1

Supplemental Data: Mitochondrial Genomes of Extinct Aurochs Survive in Domestic Cattle

A. Achilli, A. Olivieri, M. Pellecchia, C. Uboldi, L. Colli, N. Al-Zahery, M. Accetturo, M. Pala,
B. Hooshiar Kashani, U. A. Perego, V. Battaglia, S. Fornarino, J. Kalamati, M. Houshmand, R.
Negrini, O. Semino, M. Richards, V. Macaulay, L. Ferretti, H.-J. Bandelt, P. Ajmone-Marsan,
and A. Torroni
Supplemental Experimental Procedures
Testing the Molecular Clock. Assuming the HKY85 mutation model (with indels ignored, as
usual) with gamma distributed rates (approximated by a discrete distribution with 32 categories),
we used PAML 3.15 [S1] to check the molecular clock hypothesis by maximizing the likelihood
over the branch lengths and the parameters (the transition/transversion parameter in the HKY85
model) and (the heterogeneity parameter in the gamma distribution), both with and without the
clock enforced. Then we used a likelihood ratio test to compare the maximized likelihoods. This
analysis was performed on the tree (Figures 1 and S1) using two outgroups: the published Bos
grunniens sequence and the novel Bison bison mitochondrial genome. We could not reject the
null hypothesis that the molecular clock describes the evolution of these sequences. The
generalized likelihood ratio statistic was 76 which, assuming the asymptotic 2 (107) distribution
as valid, yields a P-value of 0.99, under the clock hypothesis. The maximum likelihood estimates
of and under the clock model were 36.03.7 and 0.210.02, respectively.
Mutation Rate Estimate. To calculate the mutation rate we assumed a constant mtDNA
sequence evolution rate and an estimated bifurcation between yak and bison versus cattle of 2
million years ago, based on the fossil record [S2, S3] and calibration from other molecular data
[S4, S5]. The mutation rate estimate (2.0430.099 108 base substitutions per nucleotide per
year) that was obtained was used to convert the mutational distances obtained from the ML
analysis into age estimates, whose standard errors were computed using the delta method. The
clades/subclades ages were also computed from the average distance () of the haplotypes of a
clade from the respective root haplotype, accompanied by a heuristic estimate of SE (),
following Saillard et al. [S6].

Supplemental Results
Cattle mtDNA studies have generally focused on a 240-bp sequence of the control region,
revealing five major haplotype clusters in B. taurus (T*-T1-T2-T3-T4) and two in B. indicus (I1I2) [S7-S11]. However, analogous studies of human mtDNA have taught that focussing exclusively
on a small portion of the non-coding and fast-evolving control region is often inadequate [S12].
The American bison and the yak mtDNA coding regions show a sequence divergence of ~2.9%,
a value that is less than half of that observed in the comparisons Bison bison B. taurus (~7.8%)
and B. grunniens B. taurus (~8.3%). This confirms the inadequacy of the current Bovini
taxonomy and major discrepancies between morphological, paleontological and molecular data
[S4, S13].
Compared to the sequence divergence within the human mtDNA phylogeny (38.513.70
substitutions) (~19819 ky) [S14], that within the bovine macro-haplogroup T (5.041.00
substitutions) is almost eight-fold less.
Despite the taurine phenotype, about 16% of the animals from Iranian and Iraqi breeds
harboured a B. indicus control-region motif. This allowed the inclusion of three new B. indicus
complete mtDNA sequences that formed, together with two published sequences, two clades (I1
and I2) whose control-region mutational motifs appear to encompass all B. indicus mtDNAs [S10].
In agreement with previous reports that identified haplogroup T4 only in North Eastern Asian
cattle [S8, S15], T4 mtDNAs were not observed among our samples, but they were among the
previously published sequences.
Our study reveals that at least two mitochondrial genomes of aurochs are present in modern
taurine cattle, although mtDNA due to its maternal transmission is not the best tool to evaluate
the extent of such admixture since the genetic input into the domestic stocks was most likely
mainly from aurochs bulls.

Without coding-region information, it would have been hard if not impossible to clarify the
relationship between ancient and surviving non-T aurochs mtDNA lineages. For instance, the
median network for the ancestral P, T, E [S16], and I haplotypes based on part of the control region
is quite box-like, reflecting a considerable level of noise (homoplasy). Coding-region sites are also
needed to infer haplogroup Q (or QT*) status reliably. For example, two samples from Lai et al.
[S10] and from Bollongino et al. [S17] may well belong to Q or another sister clade of T within
haplogroup QT, as suggested by the transversion at 15953. Thus, mtDNAs from other B.
primigenius populations are probably present in modern cattle. Bearing in mind that ancient
samples are unlikely to provide us with entire mtDNA genomes, we may need to learn more about
extinct aurochs mtDNA variation through these rare introgressed lineages in modern breeds.
However, the detection of these lineages not only depends on the survival of indigenous cattle
breeds, which are often endangered, but also requires molecular analyses to be expanded to the
entire mitochondrial genome [S18].

