www.sciencemag.

org/cgi/content/full/324/5926/485/DC1

Supporting Online Material for

Coat Color Variation at the Beginning of Horse Domestication

Arne Ludwig,* Melanie Pruvost, Monika Reissmann, Norbert Benecke, Gudrun A.
Brockmann, Pedro Castaños, Michael Cieslak, Sebastian Lippold, Laura Llorente, Anna-
Sapfo Malaspinas, Montgomery Slatkin, Michael Hofreiter*
*To whom correspondence should be addressed. E-mail: ludwig@izw-berlin.de (A.L.);
hofreiter@eva.mpg.de (M.H.)

Published 24 April 2009, Science 324, 485 (2009)

DOI: 10.1126/science.1172750

This PDF file includes:

Materials and Methods

Figs. S1 to S3
Tables S1 to S8
References

1
Supplementary online material

Materials and Methods

Samples
Samples were obtained from Siberia, Middle and Eastern Europe, China and the Iberian
Peninsula from different time points and cultural horizons (Table S1).
Ancient DNA Extraction and Amplification
DNA was extracted from 152 samples using between 250 mg and 400 mg bone material.
External surfaces of bones were removed 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 for 5 min at 4,000 rpm in a Universal 320 centrifuge
(Hettich), DNA was purified from the supernatant using a silica based method as
previously described (S1).
SNP amplifications were performed using multiplex PCR (S2, S3). PCR products for
SNPs varied in length between 52 bp and 78 bp (including primers; see Table S3). Four
microliters of extract were used for each multiplex PCR. The initial multiplex PCR was
performed in a 20 µl reaction volume containing 1x AmpliTaq Gold PCR buffer II (ABI),
4 mM MgCl2, 1 mg/ml Bovine Serum Albumin (BSA), 250 µM of dATP, dCTP and
dGTP, 500µM of dUTP, 150 nM of each primer and 2 U of AmpliTaq Gold (ABI). For
each sample, one PCR was performed by adding 1 U of heat-labile Uracil-DNA
Glycosylase (USB) and an initial incubation step of 15 min at 37 °C to control for carry-
over contamination. PCR products were diluted 1/30 and 5 µl (total reaction volume 20
µl) were used for the next step. Singleplex PCRs contained 1x AmpliTaq Gold PCR
Buffer II, 4 mM MgCl2, 1 mg/ml bovine serum albumin (BSA), 250 µM of dATP, dCTP
and dGTP, 500µM of dUTP, 1.5 µM of each primer and 0.5 U of AmpliTaq Gold DNA
polymerase. Both times, PCR was run under the following conditions: denaturation and
Taq activation at 94 °C for 9 min; 30 up to 35 cycles at 95 °C 20 sec; annealing
temperature depending on the primer pair (see Table S3) 30 sec; 72 °C 30 sec and final
extension 4 min 72 °C. Negative extraction controls and negative PCR controls were
used in each PCR. Amplification products were visualized on agarose gels.
Mutation analysis
Modern horses show a high variability in coat coloration. However, no color is confined
to a single breed and the same mutation is responsible for a certain color variant across
breeds, supporting the idea that mutations producing the color variants occurred prior to
breed formation during the domestication process. Unfortunately, it is currently not
possible to describe the coat color phenotype of ancient horses completely as until today
not all genes associated with coat coloration were identified. Thus for some color
phenotypes of interest (e.g. dun), there is as yet no genetic information available. We
used a set of eight SNPs in six genes (Table S2) for detecting basic coat colors (bay,
black and chestnut), two kinds of dilution (silver and cream) and three spotted or painted
colorations (overo, tobiano and sabino). SNPs were chosen according to previous studies
on modern horse populations (S4-S9); primers are listed in Table S3. Biotinylated PCR
products were prepared at the PyroMark Vacuum Prep Workstation according to the
manufacturer’s instructions. Amplicons for each SNP were sequenced using

2
pyrosequencing TM technology on a PSQTM 96MA (Biotage). The SNPs were identified
using PSQTM 96MA and automatically edited by the PSQTM 96MA SNP software. Due
to the large deletion (11bp) of the mutated non–black allele (A) of ASIP, the risk to lose
the mutated allele is relatively high for ancient DNA. For this reason, the number of
replications (n=6) was increased in order to reduce the risk of false homozygote
individuals below 1‰. Additionally, allelic separation of ASIP was verified on page gels.
The results for the color determination are summarized in Tables S4 and S5.
Allelic dropout
The probability P of a false heterozygote individual is calculated after n replicates: P = K
x (K/2)n-1 where K is the observed number of allelic dropouts divided by all heterozygous
individuals (S10). For all genes excepting ASIP we did a minimum of four replications
which reduced the risk of non-detection of a heterozygote individual to an average of 0.3
% (P = 0.0078 for KIT13, 0.0015 for KIT16 and MATP, and 0.00012 for MC1R).
Estimating the allele frequency of missed alleles
We computed the upper bound of the allele frequency of a coat color allele having been
present in the pre-domestication population but not observed in our samples assuming a
binomial sampling. Given n samples, we computed the maximum frequency f5% of a
color allele in the population so that the probability of sampling none of them is above
5% (Table S7).
Estimation of selection coefficient
We estimated the selection coefficient for each locus using Bollback et al.’s method
(S11). This method has been developed to jointly estimate the selection coefficient and
the effective population size from time-series data of allele frequencies. We binned the
Siberian East/Central European and Chinese samples into five time periods with means
about 13,100, 3,700, 2,800, 600 and 200 BC respectively. We assumed a generation time
of five years.. We used default values for all parameters of the program except for the
grid size (Ngrid) of the allelic frequencies that was set to 2000. The likelihood was
computed for selection coefficient ranging from -0.5 to 0.5 and effective population size
ranging from 10,000 to 100,000. All the loci considered are assumed to be independent,
in particular we considered only SILV9 because SILV11 seems to be in complete linkage
disequilibrium with SILV9. See Figure S3 for the change in allelic frequency for each
locus and Table S8 for the estimates of the selection parameter.
Note that, for our dataset, the maximum likelihood values are sensitive to the grid size
but that the confidence intervals seem to be robust to this parameter. Therefore we used a
bigger grid than the one suggested in (S11).
Like the above estimate of maximum allele frequencies of missed alleles, these analyses
are limited by the available sample size. This is certainly also true for our sampling of
color variations for early domesticated horses. Thus, it is possible that we missed
extremely rare variants at a certain point in time. However, detection of additional color
variants at the beginning of domestication would only strengthen our conclusions but not
change the overall picture.

