern itself to back up its
argument. This point has been made previously (33), as it has
been shown that spot motifs on reindeer (Rangifer tarandus)
depictions from Upper Paleolithic art from France are a natu-
ralistic representation of a speci
with a singleplex PCR as previously described (21). PCR products varied in
length between 52 and 78 bp (including primers) (
Table S3
). Four microliters
of extract was used for each multiplex PCR. Negative extraction controls and
negative PCR controls were used in each PCR. Ampli
cation products were
visualized on agarose gels.
Authentication. DNA sampling, extractions, and pre-PCR preparations were
carried out in a laboratory dedicated to ancient DNA analyses following the
standard procedures to avoid contamination. The multiplex and singleplex
PCRs were set up in the laboratory dedicated to ancient DNA analyses, but the
dilution of PCR products following the multiplex step and their addition to
the singleplex reactions were done in a dedicated room in an annexed
building, separate from the post-PCR laboratory, where all post-PCR analyses
were carried out. All results were replicated at least four times. Two different
primer pairs were used to detect the point mutation in the TRPMI gene
associated with the leopard spotting phenotype (
Table S4
)
. Both primer
pairs are designed for the pyrosequencing technology. Two negative PCR
controls and a blank extraction were performed for every sample. Due to
the small size of the ampli
c coat pattern found only in
females, and thus, perhaps, a deliberate indicator of the sex of
individual animals. Here, we are able to go one step further by
conrming the prehistoric occurrence of the genotype that
underlies a distinctive phenotype in Paleolithic cave art. Our
results suggest that, at least in some cases, prehistoric paintings
were closely rooted in the real-life appearance of the animals
depicted and that any symbolic or transcendental connotation, if
present at all, was not necessarily signaled by the color or pattern
of these depictions.
ed fragments, distinction between primer dimers
and positive products is sometimes dif
cult. Therefore, all negative controls
were systematically sequenced when a product was detected on the agarose
gel. All of these products found in negative controls turned out to be primer
dimers. Finally, each sample was con
rmed at least once in a second labo-
ratory also dedicated to ancient DNA analyses (
Table S1
).
Pyrosequencing. Biotinylated PCR products were prepared on the PyroMark
Vacuum Prep Workstation according to the manufacturer
s instructions.
Amplicons for each SNP were sequenced using pyrosequencing technology
on a PSQTM 96MA (Biotage). The SNPs were identi
’
ed using the PSQTM
96MA system and automatically edited by the PSQTM 96MA SNP software.
The results for the color determination, including the previous determination
of other color phenotypes (21), are summarized in Table 1 and
Table S4
.
Materials and Methods
Samples. We genotyped successfully 31 (of 69) horse (Equus caballus) bone
and teeth specimens from 14 different localities from Siberia, Eastern and
Western Europe, and the Iberian Peninsula (
Table S1
). The specimens cover
a period from the Late Pleistocene to the Copper Age and are all dated ei-
ther by the archaeological context or with
14
C dates (
Table S2
)
. All samples
were previously genotyped for eight coat-color loci in six genes (Table 1) (21).
Allelic Dropout. The probability (P) of a false heterozygote individual is cal-
culated after n replicates: P = K
1, where K is the observed number
of allelic dropouts divided by all heterozygous individuals. For all genes we
did a minimum of four replications that reduced the risk of nondetection of
a heterozygote individual to an average of 0.3%.
×
(K/2)n
−
Ancient DNA Extraction and Ampli
cation. Approximately 250 mg of bone
material was used for each extraction. External surfaces of bones were re-
moved by abrasion to minimize environmental contaminations. Each sample
was ground to powder with a freezer mill and incubated in 0.45 M EDTA
(pH 8.0) and 0.25 mg/mL proteinase K overnight at room temperature under
rotation. After centrifugation, DNA was puri
Estimating the Allele Frequency of Missed Alleles. We computed the upper
bound of the LP allele frequency having been present but not observed in
our samples assuming a binomial sampling distribution (
Table S5
;
for details,
see ref. 21).
ed from the supernatant using
a silica-based method as previously described (34, 35). Leopard complex
spotting primers were designed on the basis of the associated SNP previously
reported (20) and added to our primer set detecting coat-color SNPs.
Ampli
cations were performed in two steps using multiplex PCR combined
ACKNOWLEDGMENTS. We thank Thomas Hackmann, who provided the
photograph of the leopard spotted horse; Gloria Maria Gonzales Fortes for
reproducing a sample in York, United Kingdom; the Ministère de la Cul-
4of5
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www.pnas.org/cgi/doi/10.1073/pnas.1108982108
Pruvost et al.
ture et de la Communication, France for permission to use the photo-
graphs of the cave paintings; Serge Roussel, Bertrand Defois, Jean-Michel
Geneste, and Alain Lombard for support to get the permission for the
photographs of horse paintings from French caves; and the anonymous
reviewers and the communicating editor for improving our manuscript
with their comments. We also thank all archeologists and museums that
provided samples for DNA analysis. This study was supported by the Deut-
sche Forschungsgemeinschaft (LU 852/7-3).
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