4
Bison bison / Bos grunniens

PQTI

332.424.0 ky

248 296 518 737 1869 2953 2977 2979 2988 2990 3051 3071 3325 3335 3439 3535 3600 3793 3874 3985 4000 4562 4730 4769 4871
5285 6379 6460 6772 7330 7358 7514 7830 7851 8188 8210 8308 9005 9038 9602 9978 10039 10066 10137 10322 10849 11068 11134
11419 11842 12178 12234 12513 12622 12672 12684 12801 13056A 13275 13437 13554 13584 13689 13882 13909 14129 14138 14255
14411 14416 14897 15146 15308 15326 15579 15593 15605 15617 15629 15751 15818 16082 16084 16138 16200+A 16248

74.49.7 ky

Southern Europe
Others

QT
Q

1457 3238 3415T 7918

8318 8405 10717A
10927 11089 11476
12730 12877 13819
14108 16079

98

169 2558 5501 8370C

11000 12377 12433
12468 12675 12750
12924 14036 15134
15627 15953G 16058

68

8
9560
15396

69

7542
16022

16.03.2 ky

3965 15419
14747A 16050
16248

70

71

16050

T1a

2055+C
169 6046
9918 15082
13683

72

73

4975
6932
8667 8959 8891
15713d 12252
15964
74 16076
16115 16122

75

16074
174
1499 4953 7850
8514
5238 5632C 13201
11368
8802 16131 16058 1459 16067
15556
9383
16125 2558 16121
16008C 11287
11101
81
16122
12651
3875
82 15796 84
8523
4356 6202
15985
78
15595
8862
10325
79 80
83
11452
12651

16196

106

76

170
3940
5156T
5761
10350
13751
16050
16141

85

88

77

1323
1694
2599
3718
4781
7398
10601
15157
16058
16248
16301

89

35
735
12728
7571
1520
7610
5072
1481 169
9176
6080
8166A
8169
14126 11529C
16110 15939 9020 10888
10385 14132
16248
14

15

173
1491
6930
9729
13173
16301

16

12730

17

169
119 1553
2085
120 4975
3190
166 6205
9987
3349 8860
11734
3850 16139
14579
6403
8475
91 169h 2747
9308
10660
9480 6314
11830
13396 8984G
12292
15194
12504
93
12765
92
13480
15883d
16080
16231

169
8024
9089
9232
10879
15882
16197

94

T5

I1
9894A
7658

96

204
1290T
8045
16137

97

12780

363+2C
3355
7892
15587
16143d@

2321A

102

39
3316
10888
10933
14369
16085
16141
16232

10.93.5 ky

I2
173
16084
363+C
5273T
6496
4310 7946
14232 7499
14831 10688 105
16050A 12339

103

104

101

95

16255

90

T3

3379
13524
10741
16057
16058
1066+A
12908
1290T 16133 3341 13878 8
351+G 8240
12173-12175d
9447
7622 14153 513
6017
1858+A11185
14745
4820 10889
14078
16119
5102 13494 16231
814 106
1290 312A 8898 58 12023
6688 11800
3334 6573
16086
8
16113 5944
14561 14594
12173 61 15092 15910 106 7192 16108
7776
2145 169
6499 8880 13470
3850A6994C
10083 34 1193
16066 16055 3727 14366 16302
15215 16058 50
14036
11035
3302 3544
106 9684 6946 10870 14178
4769
3718
14580 16301
30 13429
15964
16085
16113
12684
2078 169 1552
3871
7799 16042
8710
16042
11734
43
44
5220A
16042
16228
16109
19 16093
2700+C11672 4337 54 16133
57
8925 46 14522 2220
9480
12223
67
10991
3086 1132 16093
6133 12159 4385
13401
16250
16165h
16049
16127
15419 14228 49
56
15329 8286
66
7779
8666G 5681
8652 13695 5141
62 3796 2569
169 16119
15102
59
15510
16067 13628
12158 7373G 45
31
9096 6619 65
9515 13743 14291
9778T
8
15386
60
55
47
18
12684h
9729
12209 16074 16127
3856
169
16051 13029 12938h
16022
16302
14464 16140 16247
15758
32
28 29
16200
63 16112
35 36
16167
16229 16250
27
14063