3
Supplementary figures

Figure S1: Map of Eurasia showing the archaeological sites (black dots) from which the
horse samples that were successfully analyzed for SNPs originate. The diagrams show
the proportion of the different coat color phenotypes observed. Numbers show the total
number of samples showing the indicated phenotype. From top to bottom: samples from
the Pleistocene; the Neolithic/Copper Age; the Bronze Age; the Iron Age.

4
5
Fig. S2: Timeline for the first occurrence of the observed coat color phenotypes in
Siberia, Europe and the Iberian Peninsula, respectively. Above the timescale, the timing
of the first depictions of various horse-related implements is shown. Note that the
timeline is interrupted between 12,000 and 5,000 BC.

6
Figure S3: Change in allelic frequency for each locus. The number of chromosomes for
each time point is 22, 20, 20, 36, 38 from left to right (see Table S5). The earliest
Pleistocene samples were excluded. Note that for two genes (ASIP and MC1R), the allele
frequencies change drastically.

7
Supplementary tables
Table S1: Samples analyzed for this study. The samples highlighted in grey are the
samples that gave a complete and reproducible genotype for the coat coloration (Typing).
The extraction and amplification of the samples (Ext/Amp) and the reproduction (Rep)
were performed in two different institutes by Melanie Pruvost (MP) and Michael
Cieslack (MC) at the Humboldt University in Berlin and Sebastian Lippold (SL) and
Melanie Pruvost (MP) at the Max Planck Institute in Leipzig. The Pleistocene samples
are not directly dated, but estimated from context should be around 20,000 years old. The
remaining dates are either calibrated carbon dates or derived from archaeological context.

Siberia
Geographical
Sample Ext/Amp Rep. Det. Excavation Date Typing
location
SP1181A MP/MP W(ild) Maliy Lyakhovsky Isl. North Siberia Pleistocene Yes
SP1181B MP/MP W Bol'shoy Lyakhovsky Isl. North Siberia Pleistocene Yes
PLEISTOCENE

SP1181C MP/MP W Bol'shoy Lyakhovsky Isl. North Siberia Pleistocene Yes

SP1181D MP/MP W Bol'shoy Lyakhovsky Isl. North Siberia Pleistocene No
SP1181E MP/MP W Oyagosskiy Yar, Kondrat'evo R., mouth Siberia Pleistocene Yes
SP1181F MP/MP W Kotel'niy Isl., Anisiy Cape Siberia Pleistocene Yes
BER 001 MP/MP D(om) Denisova‐Pescera Siberia (Altai) 3000BC Yes
TAR 001 MP/MP SL D Tartas1 West Siberia 2500‐3000BC Yes
TAR 002 MP/MP SL D Tartas1 West Siberia 2500‐3000BC Yes
TAR 004 MP/MP D Tartas1 West Siberia 2500‐3000BC Yes
EARLY BRONZE AGE

TAR 005 MP/MP SL D Tartas1 West Siberia 2500‐3000BC Yes

TAR 006 MP/MP D Tartas1 West Siberia 2500‐3000BC No
TAR 007 MP/MP SL D Tartas1 West Siberia 2500‐3000BC Yes
TAR 008 MP/MP D Tartas1 West Siberia 2500‐3000BC Yes
TAR 009 MP/MP D Tartas1 West Siberia 2500‐3000BC No
TAR 010 MP/MP D Tartas1 West Siberia 2500‐3000BC Yes
TAR 011 MP/MP D Tartas1 West Siberia 2500‐3000BC Yes
BAR 002 MP/MP D Preobrazhenka‐‐/2005 Siberia 2000‐2500BC No
BER 002 MP/MP SL D Om‐1 Siberia (Altai) 900BC Yes
Arz 1‐2 CW/MP D Arzan1 South Siberia (Tuva) 800BC Yes
Arz 1‐3 CW/MP D Arzan1 South Siberia (Tuva) 800BC Yes
Arz 2‐1 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes
Arz 2‐2 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes
IRON AGE

Arz 2‐3 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes

Arz 2‐4 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes
Arz 2‐5 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes
Arz 2‐6 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes
Arz 2‐7 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes
Arz 2‐8 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes
Arz 2‐9 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes

8
 Arz 2‐10 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes
Arz 2‐11 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes
Arz 2‐12 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes
Arz 2‐13 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes
Arz 2‐14 CW/MP MP D Arzan2 South Siberia (Tuva) 619‐608 BC Yes
Bars1A CW/MP D Barsucij Log South Siberia (Tuva) 370‐150 BC Yes
Bars1B CW/MP D Barsucij Log South Siberia (Tuva) 400‐200 BC Yes
OKG 001 MP/MP D Olon‐Kurin‐Gol 10 Siberia (Mongolia) 400‐300 BC Yes
OKG 002 MP/MP D Olon‐Kurin‐Gol 10 Siberia (Mongolia) 400‐300 BC Yes
OKG 003 MP/MP SL D Olon‐Kurin‐Gol 10 Siberia (Mongolia) 400‐300 BC Yes

East European steppe

Sample Ext/Amp Rep. Det. Excavation Geographical location Date Typing
PET1 SL/MP W Petersfels South Germany 14000‐11000 BC Yes
PET2 SL/MP W Petersfels South Germany 14000‐11000 BC Yes
LATE GLACIATION‐MESOLITHIC

PET3 SL/MP W Petersfels South Germany 14000‐11000 BC Yes

PET5 SL/MP MP W Petersfels South Germany 14000‐11000 BC Yes
PET6 SL/MP W Petersfels South Germany 14000‐11000 BC Yes
Kg1 SL/MP W Kniegrotte Germany (Thuringia) 15000‐14000 BC Yes
Kg2 SL/MP MP W Kniegrotte Germany (Thuringia) 15000‐14000 BC Yes
Kg3 SL/MP W Kniegrotte Germany (Thuringia) 15000‐14000 BC Yes
Kg4 SL/MP W Kniegrotte Germany (Thuringia) 15000‐14000 BC Yes
Kg5 SL/MP W Kniegrotte Germany (Thuringia) 15000‐14000 BC Yes
Spa 1 CW/MP SL W Span‐Koba Ukraine (Peninsula Crimea) 9390‐9210 BC Yes
TRE1 MP/MP W Trestiana Romania 5700‐5600 BC No
TRE2 MP/MP W Trestiana Romania 5700‐5600 BC No
TRE3 MP/MP W Trestiana Romania 5700‐5600 BC No
BUP1 MP/MP W Bucsani Pod Romania 5500‐5000 BC No
ISA1 MP/MP W Isaia Romania 5500 BC No
ISA2 MP/MP W Isaia Romania 5500 BC No
HAR1 MP/MP W Harsova Romania 5000‐5500 BC No
ENEOLITHIC

HAR2 MP/MP W Harsova Romania 4500‐2000 BC No

HAR3 MP/MP W Harsova Romania 4500‐2000 BC No
Pie7 MP/MP W Pietrele Romania 4300 BC No
Pie9 MP/MP W Pietrele Romania 4300 BC Yes
Pie11 MP/MP W Pietrele Romania 4300 BC No
VIT1 MP/MP W Vitanesti Romania 4300‐4220 BC No
VIT2 MP/MP W Vitanesti Romania 4350‐4220 BC Yes
VIT3 MP/MP W Vitanesti Romania 4300‐4220 BC No
VIT4 MP/MP W Vitanesti Romania 4360‐4220 BC Yes
ORL1 MP/MP W Orlovka Moldova 4000 BC No

9
 ORL2 MP/MP W Orlovka Moldova 4000 BC No
ORL3 MP/MP W Orlovka Moldova 4000 BC No
ORL4 MP/MP W Orlovka Moldova 4000 BC No
CAS1 MP/MP W Cascioarele Romania 3700‐3380 BC Yes
MAY1 MP/MP W Mayaki Ukraine 3600‐3100 BC No
MAY2 MP/MP W Mayaki Ukraine 3600‐3100 BC No
MAY3 MP/MP W Mayaki Ukraine 3640‐3490 BC Yes
MAY4 MP/MP W Mayaki Ukraine 3600‐3100 BC No
MAY5 MP/MP W Mayaki Ukraine 3250‐3100 BC Yes
COPPER AGE

MAY6 MP/MP W Mayaki Ukraine 3520‐3330 BC Yes

MAY7 MP/MP W Mayaki Ukraine 3520‐3380 BC Yes
MAY8 MP/MP W Mayaki Ukraine 3600‐3100 BC No
MAY9 MP/MP W Mayaki Ukraine 3600‐3100 BC No
MAY10 MP/MP W Mayaki Ukraine 3650‐3500 BC Yes
MOL5 MP/MP W? Molyukhov Bugor Ukraine 3720‐3630 BC Yes
MOL7 MP/MP W? Molyukhov Bugor Ukraine 3720‐3630 BC No
MOL8 MP/MP W? Molyukhov Bugor Ukraine 3720‐3630 BC No
GRO8 MP/MP D Großobringen Germany 3000‐2500 BC No
GRO9 MP/MP D Großobringen Germany 3000‐2500 BC No
GRO10 MP/MP D Großobringen Germany 3000‐2500 BC No
GRO11 MP/MP D Großobringen Germany 3000‐2500 BC Yes
Gar1 MP/MP D Garbovat Romania 1500‐1000 BC No
Gar2 MP/MP D Garbovat Romania 1500‐1000 BC Yes
Gar3 MP/MP D Garbovat Romania 1500‐1000 BC Yes
Gar4 MP/MP D Garbovat Romania 1500‐1000 BC Yes
BRONZE AGE

Bar1 MP/MP D Garbovat Romania 1500‐1000 BC No

Bar3 MP/MP D Garbovat Romania 1500‐1000 BC No
Lch 1 CW/MP D Lchashen Armenia 1410‐1250 BC Yes
Lor 1 CW/MP D Lori‐Berd North Armenia 1950‐1750 BC Yes
Mic1 MP/MP D Miciurin Moldova 1500‐1000 BC Yes
Mic2 MP/MP D Miciurin Moldova 1500‐1000 BC Yes
Mic3 MP/MP D Miciurin Moldova 1500‐1000 BC Yes
Mic4 MP/MP D Miciurin Moldova 1500‐1000 BC Yes
Mic5 MP/MP D Miciurin Moldova 1500‐1000 BC Yes
Shi 1 CW/MP SL D Shirakavan Armenia 895‐795 BC Yes

China
Sample Ext/Amp Rep. Det. Excavation Geographical location Date Typing
Fen 1 CW/MP D Fengtai China (Qinghai) 905‐800 BC Yes
IRON
AGE

Fen 2 CW/MP D Fengtai China (Qinghai) 1000‐800 BC Yes

10
 Fen 3 CW/MP D Fengtai China (Qinghai) 1000‐800 BC Yes
Fen 4 CW/MP D Fengtai China (Qinghai) 1000‐800 BC Yes

Spain
Sample Extraction Rep. Det. Excavation Geographical location Date Typing
41 MC/MC SL W Atxoste Iberian Peninsula (Spain) 5500‐4950 BC No
42 MC/MC SL W Atxoste Iberian Peninsula (Spain) 5500‐4950 BC No
43 MC/MC SL W Atxoste Iberian Peninsula (Spain) 5500‐4950 BC No
44 MC/MC SL W Atxoste Iberian Peninsula (Spain) 5500‐4950 BC Yes
45 MC/MC SL W Atxoste Iberian Peninsula (Spain) 5500‐4950 BC Yes
MESOLITHIC‐NEOLITHIC

1 MC/MC W Cueva Fosca ‐Valencia‐Cartellon Iberian Peninsula (Spain) 5200 ‐4900BC No

2 MC/MC W Cueva Fosca ‐Valencia‐Cartellon Iberian Peninsula (Spain) 5200 ‐4900BC No
3 MC/MC W Cueva Fosca ‐Valencia‐Cartellon Iberian Peninsula (Spain) 5200‐4900 BC Yes
31 MC/MC W Cueva Fosca ‐Valencia‐Cartellon Iberian Peninsula (Spain) 5210‐4910 BC Yes
32 MC/MC W Cueva Fosca ‐Valencia‐Cartellon Iberian Peninsula (Spain) 5220‐4980 BC Yes
33 MC/MC W Cueva Fosca ‐Valencia‐Cartellon Iberian Peninsula (Spain) 5220‐4900 BC No
34 MC/MC W Cueva Fosca ‐Valencia‐Cartellon Iberian Peninsula (Spain) 5220‐4900 BC Yes
35 MC/MC W Cueva Fosca ‐Valencia‐Cartellon Iberian Peninsula (Spain) 5380‐5210BC No
36 MC/MC W Cueva Fosca ‐Valencia‐Cartellon Iberian Peninsula (Spain) 5070‐4840BC No
37 MC/MC W Cueva Fosca ‐Valencia‐Cartellon Iberian Peninsula (Spain) 5210‐4910 BC Yes
17 MC/MC SL W Cueva De La Vaquera‐Segovia Iberian Peninsula (Spain) 5210‐4940 BC Yes
27 MC/MC D El Caprichio‐Madrid Iberian Peninsula (Spain) 4300‐2200 BC No
COPPER AGE