12158

16119

16122

T3a

T4

11174
169
11008 5376 352G 16116
7211 3343 11248
7865
10467 5597
15556 8710 12879
6688 9119 16049 23 13509
12165
26
15595
7703 13231 16057
14057 25
9 14906
16104
15635 11 8210 16074
16164
12492
8
16247
24
14907C 21

10

5.32.6 ky

10.33.1 ky

5146
1651
9500
16128 173
14402
363+C
16062
6202
16247 87
8245
16301
10259
16049
16113
86

32.46.7 ky

173
722
2575
2634
4988A
8194
8514
9302
9480
10071
13101A
13371
13628

163
4252
12981

T2

11.30.9 ky
2125 5471C
587+C
169
3784 12234
2536A 3302
16248 15207
9682C 6820
16069
13310C 9257 163
564
12
1090
13140 10696
13
BRS 15877 11026 119
12450
5542+A
4094
1 16022 9068 6568 12705
16042
11749
16141
13164
2
13401
3727 173
3 13830 16302
2027
13854
4000
15836
15354
16042 5 15960
16122
16228
4

6202 6742 10708

16057C 16185

10.50.9 ky

T1

100

99

T123

16113

173 190 222 301 1128

1481 2171 2585 3379
4252 4293 4676 5156
5681 5899 6160 7952
7994 8236 8358 10126
11140 11468 12016
12433 12738 12924
13821 14129T 14873
15627 15673 15714h
16051 16085 16231
16264

52.28.0 ky

12.00.8 ky 13005
9.42.4 ky

PQT

106 166 249 300 3550 5743 5890

6436 7356 10691G 15951 15994
16049 16074 16122 16247

Near East

3709T
8638
9303
16074

8 206 232 233 761 816 1158 1474 1492 1677 1824 1860A 2016 2099 2989
3136 3145 3241 3379 3829 3931 3976 4328 4442 4733 4937 5531 5614 5783
5917 5998 6115T 6235 6340 6367 6727 6881 6922 7304 7361 8045 8168
8285 8466 8494 8503 8571 8749 8984 9068 9245 9581 9767 9891 9930C
10153 10268 10331 10445 10590 10621 11035 11200 11266 11329 11407
11803 12135 12469 12900 12923T 13098 13104 13380 13433 13564 13677
13692 13908A 14066 14120 14315 14503 14606 14825 14858 15105 15287
15563 15741 15959 16022 16057 16102 16109 16113 16116 16117 16119
16121 16130 16137 16143d 16147 16196 16229 16255 16300 16301

20

33

37

16247

41 42

48

51

52 53

64

38 39 40

22 6.91.8 ky

Supplemental Figure S1. Tree of Complete mtDNA Sequences from Cattle

This tree encompasses 106 sequences and was rooted by using a published yak sequence and a
novel sequence from American bison. Mutations are shown on the branches and are numbered
according to the BRS [S19]; they are transitions unless a base is explicitly indicated. Suffixes
indicate transversions (to A, G, C, or T), indels (+, d), heteroplasmy (h) or indels reversion (@).
Recurrent mutations are underlined. The 56 novel sequences are all from local breeds and their
geographic origins are as follows: Italy (1-6, 11, 47-53, 55-58, 68-69, 71-76, 82-83, 85-87, 93-95,
98-99); Spain (59-60); Iraq (61-63, 67, 77, 88, 96-97, 102-103); Iran (66, 89, 90-92, 104); Greece
(81, 84). The phylogeny construction was performed as described elsewhere [S20]. The tree
shown is only one out of many nearly most parsimonious trees, which mainly differ in the
different reconstruction of mutations at highly recurrent sites in the control region. Mitochondrial

DNAs to be completely sequenced were selected, as previously described [S20-S22], through a

preliminary sequence analysis of the control region in order to include the widest possible range
of mtDNA variation. The hypervariable insertion of a G at np 364, the length variations in the C
tract scored at np 221 (221+C, 221+2C) and the A tract scored at np 1600 (1600+A, 1600+2A,
1600d) were not used for the phylogeny construction. Variants at those positions, relative to the
BRS, are present in the following sequences: 221+C (1-3, 5, 7-46, 49-52, 54, 56, 61, 64-65, 6770, 75-80, 82, 84, 90, 95-97, 100-105); 221+2C (4); 364+G (1-4, 47-49, 52, 55); 1600+A (4);
1600+2A (76); 1600d (11, 47-50, 53, 55-58, 62-63, 66-68, 86, 89, 101-105). The Italian breeds
are as follows: Cabannina (68, 82-83, 98-99); Chianina (2, 4-6, 47, 50, 53, 69, 74-75, 85-87);
Cinisara (48, 71); Italian Friesian (11, 51-52, 76); Maremmana (49, 55, 73); Modicana (57);
Pettiazza (58); Piedmontese (1, 95); Italian Podolian (55, 72); Rendena (93); Valdostana (3, 94).
The Spanish breed is the Betizuak (59-60) from Navarra.