28 MC/MC D Carmona‐Sevillia/Andalusia Iberian Peninsula (Spain) 4300‐2200 BC No

19 MC/MC SL D Las Pozas ‐Zamora Iberian Peninsula (Spain) 4300 ‐2200 BC No
20 MC/MC SL D Cueva Rubia‐Valmayor/Madrid Iberian Peninsula (Spain) 2880‐2570 BC No
21 MC/MC SL D Cueva Rubia‐Valmayor/Madrid Iberian Peninsula (Spain) 2900‐ 2500 BC No
22 MC/MC SL D Cueva Rubia‐Valmayor/Madrid Iberian Peninsula (Spain) 1350 BC Yes
23 MC/MC D Cueva Rubia‐Valmayor/Madrid Iberian Peninsula (Spain) 1350 BC No
24 MC/MC D Cueva Rubia‐Valmayor/Madrid Iberian Peninsula (Spain) 1350 BC Yes
25 MC/MC D Cueva Rubia‐Valmayor/Madrid Iberian Peninsula (Spain) 1350 BC Yes
26 MC/MC D Cueva Rubia‐Valmayor/Madrid Iberian Peninsula (Spain) 1350 BC No
38 MC/MC D El Acequion Iberian Peninsula (Spain) 2200‐800 BC No
BRONZE AGE

39 MC/MC D El Acequion Iberian Peninsula (Spain) 2200‐800 BC Yes

40 MC/MC D El Acequion Iberian Peninsula (Spain) 2200‐800 BC Yes
8 MC/MC SL D Peñalosa‐Jaén ‐Andalucia Iberian Peninsula (Spain) 2200 ‐ 800 BC No
9 MC/MC SL D Peñalosa‐Jaén ‐Andalucia Iberian Peninsula (Spain) 2200 ‐ 800 BC No
10 MC/MC SL D Peñalosa‐Jaén ‐Andalucia Iberian Peninsula (Spain) 2200 ‐ 800 BC No
11 MC/MC SL D Peñalosa‐Jaén ‐Andalucia Iberian Peninsula (Spain) 2200 ‐ 800 BC No
12 MC/MC SL D Peñalosa‐Jaén ‐Andalucia Iberian Peninsula (Spain) 2200 ‐ 800 BC No
13 MC/MC SL D Peñalosa‐Jaén ‐Andalucia Iberian Peninsula (Spain) 2200 ‐ 800 BC No

11
 14 MC/MC SL D Peñalosa‐Jaén ‐Andalucia Iberian Peninsula (Spain) 2200 ‐ 800 BC No
15 MC/MC SL D Peñalosa‐Jaén ‐Andalucia Iberian Peninsula (Spain) 2200 ‐ 800 BC No
18 MC/MC SL D Morra Del Quintanar‐ Albacete Iberian Peninsula (Spain) 2200 ‐ 800 BC No
16 MC/MC SL D La Mota ‐Medina Del Campo Iberian Peninsula (Spain) 800 BC ‐ 6 AD No
4 MC/MC SL W? Soto de Medinilla ‐Valladolid Iberian Peninsula (Spain) 800 BC ‐ 6 AD Yes
IRON AGE

5 MC/MC SL W? Soto de Medinilla ‐Valladolid Iberian Peninsula (Spain) 800 BC ‐ 6 AD No

6 MC/MC SL W? Soto de Medinilla ‐Valladolid Iberian Peninsula (Spain) 800 BC ‐ 6 AD No
7 MC/MC SL W? Soto de Medinilla ‐Valladolid Iberian Peninsula (Spain) 800 BC ‐ 6 AD No
MEDIEV

29 MC/MC D Mucientes‐Valladolid Iberian Peninsula (Spain) 660‐780 AD Yes

AL

30 MC/MC D Mucientes‐Valladolid Iberian Peninsula (Spain) 680‐890 AD Yes

12
Table S2. Genes associated with coat color variation typed in this study. Shown are gene
name, GenBank accession number, position and type of mutation, the associated color of both
the wildtype and the derived state and the reference in which the mutation was first described.

Gene AccNo Mutation Associated color Reference

MC1R X98012 g.201C>T C: non-chestnut (E) S8
T: chestnut (e)
ASIP AF288358 g.2183_2193del -: non-black (A) S9
Deletion 11 bp: black (a)
MATP AY187093 g.72G>A G: non-cream (C) S7
(SLC45A2) A: cream (cr)
KIT AY048669 g.786G>C G: tobiano pattern (KM1) S5
C: non-tobiano pattern (KM0)
AY874542 g.1120T>A T: non-sabino spotted (sb1) S4
A: sabino spotted (SB1)
SILV DQ665301 g.1457C>T C: non-silver S12
T: silver

g.697A>T A: non-silver, linked with

g.1457C
T: silver, coupled with g.1457T
EDNRB AF038900 c.323_333TC>AG TC: non-overo pattern S13
AG: overo pattern

Table S3: Primers used for the coat color SNPs amplification. An annealing temperature of
57 °C was used for all of the primer pairs.

Gene Name Sequence 5’ to 3’ bp Modif. Length

KIT Pk-KIT-I13 F CGT CAT GAC TCA TTC GTG AGA A 22 63 bp
Pk-KIT-I13 R GCT CTG AAG GTA ACA AGC AAC TAA 24 5’-Biotin
Pk-KIT-I13 S TGA GAA ATT TCC GCC 15
EDNRB P-EDNRB-E1 F CAG TAG TGT CCT GCC TAG TGT TCG 24 5’-Biotin 63bp
P-EDNRB-E1 S TCA GCA GTG TGG AGT TT 17
Pk-EDNRB-E1 R TGA TTC TCA GCA GTG TGG AGT TT 23
KIT Pk-KIT-I16 F TTT AAA TGG CTT TCT TTT CTC C 22 5’-Biotin 59 bp
Pk-KIT-I16 R TGC CAA GTC CCT ATG AAT ACA C 22
Pk-KIT-I16 S CTA TGA ATA CAC TAT TAG GA 20
MATP P-MATP-E2F GCC ATA ACC ATC ACC ATG ATA G 22 5’-Biotin 65bp
Pk-MATP-E2 R GGC CCA TCA ATG AAG TCA G 19
P-MATP-E2 S GAA GTC AGC AGC AAA A 16
MC1R Pk-MC1R-E1 F GCA CTC ACC CAT GTA CTA CTT CAT 24 71 bp
Pk-MC1R-E1 R GCA CGT TGC TCA TGC TCA C 19 5’-Biotin
P-MC1R-E1 S CTG CTG CCT GGC CGT 15
SILV Pk-SILV-E11 F TCC TTC TTC TTC TCC CAA ATC A 22 5’-Biotin 52 bp
P-SILV-E11 R GAG CTG AGC CCT GCT TCA TAA 21
P-SILV-E11 S GCC CTG CTT CAT AAG TC 17
SILV Pk-SILV-I9 F AGG ATG AAG GGG AGT GGG 18 62 bp
Pk-SILV-I9 R GGC ACA GCT TCA GTC AGT GTC T 22 5’-Biotin

13
 Pk-SILV-I9 S GGG GAG TGG GCA GAG 15
ASIP Pk-ASIP-E2 F CAA GAA ATC CAA AAA GAT CAG C 22 5’-Biotin 67/78 bp
Pk-ASIP-E2 R ATG AGA AGT CCA AGG CCT ACC T 22
P-ASIP-E2 S CCT ACC TTG GAA GAT CTC 18

14
Table S4. Sample information for the horse fossils successfully typed for the coat color SNPs. The table contains information on geographic
location, excavation, dating, and color phenotype of the samples. Grey shaded dates indicate samples directly dated by radiocarbon dating (see also
Table S6). All radiocarbon ages are given in calibrated years (cal.). The Late Pleistocene samples were estimated at around 20,000 years based on
context.
Siberia
Sample Designation Location Excavation Date Phenotype
SP1181A W(ild) North Siberia Maliy Lyakhovsky Isl. Late Pleistocene Bay
PLEISTOCENE

SP1181B W North Siberia Bol'shoy Lyakhovsky Isl. Late Pleistocene Bay

SP1181C W North Siberia Bol'shoy Lyakhovsky Isl. Late Pleistocene Bay
SP1181E W Siberia Oyagosskiy Yar, Kondrat'evo River mouth Late Pleistocene Bay
SP1181F W Siberia Kotel'niy Isl., Anisiy Cape Late Pleistocene Bay
BER 001 D(omestic) Siberia (Altai) Denisova-Pescera 3000BC Chestnut
EARLY BRONZE AGE

TAR 001 D West Siberia Tartas1 2500-3000BC Bay

TAR 002 D West Siberia Tartas1 2500-3000BC Chestnut
TAR 004 D West Siberia Tartas1 2500-3000BC Bay
TAR 005 D West Siberia Tartas1 2500-3000BC Bay Sabino
TAR 007 D West Siberia Tartas1 2500-3000BC Black
TAR 008 D West Siberia Tartas1 2500-3000BC Bay
TAR 010 D West Siberia Tartas1 2500-3000BC Black
TAR 011 D West Siberia Tartas1 2500-3000BC Black
BER 002 D Siberia (Altai) Om-1 900BC Bay
Arz 1-2 D South Siberia (Tuva) Arzan1 800BC Chestnut
Arz 1-3 D South Siberia (Tuva) Arzan1 800BC Black Silver
Arz 2-1 D South Siberia (Tuva) Arzan2 619-608 BC
IRON AGE

Bay
Arz 2-2 D South Siberia (Tuva) Arzan2 619-608 BC Black
Arz 2-3 D South Siberia (Tuva) Arzan2 619-608 BC Chestnut
Arz 2-4 D South Siberia (Tuva) Arzan2 619-608 BC Bay
Arz 2-5 D South Siberia (Tuva) Arzan2 619-608 BC Chestnut
Arz 2-6 D South Siberia (Tuva) Arzan2 619-608 BC Bay
Arz 2-7 D South Siberia (Tuva) Arzan2 619-608 BC Black

15
 Arz 2-8 D South Siberia (Tuva) Arzan2 619-608 BC Buckskin
Arz 2-9 D South Siberia (Tuva) Arzan2 619-608 BC Buckskin
Chestnut
Arz 2-10 D South Siberia (Tuva) Arzan2 619-608 BC
Tobiano
Arz 2-11 D South Siberia (Tuva) Arzan2 619-608 BC Chestnut
Arz 2-12 D South Siberia (Tuva) Arzan2 619-608 BC Chestnut
Arz 2-13 D South Siberia (Tuva) Arzan2 619-608 BC Chestnut
Arz 2-14 D South Siberia (Tuva) Arzan2 619-608 BC Bay
Bars1A D South Siberia (Tuva) Barsucij Log cal. 370-150 BC Bay Tobiano
Bars1B D South Siberia (Tuva) Barsucij Log cal. 400-200 BC Bay Tobiano
OKG 001 D Siberia (Mongolia) Olon-Kurin-Gol 10 400-300 BC Chestnut
OKG 002 D Siberia (Mongolia) Olon-Kurin-Gol 10 400-300 BC Bay Tobiano
OKG 003 D Siberia (Mongolia) Olon-Kurin-Gol 10 400-300 BC Buckskin

Eastern Europe
PET1 W South Germany Petersfels 14000-11000 BC Bay
GLACIATION - MESOLITHIC

PET2 W South Germany Petersfels 14000-11000 BC Bay

PET3 W South Germany Petersfels 14000-11000 BC Bay
PET5 W South Germany Petersfels 14000-11000 BC Bay
PET6 W South Germany Petersfels 14000-11000 BC Bay
Kg1 W Germany (Thuringia) Kniegrotte 15000-14000 BC Bay
Kg2 W Germany (Thuringia) Kniegrotte 15000-14000 BC Bay
Kg3 W Germany (Thuringia) Kniegrotte 15000-14000 BC Bay
Kg4 W Germany (Thuringia) Kniegrotte 15000-14000 BC Bay
Kg5 W Germany (Thuringia) Kniegrotte 15000-14000 BC Bay
Ukraine (Peninsula
Spa 1 W Span-Koba cal. 9390-9210 BC Bay
Crimea)

16
 Pie9 W Romania Pietrele 4300 BC Black
ENEOLITHIC-COPPER AGE

CAS1 W Romania Cascioarele cal. 3700-3380 BC Black

VIT2 W Romania Vitanesti cal. 4350-4220 BC Black
VIT4 W Romania Vitanesti cal. 4360-4220 BC Black
MAY3 W Ukraine Mayaki cal. 3640-3490 BC Bay
MAY5 W Ukraine Mayaki cal. 3250-3100 BC Bay
MAY6 W Ukraine Mayaki cal. 3520-3330 BC Bay
MAY7 W Ukraine Mayaki cal. 3520-3380 BC Bay
MAY10 W Ukraine Mayaki cal. 3650-3500 BC Bay
MOL5 W? Ukraine Molyukhov Bugor cal. 3720-3630 BC Black
Gar2 D Romania Garbovat 1500-1000 BC Black
Gar3 D Romania Garbovat 1500-1000 BC Bay
Gar4 D Romania Garbovat 1500-1000 BC Bay
GRO11 D Germany Großobringen 3000-2500 BC Black
BRONZE AGE