Supplemental Information for Figure S1

The Bison bison and B. grunniens complete mtDNA sequences share the following 666 mutations
(612 in the coding region) from the PQTI node of Fig. S1. The suffix @ indicates a reversion
from the PQTI node to BRS. Mutations: 172C 177d 179 221+C 223 234d 249@ 255 268 279T
315A 327T 329 458 555 649 709 739 756 760 817 824 909 923 1017 1083 1087 1132 1166 1190
1191 1296 1308 1457 1459 1480 1481 1495 1499 1550+G 1552 1555 1600d 1610 1684 1694
1716 1729 1822 1823T 1833T 1871 1879 1981 1987 2021 2026 2031 2037 2039 2077T 2087
2117 2145 2165 2172 2173 2185 2206 2208 2220 2318 2321 2335 2434 2504 2553 2576 2642
2877 2965 2971 3127T 3160 3163 3169 3190 3223 3238 3271 3281 3289 3292 3295 3328
3329T 3388 3448 3526 3551 3574 3620 3637 3668 3691C 3698 3805 3808A 3821 3826 3844
3879 3883 3892 3895 3907A 3940 3979 3988 4006 4018A 4021 4071A 4108 4139 4168 4248
4252 4285 4293 4296 4316 4361 4391 4406 4448 4530 4592 4625 4647 4649A 4740 4743 4823
4826 4862 4883 4889 4892 4907 4913 4943 4949 4989 4997 5000 5039A 5045 5072 5084 5096
5156 5166 5180 5186 5225 5237 5272 5279 5292 5300 5493 5500 5504+T 5506A 5604 5670
5731 5743@ 5755A 5758 5824 5899 5905 5938 5950 5962 5968 5980 6013 6049 6109 6127
6157T 6220 6269 6274C 6280 6290 6304 6313 6314 6352 6370 6374 6403 6427 6451 6457
6499 6508 6511 6533 6553 6568A 6610 6637 6646 6656 6758 6763 6814 6937 6955 7027 7043
7054 7069 7081 7135 7145 7219 7356@ 7359 7397C 7409T 7448 7499 7520 7526 7538 7575
7583 7586 7589 7655 7661 7668 7670A 7700 7769 7770 7805 7829 7847 7865 7871 7883 7892
7910 7932A 7944 7970 7976 7985 7994 8009 8015 8021 8027 8109A 8134 8164 8194 8204
8230 8231 8249 8326 8343 8352 8394 8398 8403 8405 8475 8476 8478 8514 8517 8542 8550
8607 8656 8691A 8721 8775 8793 8806 8817 8870 8892 8898 8937 8996 9020 9033 9035 9060
9063 9071 9077 9088 9089 9090 9119 9146 9150 9155 9167 9221 9224 9242C 9269A 9278
9296 9299 9335 9344 9386 9431 9440C 9488 9509 9524 9563 9626 9636 9650 9713 9770 9771
9809 9829 9864 9873 9879 9912 9921 9993 10047 10071 10113G 10134 10155 10216 10251
10259 10275 10304 10349 10350 10373 10388 10418 10433 10466 10493 10505 10514 10521
10540 10576 10586 10587 10597 10600 10624A 10645 10651 10657 10666 10672 10678
10691A 10702 10747 10750 10777 10813 10822 10825 10830 10867 10879C 10888 10909A
10918 10936 10954 10988 11037 11063 11065C 11086G 11095 11105 11107 11158 11179
11228 11230 11245 11287 11324 11338 11386 11395 11428 11440 11452 11470 11473 11485
11551 11627 11633 11672 11746 11755 11758 11785 11809 11824 11843 11845 11852 11857
11867 11870T 12016 12017 12021 12057d 12058 12079 12127 12148 12156A 12157 12173
12193 12223 12273 12313 12318 12327 12334 12339 12376 12377@ 12429 12486 12493A
12495A 12519A 12525 12546 12597 12603 12624 12654 12657 12658 12687A 12699 12721
12728 12730 12758 12760 12849 12894 12897 12903A 12915 12927 12946 12964 12975 12984
13029 13050 13074 13083A 13098 13128 13146A 13173 13176 13206 13215 13350 13368
13374 13386 13389 13392 13401 13419 13420 13429 13455 13458 13479 13485 13509 13512
13521 13527 13537 13539 13563 13604 13620 13623 13626 13638 13646T 13671 13677C
13698 13732 13791 13806 13815 13839 13857 13878 13887 13893 13896 13955A 13988 14009
14027 14098 14105 14108 14161 14198 14204 14213 14273 14279 14287 14291 14294 14309
14321 14348 14387 14390 14396A 14402 14405 14468 14531 14537 14561 14570 14582 14609
14615 14630A 14634 14687 14708 14712 14780 14783 14817 14837 14840 14873 14876
14909A 14933 14939 14951 14961 15005 15011 15038 15059 15077 15080 15167 15173 15194
15206 15210 15212A 15227 15239 15249 15290A 15311 15353 15371 15413 15425 15492
15494 15512 15527 15559 15581 15582A 15590 15627@ 15639 15678 15698 15740 15795
15829 15831 15846 15847 15854 15871 15873C 15878 15881 15917d 15921d 15922d 15927d
15931d 15932d 15933d 15937d 15944C 15953A 15954 15956d 15957 15994@ 15995T 16047