Shi 1 D Armenia Shirakavan cal. 895-795 BC Chestnut

Chestnut
Lch 1 D Armenia Lchashen cal. 1410-1250 BC
Sabino
Lor 1 D North Armenia Lori-Berd cal. 1950-1750 BC Bay
Mic1 D Moldova Miciurin 1500-1000 BC Bay Tobiano
Mic2 D Moldova Miciurin 1500-1000 BC Chestnut
Mic3 D Moldova Miciurin 1500-1000 BC Black
Mic4 D Moldova Miciurin 1500-1000 BC Bay
Mic5 D Moldova Miciurin 1500-1000 BC Bay Sabino

China
IRON AGE Fen 1 D China (Qinghai) Fengtai cal. 905-800 BC Bay Tobiano
Fen 2 D China (Qinghai) Fengtai cal. 1000-800 BC Bay
Fen 3 D China (Qinghai) Fengtai cal. 1000-800 BC Bay

17
 Fen 4 D China (Qinghai) Fengtai cal. 1000-800 BC Bay

Iberian Peninsula
44 W Spain Atxoste 5500- 4950 BC Bay
45 W Spain Atxoste 5500- 4950 BC Bay
MESOLITHIC-
NEOLITHIC

3 W Spain Cueva Fosca - Valencia-Cartellon cal. 5200-4900 BC Black

31 W Spain Cueva Fosca - Valencia-Cartellon cal. 5210-4910 BC Bay
32 W Spain Cueva Fosca - Valencia-Cartellon cal. 5220-4980 BC Black
34 W Spain Cueva Fosca - Valencia-Cartellon cal. 5220-4900 BC Black
37 W Spain Cueva Fosca - Valencia-Cartellon cal. 5210-4910 BC Bay
17 W Spain Cueva De La Vaquera - Segovia cal. 5210-4940 BC Black
39 D Spain El Acequion 2200 BC - 800 AD Bay
BRONZE

40 D Spain El Acequion 2200 BC - 800 AD Bay

AGE

24 D Spain Cueva Rubia - Valmayor/Madrid 1350 BC Bay

22 D Spain Cueva Rubia - Valmayor/Madrid 1350 BC Black
25 D Spain Cueva Rubia - Valmayor/Madrid 1350 BC Black
IRON AGE 4 W? Spain Soto de Medinilla - Valladolid 800 BC - 6 AD Black
29 D Spain Mucientes - Valladolid cal. 660-780 AD Chestnut
MEDIEVAL
30 D Spain Mucientes - Valladolid cal. 680-890AD Chestnut

18
Table S5. Results for SNP typing. In addition to the phenotype, the genotypes for all eight SNPs investigated are shown. Grey shading indicates that
at least one allele differs from the wildtype. We did not detect any variation at the EDNRB locus.

Sample Designation Phenotype nuclear Genes

Siberia ASIP EDNRB KIT13 KIT16 MATP MC1R SILV9 SILV11
SP1181A W(ild)
PLEISTOCENE

Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z

SP1181B W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
SP1181C W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
SP1181E W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
SP1181F W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
BER 001 D(omestic) Chestnut A/A ov/ov KM0/KM0 sb1/sb1 C/C e/e z/z z/z
EARLY BRONZE AGE

TAR 001 D Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
TAR 002 D Chestnut a/a ov/ov KM0/KM0 sb1/sb1 C/C e/e z/z z/z
TAR 004 D Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
TAR 005 D Bay Sabino A/A ov/ov KM0/KM0 SB1/sb1 C/C E/e z/z z/z
TAR 007 D Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
TAR 008 D Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
TAR 010 D Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
TAR 011 D Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/e z/z z/z
BER 002 D Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/e z/z z/z
Arz 1-2 D Chestnut A/a ov/ov KM0/KM0 sb1/sb1 C/C e/e z/z z/z
Arz 1-3 D Black Silver a/a ov/ov KM0/KM0 sb1/sb1 C/C E/e Z/z Z/z
Arz 2-1 D Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
IRON AGE

Arz 2-2 D Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/e z/z z/z
Arz 2-3 D Chestnut A/A ov/ov KM0/KM0 sb1/sb1 C/C e/e z/z z/z
Arz 2-4 D Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/e z/z z/z
Arz 2-5 D Chestnut A/a ov/ov KM0/KM0 sb1/sb1 C/C e/e z/z z/z
Arz 2-6 D Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/e z/z z/z
Arz 2-7 D Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/e z/z z/z
Arz 2-8 D Buckskin A/a ov/ov KM0/KM0 sb1/sb1 C/cr E/e z/z z/z

19
 Arz 2-9 D Buckskin A/a ov/ov KM0/KM0 sb1/sb1 C/cr E/e z/z z/z
Arz 2-10 D Chestnut Tobiano A/a ov/ov KM0/KM1 sb1/sb1 C/C e/e z/z z/z
Arz 2-11 D Chestnut A/A ov/ov KM0/KM0 sb1/sb1 C/C e/e z/z z/z
Arz 2-12 D Chestnut A/a ov/ov KM0/KM0 sb1/sb1 C/C e/e z/z z/z
Arz 2-13 D Chestnut A/a ov/ov KM0/KM0 sb1/sb1 C/C e/e z/z z/z
Arz 2-14 D Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/e z/z z/z
Bars1A D Bay Tobiano A/a ov/ov KM0/KM1 sb1/sb1 C/C E/e z/z z/z
Bars1B D Bay Tobiano A/a ov/ov KM0/KM1 sb1/sb1 C/C E/e z/z z/z
OKG 001 D Chestnut A/a ov/ov KM0/KM0 sb1/sb1 C/C e/e z/z z/z
OKG 002 D Bay Tobiano A/a ov/ov KM0/KM1 sb1/sb1 C/C E/E z/z z/z
OKG 003 D Buckskin A/a ov/ov KM0/KM0 sb1/sb1 C/cr E/e z/z z/z

Eastern Europe
Sample Designation Phenotype nuclear Genes
ASIP EDNRB KIT13 KIT16 MATP MC1R SILV9 SILV11
PET1 W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
GLACIATION - MESOLITHIC

PET2 W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
PET3 W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
PET5 W Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
PET6 W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
Kg1 W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
Kg2 W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
Kg3 W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
Kg4 W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
Kg5 W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
Spa 1 W Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
Pie9 W Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/e z/z z/z
CAS1 W Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
VIT2 W Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z

20
 VIT4 W Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
COPPER AGE
ENEOLITHIC-

MAY3 W Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
MAY5 W Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
MAY6 W Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
MAY7 W Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
MAY10 W Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
MOL5 W? Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
Gar2 D Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/e z/z z/z
Gar3 D Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
Gar4 D Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
GRO11 D Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
BRONZE AGE

Shi 1 D Chestnut A/A ov/ov KM0/KM0 sb1/sb1 C/C e/e z/z z/z
Lch 1 D Chestnut Sabino A/a ov/ov KM0/KM0 SB1/sb1 C/C e/e z/z z/z
Lor 1 D Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
Mic1 D Bay Tobiano A/a ov/ov KM0/KM1 sb1/sb1 C/C E/E z/z z/z
Mic2 D Chestnut A/a ov/ov KM0/KM0 sb1/sb1 C/C e/e z/z z/z
Mic3 D Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
Mic4 D Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
Mic5 D Bay Sabino A/a ov/ov KM0/KM0 SB1/sb1 C/C E/e z/z z/z

China
Sample Designation Phenotype nuclear Genes
ASIP EDNRB KIT13 KIT16 MATP MC1R SILV9 SILV11
Fen 1 D Bay Tobiano A/A ov/ov KM0/KM1 sb1/sb1 C/C E/E z/z z/z
Fen 2 D Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/e z/z z/z
IRON AGE
Fen 3 D Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
Fen 4 D Bay A/A ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z

21
Iberian
Peninsula
44 W Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
45 W Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
MESOLITHIC-
NEOLITHIC

3 W Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z

31 W Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
32 W Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
34 W Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
37 W Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
17 W Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
39 D Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
BRONZE

40 D Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z

AGE

24 D Bay A/a ov/ov KM0/KM0 sb1/sb1 C/C E/e z/z z/z

22 D Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
25 D Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
IRON AGE 4 W?* Black a/a ov/ov KM0/KM0 sb1/sb1 C/C E/E z/z z/z
29 D Chestnut a/a ov/ov KM0/KM0 sb1/sb1 C/C e/e z/z z/z
MEDIEVAL
30 D Chestnut A/A ov/ov KM0/KM0 sb1/sb1 C/C e/e z/z z/z
* Based on archaeological context.

22
 Table S6. Detailed information on the ages of the samples investigated.

Siberia

Geographical Stratigraphic Calibrated

Sample Excavation Age 14C Lab. No. Details
location date date
PLEISTOCENE

SP1181A North Siberia Maliy Lyakhovsky Isl. Pleistocene

SP1181B North Siberia Bol'shoy Lyakhovsky Isl. Pleistocene
SP1181C North Siberia Bol'shoy Lyakhovsky Isl. Pleistocene
Oyagosskiy Yar, Kondrat'evo R.,
SP1181E Siberia mouth Pleistocene
SP1181F Siberia Kotel'niy Isl., Anisiy Cape Pleistocene
BER 001 Siberia (Altai) Denisova-Pescera 3000BC
EARLY BRONZE AGE

TAR 001 West Siberia Tartas1 2500-3000BC

TAR 002 West Siberia Tartas1 2500-3000BC
TAR 004 West Siberia Tartas1 2500-3000BC
TAR 005 West Siberia Tartas1 2500-3000BC
TAR 007 West Siberia Tartas1 2500-3000BC
TAR 008 West Siberia Tartas1 2500-3000BC
TAR 010 West Siberia Tartas1 2500-3000BC
TAR 011 West Siberia Tartas1 2500-3000BC
BER 002 Siberia (Altai) Om-1 900BC
Arz 1-2 South Siberia (Tuva) Arzan1 800BC
Arz 1-3 South Siberia (Tuva) Arzan1 800BC
IRON AGE

Arz 2-1 South Siberia (Tuva) Arzan2 619-608 BC

Arz 2-2 South Siberia (Tuva) Arzan2 619-608 BC
Arz 2-3 South Siberia (Tuva) Arzan2 619-608 BC
Arz 2-4 South Siberia (Tuva) Arzan2 619-608 BC
Arz 2-5 South Siberia (Tuva) Arzan2 619-608 BC
Arz 2-6 South Siberia (Tuva) Arzan2 619-608 BC

23
 Arz 2-7 South Siberia (Tuva) Arzan2 619-608 BC
Arz 2-8 South Siberia (Tuva) Arzan2 619-608 BC
Arz 2-9 South Siberia (Tuva) Arzan2 619-608 BC
Arz 2-10 South Siberia (Tuva) Arzan2 619-608 BC
Arz 2-11 South Siberia (Tuva) Arzan2 619-608 BC
Arz 2-12 South Siberia (Tuva) Arzan2 619-608 BC
Arz 2-13 South Siberia (Tuva) Arzan2 619-608 BC
Arz 2-14 South Siberia (Tuva) Arzan2 619-608 BC
Bars1A South Siberia (Tuva) Barsucij Log 2170 ± 30 BP cal. 370-150 BC Poz-22611 3.3%N 11.5%C
Bars1B South Siberia (Tuva) Barsucij Log cal. 400-200 BC
OKG 001 Siberia (Mongolia) Olon-Kurin-Gol 10 400-300 BC
OKG 002 Siberia (Mongolia) Olon-Kurin-Gol 10 400-300 BC
OKG 003 Siberia (Mongolia) Olon-Kurin-Gol 10 400-300 BC

East Europe

Geographical Stratigraphic Calibrated

Sample Excavation Age 14C Lab. No. Details
location date date
PET1 South Germany Petersfels 14000-11000 BC
PET2 South Germany Petersfels 14000-11000 BC
LATE GLACIATION

PET3 South Germany Petersfels 14000-11000 BC

PET5 South Germany Petersfels 14000-11000 BC
PET6 South Germany Petersfels 14000-11000 BC
Kg1 Germany (Thuringia) Kniegrotte 15000-14000 BC
Kg2 Germany (Thuringia) Kniegrotte 15000-14000 BC
Kg3 Germany (Thuringia) Kniegrotte 15000-14000 BC
Kg4 Germany (Thuringia) Kniegrotte 15000-14000 BC
Kg5 Germany (Thuringia) Kniegrotte 15000-14000 BC

24
 Ukraine (Peninsula cal. 9390-9210
Spa 1 Crimea) Span-Koba BC
Pie9 Romania Pietrele 4300 BC
cal. 3700-3380 1.0%N 7.6%C
ENEOLITHIC-COPPER AGE