16056T 16067 16074A 16094 16124d 16126d 16128d 16131d 16132 16143d 16228 16260
16264 16294 16302C.
There are the following 220 private mutations (182 in the coding region) in the Bison bison
mtDNA sequence: 8 143 106@ 163 166@ 190 202d 203 205 256 283 701 807 1157 1470d 1474
1492 1496 1497 1627 1892 1948A 1969 2098 2146 2582A 2675 2700A 2952 2991 3181 3334
3385 3400T 3481 3559 3593 3754 3827 3863 4048 4271 4325 4331 4382 4388 4421 4446 4451
4454 4541 4542 4550C 4658 4679A 4722 4728 4734 4799 5003 5009 5108 5177 5224 5240
5375C 5393 5498 5803 5959 6133 6142 6181 6235C 6283 6289 6301 6322 6373 6418 6634A
6661 6859 7051T 7084 7297+T 7297+C 7307 7382 7433 7634 7654 7877 7949 7952 8064 8228
8245T 8436 8512 8646 8730 8748 8766 8872 8890 8925 9095 9176 9185 9233 9416 9480 9497
9644 9901 10125 10577 10621 10681 10717 10840 10843 10903 10924 11014 11020 11104
11305 11413 11468 11651 11662 11692 11716 11722 11752 11789 11815A 11858 12026 12195
12297 12363 12367 12399 12543 12690 12702 12727 12745 12750A 12807 12886 12963 12999
13005@ 13023 13038 13041 13260 13453 13467 13470 13594 13663 13710 13725 13869 14039
14042 14102T 14136 14144 14351 14372 14523C 14558 14638 14702 14834 14990 15105
15156 15201 15269 15302 15419 15422C 15443 15461T 15570A 15706 15895 15914 15916d
15923 15926d 15929C 15939d 15958d 15962G 15966 15989 16045T 16048T 16049@ 16057+A
16058@ 16076 16109 16110 16112 16113 16118d 16119d 16120d 16133 16301 16304.
There are the following 263 private mutations (226 in the coding region) in the B. grunniens
mtDNA sequence: 201 281 292A 379 380 418 525 526 726 740 968 987 1082 1158 1169+T
1193 1226 1328 1470 1489T 1553 1599d 1718 1858+A 1948 2016 2570 2700 2748 2967 2981
3130 3136 3140 3145 3302 3313 3322 3377 3382 3421A 3615 3697 3832 3850 3906 3931 3955
3982 4129 4286 4288 4364 4385 4550T 4622 4634 4853 4859 4878 4894 5087 5102 5105 5259
5267 5288 5395 5614 5631 5983 6019 6229 6232 6235 6256 6565 6607 6658 6748 6793 6799
6877 6895 6904 7015 7114 7132 7276 7367 7418 7509 7523 7565 7643 7697 7868 7946 7991
8000 8110 8114 8245 8332 8406 8421 8439 8529 8838 8845 8868 8871 8934 9092 9096 9420
9422 9479 9518 9545 9590 9867 9927 9945 10002 10020 10119 10253 10293 10343 10577A
10601 10613 10636 10660 10714 10753 10798 10828 10843 10864 11009 11053 11071 11084
11153 11209 11212 11248 11422 11581 11632 11710 11720 11764 11782 11791 11851 11878
12035 12144 12170 12207 12228 12274 12292 12330 12375 12469 12492 12528 12555 12579
12582 12612 12738 12740 12864 12867 12879 12900 12922 12924@ 12945 12969 13086 13143
13255 13336 13434 13503 13506 13692 13899 14051 14063 14102C 14120 14192 14243 14522
14603 14624 14628 14747 14756 14801 14813 14867 14888 14972 15002 15014 15068 15082
15157 15236T 15275 15386 15398 15422T 15434 15440 15461C 15554 15557 15563 15656
15711 15819 15915d 15923A 15929d 15939 15948 15949 15962A 15963T 15965 15974 16050
16051 16057+G 16073 16085 16093 16096 16108d 16109d 16111C 16113A 16116d 16117C
16118C 16121 16137 16147 16164 16199 16204 16205 16232 16308.

Supplemental Tables
Table S1. Sources and Haplogroup Affiliation for the mtDNA Complete Sequences
Sample ID
BRSa
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52

Haplogroup
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T4
T4
T4
T4
T4
T4
T4
T3a
T3a
T3a
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3

GenBank ID
V00654
EU177815
EU177816
EU177817
EU177818
EU177819
EU177820
DQ124415
DQ124407
DQ124408
DQ124413
EU177821
DQ124406
DQ124414
DQ124404
DQ124405
DQ124409
DQ124410
DQ124385
DQ124416
DQ124372
DQ124375
DQ124377
DQ124392
DQ124400
DQ124401
DQ124412
DQ124418
DQ124417
DQ124371
DQ124373
DQ124378
DQ124391
DQ124397
DQ124379
DQ124374
DQ124380
DQ124381
DQ124388
DQ124394
DQ124398
DQ124382
DQ124402
DQ124387
DQ124395
DQ124390
DQ124411
EU177822
EU177823
EU177824
EU177825
EU177826
EU177827

9
53
T3
54
T3
55
T3
56
T3
57
T3
58
T3
59
T3
60
T3
61
T3
62
T3
63
T3
64
T3
65
T3
66
T3
67
T3
68
T1'2'3
69
T1
70
T1
71
T1
72
T1a
73
T1a
74
T1a
75
T1a
76
T1a
77
T1a
78
T2
79
T2
80
T2
81
T2
82
T2
83
T2
84
T2
85
T2
86
T2
87
T2
88
T2
89
T2
90
T2
91
T2
92
T2
93
T2
94
T5
95
T5
96
T5
97
T5
98
Q
99
Q
100
P
101
I1
102
I1
103
I2
104
I2
105
I2
Bison bison
Bos grunniens
a
Bovine reference sequence [S19]

EU177828
DQ124384
EU177829
EU177830
EU177831
EU177832
EU177833
EU177834
EU177835
EU177836
EU177837
DQ124376
DQ124386
EU177838
EU177839
EU177840
EU177841
DQ124399
EU177842
EU177843
EU177844
EU177845
EU177846
EU177847
EU177848
DQ124383
DQ124393
DQ124396
EU177849
EU177850
EU177851
EU177852
EU177853
EU177854
EU177855
EU177856
EU177857
EU177858
EU177859
EU177860
EU177861
EU177862
EU177863
EU177864
EU177865
EU177866
EU177867
DQ124389
NC_005971
EU177868
EU177869
EU177870
AF492350
EU177871
NC_006380

10

Table S2. Divergence of Relevant Haplogroups/Subhaplogroups in the Cattle mtDNA Phylogeny

Maximum Likelihood
c Statistics
Haplogroups/
No. of
Subhaplogroups mtDNAs Substitutions
T
T
T (ya) a
S.E.
T (ya) a

per site
(ya) b
(ya) b
PQTI
106
0.006793
0.000490 332,400 24,000 114.052 7.299 361,800 25,800
PQT
101
0.001520
0.000198 74,400 9,700 25.503 3.303 80,900 10,900
QT
100
0.001067
0.000163 52,200 8,000 16.167 2.650 51,300 8,600
T
98
0.000327
0.000065 16,000 3,200
5.380 0.937 17,100 3,100
T5
4
0.000211
0.000063 10,300 3,100
3.500 1.225 11,100 3,900
T123
94
0.000245
0.000016 12,000
800
4.340 0.413 13,800 1,400
T1 d
9
0.000192
0.000049 9,400
2,400
2.556 0.556 8,100
1,800
T2
16
0.000214
0.000019 10,500
900
4.812 0.634 15,300 2,100
T3 d, e
68
0.000231
0.000019 11,300
900
3.779 0.362 12,000 1,300
T4 d
7
0.000142
0.000037 6,900
1,800
2.286 0.606 7,300
1,900
I
5
0.000663
0.000137 32,400 6,700
8.944 1.900 28,400 6,100
I1
2
0.000108
0.000053 5,300
2,600
2.000 1.000 6,300
3,200
I2
3
0.000223
0.000072 10,900 3,500
3.000 1.000 9,500
3,200
a