CAS1 Romania Cascioarele 4820 ± 40 BP BC Poz-24925 carbonate

cal. 4350-4220
VIT2 Romania Vitanesti 5400 ± 40 BP BC Poz-24899 1.5%N 6.0%C
cal. 4360-4220
VIT4 Romania Vitanesti 5430 ± 40 BP BC Poz-24900 1.7%N 6.5%C
cal. 3640-3490 0.6%N 2.8%C
MAY3 Ukraine Mayaki 4745 ± 35 BP BC Poz-24926 carbonate
cal. 3250-3100
MAY5 Ukraine Mayaki 4550 ± 35 BP BC Poz-24826 2.4%N 9.8%C
cal. 3520-3330
MAY6 Ukraine Mayaki 4605 ± 35 BP BC Poz-24849 1.6%N 6.7%C
cal. 3520-3380
MAY7 Ukraine Mayaki 4640 ± 35 BP BC Poz-24850 2.6%N 10.4%C
cal. 3650-3500 0.5%N 3.4%C
MAY10 Ukraine Mayaki 4770 ± 40 BP BC Poz-24927 carbonate
MOL5 Ukraine Molyukhov Bugor cal. 3720-3630 BC
Gar2 Romania Garbovat 1500-1000 BC
Gar3 Romania Garbovat 1500-1000 BC
Gar4 Romania Garbovat 1500-1000 BC
GRO11 Germany Großobringen 3000-2500 BC
BRONZE AGE

Shi 1 Armenia Shirakavan 2670 ± 30 BP cal. 895-795 BC Poz-22615 1.5%N 5.5%C

cal. 1410-1250
Lch 1 Armenia Lchashen 3050 ± 30 BP BC Poz-22613 3.7%N 12.4%C
cal. 1950-1750
Lor 1 North Armenia Lori-Berd 3525 ± 35 BP BC Poz-22701 1.1%N 4.6%C
Mic1 Moldova Miciurin 1500-1000 BC
Mic2 Moldova Miciurin 1500-1000 BC
Mic3 Moldova Miciurin 1500-1000 BC
Mic4 Moldova Miciurin 1500-1000 BC
Mic5 Moldova Miciurin 1500-1000 BC

25
China

Geographical Stratigraphic Calibrated

Sample Excavation Age 14C Lab. No. Detail
location date date
Poz-22612 3.4%N 11.0%C
IRON AGE

Fen 1 China (Qinghai) Fengtai 2695 ± 30 BP cal. 905-800 BC

Fen 2 China (Qinghai) Fengtai 905-800 BC
Fen 3 China (Qinghai) Fengtai 905-800 BC
Fen 4 China (Qinghai) Fengtai 905-800 BC

Iberian Peninsula

Geographical Stratigraphic Calibrated

Sample Excavation Age 14C Lab. No. Detail
location date date
44 Iberian Peninsula (Spain) Atxoste 5500- 4950 BC
MESOLITHIC-NEOLITHIC

45 Iberian Peninsula (Spain) Atxoste 5500- 4950 BC

3 Iberian Peninsula (Spain) Cueva Fosca - Valencia-Cartellon cal. 5200-4900
BC
Iberian Peninsula (Spain) Cueva Fosca - Valencia-Cartellon cal. 5210-4910
31 6100 ± 40 BP BC Poz-24720 3.5%N 15.4%C
cal. 5220-4980
Iberian Peninsula (Spain)
32 Cueva Fosca - Valencia-Cartellon 6135 ± 35 BP BC Poz-24743 3.4%N 17.2%C
34 Iberian Peninsula (Spain) Cueva Fosca - Valencia-Cartellon cal. 5200-4900 BC
cal. 5210-4910
Iberian Peninsula (Spain)
37 Cueva Fosca - Valencia-Cartellon 6100 ± 40 BP BC Poz-24745 2.6%N 11.9%C
cal. 5210-4940
Iberian Peninsula (Spain)
17 Cueva De La Vaquera - Segovia 6110 ± 40 BP BC Poz-24721 2.7%N 13.9%C
39 Iberian Peninsula (Spain) El Acequion 2200 BC - 800 AD
BRONZE AGE

40 Iberian Peninsula (Spain) El Acequion 2200 BC - 800 AD

24 Iberian Peninsula (Spain) Cueva Rubia - Valmayor/Madrid cal. 2880-2570BC

22 Iberian Peninsula (Spain) Cueva Rubia - Valmayor/Madrid cal. 2880-2570BC
25 Iberian Peninsula (Spain) Cueva Rubia - Valmayor/Madrid cal. 2880-2570BC

26
IRON AGE 4 Iberian Peninsula (Spain) Soto de Medinilla – Valladolid 800 BC - 6 AD
29 Iberian Peninsula (Spain) Mucientes - Valladolid 1275 ± 25 BP cal. 660-780 AD Poz-24740 1.2%N 6.9%C
MEDIEVAL
30 Iberian Peninsula (Spain) Mucientes - Valladolid 1230 ± 30 BP cal. 680-890AD Poz-24739 3.4%N 15%C

27
Table S7. Upper bound of the frequency f5% a colour allele could have had in the horse
population prior to domestication having a chance to be missed of p ≤ 0.05 (n = number
of alleles).
Population n f5%
Siberia 10 0.26
East Europe 42 0.07
Iberia 16 0.15
Siberia + East Europe 52 0.06
Siberia + East Europe + Iberia 68 0.04

Table S8. Maximum likelihood estimates of the selection coefficient for each of the loci.
The results were obtained using Bollback et al’s method (S11), excluding the Pleistocene
samples. To get the 95% confidence interval we used the profile likelihood. For ASIP and
MC1R loci the selection coefficient is significantly different than 0. For the other loci, the
data is compatible with s=0. Therefore, the observed data are difficult to explain by
genetic drift alone under a standard neutral model (for review see S14).

Note that we had no power to estimate the effective population size. Indeed, the
confidence interval for the effective population size spans all the values of the grid we
used to compute the likelihood, that is 10,000 to 100,000. This is not surprising since
Bollback et al. (S11) noted that their method has little power to co-estimate the effective
population size and the selection coefficient.

Gene Selection coefficient, maximum likelihood estimate

and 95% confidence interval
ASIP 0.0007 (0.0001, 0.0015)
KIT13 0.0003 (-0.0007, 0.007)
KIT16 -0.0003 (-0.0013, 0.0011)
MATP 0.000 (-0.001, 0.001)
MC1R 0.0019 (0.0007, 0.0035)
SILV19 -0.0003 (-0.0015, 0.0013)

Supplementary references
S1. N. Rohland, M. Hofreiter, Nature Protocols 2, 1756 (2007).
S2. J. Krause et al., Nature 439, 724 (Feb 9, 2006).
S3. H. Römpler et al., Nat. Protocols 1, 720 (2006).
S4. S. A. Brooks, E. Bailey, Mamm Genome 16, 893 (Nov, 2005).
S5. S. A. Brooks, R. B. Terry, E. Bailey, Anim Genet 33, 301 (Aug, 2002).
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