Estimate of the time to the most recent common ancestor of each cluster, using an evolutionary rate estimate of 2.043 0.099
10-8 substitutions per site per year in the coding region, corresponding to 3,172 years per substitution in the whole coding
region (15,428 bp). This rate is about 63% faster than the corresponding human rate [S14], in agreement with previous
observation in other domesticated mammals [S23, S24].
b
We used the delta method to compute the standard errors of haplogroup/subhaplogroup ages.
c
The average number of base substitutions in the mtDNA coding region (between nps 364 and 15791) from the ancestral
sequence type. For haplogroups PQTI, PQT, QT, T and I the contribution to from each subclade is weighted based on their
individual standard errors.
d
The ML age for this haplogroup, defined only by a control-region motif, was calculated separately.
e
Including T4 mtDNAs.

11

Table S3. Oligonucleotide Pairs Used to Amplify the Entire Bovine mtDNA in 11 Overlapping PCR
Fragments
OLIGONUCLEOTIDEa
PCR ID
Number
1
2
3
4
5
6
7
8
9
10
11

Fragment
Length
(bp)
1,138
1,935
1,843
1,861
1,869
1,901
1,849
1,881
1,890
1,876
1,751

Melting
Temperature
(C)
59.95

Name

5 np

3 np

Length
(nt)

15718for

15718

15741

24

CCTAAGACTCAAGGAAGAAACTGC

517rev

517

496

22

AACCTAGAGGGCATTCTCACTG

59.77

312for

312

335

24

CAATTTAGCACTCCAAACAAAGTC

59.27

2246rev

2246

2225

22

TTTCCTTAGATGCACTCCTGTG

59.38

1807for

1807

1828

22

TAGCTGGTTGTCCAGAAAATGA

59.75

3649rev

3649

3630

20

TATTGCTAGAGGCCATGCTG

59.04

3339for

3339

3360

22

GCCTAGCCTTAACCATGTGAAT

59.53

5199rev

5199

5178

22

TTATGTTGTTTGTGGAGGGAAA

59.36

4922for

4922

4943

22

TATAGCCAATTCCACCACCACT

60.61

6790rev

6790

6768

23

GTGAAGAACAATATCGAGGGAAG

59.15

6525for

6525

6546

22

TCGGATTTCTAGGTTTCATCGT

59.97

8425rev

8425

8406

20

GGAGGGTTACAAAGCGATTG

59.57

8109for

8109

8132

24

CATATACTCTCCTTGGTGACATGC

59.92

9957rev

9957

9938

20

TCCTATGGGGTCAAATCCAC

59.61

9599for

9599

9620

22

TGGGTCCACCTTCTTAATTGTC

60.22

11479rev

11479

11460

20

AATCATAAGGGCGGTTGCTC

61.33

11201for

11201

11220

20

ATCGCAGGCTCCATAGTCCT

61.15

13090rev

13090

13069

22

GGAGGAAAGCTAGGTAAGGTTG

58.48

12750for

12750

12771

22

TGGACTAGCATTAGCTGCAACC

61.65

14625rev

14625

14608

18

CCAGGAGGGAACCGAAAT

59.86

14210for

14210

14231

22

TTCTGTAGCCATAGCCGTTGTA

59.80

15960rev

15960

15939

22

CCCTTGCGTAGGTAATTCATTC

59.86

Sequence (53)

The annealing temperature for all PCR reactions is 55C. Nucleotide positions correspond to the bovine reference sequence
BRS [S19]

12

Table S4. Oligonucleotides Used for Sequencing the Entire Bovine mtDNA
a

SEQUENCING OLIGONUCLEOTIDE
Template
PCR ID
number
1

Name

5np

3np

Length (nt)

15757for

15757

15776

20

CCCCAAAGCTGAAGTTCTAT

Melting
Temperature
(C)
56.05

16272for

16272

16291

20

TTCTTTCTTCAGGGCCATCT

58.86

1026rev

1026

1007

20

TAGCAAGAATTGGTGAGGTT

54.96

812for

812

829

18

TACAATAGCCGACGCACT

55.33

2163rev

2163

2142

22

GGTCAGAGTATTATCAGGCACT

55.11

1915for

1915

1934

20

GAAACGGATACAACCTTGAC

54.61

2435for

2435

2454

20

CACGAGGGTTTTACTGTCTC

54.85

2902for

2902

2921

20

ACGTATTTCTCCCAGTACGA

54.83

3420for

3420

3439

20

CCATATCAAGCCTAGCCGTA

57.90

3923for

3923

3940

18

GCATCCTACCCTCGATTT

55.03

4450for

4450

4470

21

CTCAATCAACAGCCTCAATAC

54.97

4999for

4999

5016

18

TTGCCACTCTCCTATCCA

54.97

5506for

5506

5525

20

TGGCTTCAATCTACTTCTCC

54.57

6011for

6011

6030

20

TTCCTACTACTCCTCGCATC

54.61

6594for

6594

6613

20

CCTACTTCACATCAGCCACT

55.31

7055for

7055

7074

20

GGCTCATTCATTTCCCTAAC

56.22

7525for

7525

7543

19

CCCATACAAGCACGATAGA

54.62

8180for

8180

8201

22

TTGACCCTTTTTATCATCTTTC

55.16

8684for

8684

8703

20

GAACACCCACTCCACTAATC

54.40

9133for

9133

9152

20

ACCAATGATGACGAGATGTT

55.31

9681for

9681

9700

20

GGTGCCTGATACTGACATTT

55.06

10143for

10143

10164

22

TCAAAAAGGACTAGAATGAACC

55.22

10738for

10738

10757

20

ATGGCTCCTCCCTCTAATAC

54.92

11275for

11275

11295

21

TCTAAACCCTATGACCGACTT

55.51

11803for

11803

11822

20

TATCTCGCCTTCCTTTACAC

54.64

12297for

12297

12318

22

TTCAAACTGACACTGACTAACC

54.53

10

12842for

12842

12861

20

CACTACTCCATTCAAGCACA

54.75

10

13373for

13373

13392

20

ACAGCACCCGTATTATTTTT

54.01

10

13833for

13833

13850

18

CCTGGTCACAAACCAAAA

55.26

11

14353for

14353

14372

20

CCCCATAAATAGGTGAAGGT

55.54

11

14872for

14872

14891

20

TGCTCACAGTAATAGCCACA

55.41

11

15300for

15300

15317

18

AACACACCCCCTCACATC

56.47

Sequence (53)

np corresponds to the bovine reference sequence BRS [S19]

13

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Yang, Z. (1997). PAML: a program package for phylogenetic analysis by

maximum likelihood. Comput. Appl. Biosci. 13, 555556.

S2.

Flerow, C.C. (1980). Zur geographischen verbreitung der gattung Poephagus im

Pleistozn und Holozn. Quartrpalontolog 4, 123126.

S3.

The Paleobiology Database: http://paleodb.org.

S4.

Hassanin, A., and Ropiquet, A. (2004). Molecular phylogeny of the tribe Bovini
(Bovidae, Bovinae) and the taxonomic status of the Kouprey, Bos sauveli Urbain
1937. Mol. Phylogenet. Evol. 33, 308324.

S5.

Ritz, L.R., Glowatzki-Mullis, M.-L., MacHugh, D.E., and Gaillard, C. (2000).

Phylogenetic analysis of the tribe Bovini using microsatellites. Anim. Genet. 31,
178185.

S6.

Saillard, J., Forster, P., Lynnerup, N., Bandelt, H.-J., and Norby, S. (2000).
MtDNA variation among Greenland Eskimos: the edge of the Beringian
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Troy, C.S., MacHugh, D.E., Bailey, J.F., Magee, D.A., Loftus, R.T., Cunningham,
P., Chamberlain, A.T., Sykes, B.C., and Bradley, D.G. (2001). Genetic evidence
for Near-Eastern origins of European cattle. Nature 410, 10881099.

S8.

Mannen, H., Kohno, M., Nagata, Y., Tsuji, S., Bradley, D.G., Yeo, J.S.,
Nyamsamba, D., Zagdsuren, Y., Yokohama, M., Nomura, K., et al. (2004).
Independent mitochondrial origin and historical genetic differentiation in North
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S9.

Beja-Pereira, A., Caramelli, D., Lalueza-Fox, C., Vernesi, C., Ferrand, N., Casoli,
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European cattle: evidence from modern and ancient DNA. Proc. Natl. Acad. Sci.
USA 103, 81138118.

S10. Lai, S.-J., Liu, Y.-P., Liu, Y.-X., Li, X.-W., and Yao, Y.-G. (2006). Genetic
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Maternal and paternal lineages in cross-breeding bovine species. Has wisent a
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Brandon, M., Easley, K., Chen, E., Brown, M.D., et al. (2003). Natural selection
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14

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