Supplementary Material
Sections:
I
II
III
IV
V
VI
VII
VIII
Primer Design
PCR Protocol
Sequence Assembly
Identification of Heterozygous Sites and Sequence Alignment
Primate Samples
EvoNC hypothesized positively selected sites
Supplementary tables
References
I─Primer Design
To design primers, we searched for conserved regions using the human, chimpanzee and
rhesus macaque DARC sequences downloaded from GenBank (see figure 1 below).
II ─ PCR Protocol
DNA was divided into 1µL aliquots and mixed with 2.5µL Biotools buffer, 2µL 10mM
dNTPs, 1.5µL 20µM Forward and Reverse primers, 1µL of Taq (Biotools) and H2O to 20µL.
PCR reactions were run – 1x(94°C-4min), 1x(annealing temperature – Table1), 35x(72°C-50sec,
94°C-40sec, annealing temperature-40sec) 1x(72°C-10min) 1x(4°C-forever).
Annealing temperature for each pair of primers
Primer
DARCp1
DARCp2
DARCp3
DARCp4
DARCp5
DARCp6
DARCp7
DARCp8
Annealing Temperature (°C)
59
58
59
57
58
58
61
58
III ─ Sequence Assembly
The sequence assembly was done using the software programs phred (Ewing and Green
1998; Ewing et al. 1998) and phrap (Gordon et al. 2001). Reference sequences from NCBI in
1
FASTA format were used to guide the assembly. These sequences were converted into PHD files
(phred format file) and each base received the value 18 as phred quality score.
All Old World monkeys samples were assembled using a genomic reference sequence
from Macacamulatta and New-World monkeys samples were assembled using a
Saimiriboliviensissequence from Genbank (accession number:AF3119180). Cebusapella and
Gorilla gorilla samples were assembled without reference sequences.
IV─Identification of Heterozygous Sites and Sequence Alignment
The identification of heterozygous sites using chromatogram files was done by PolyPhred 6.11
(Nickerson et al. 1997) andPolyscan 3.0 (Chen et al. 2007). Results were then manually inspected
with the Consed program (Gordon et al. 1998) and sites with heterozygote chromatogram profiles
were substituted by ambiguity symbol defined by IUPAC.
Multiple global alignments were implementedusingClustalW (Larkin et al. 2007) using default
parameters.
2
V ─Primate Samples
Species
Source of DNA
Saimirisciureus
Department of Genetics and Molecular Biology, Federal University of Pará,Pará State, Brazil
Saimiriustus
Department of Genetics and Molecular Biology, Federal University of Pará, Pará State, Brazil
Cebusapella
Department of Genetics and Molecular Biology, Federal University of Pará, Pará State, Brazil
Gorilla gorilla
Department of Genetics and Molecular Biology, Federal University of Pará, Pará State, Brazil
Macacafascicularis
Nonhuman primate tissue samples from Covance, Inc., Princeton, NJ, USA
Macacamulatta
Nonhuman primate tissue samples from Covance, Inc., Princeton, NJ, USA
Macacanemestrina
Coriell Institute, IPBIR, Camden, NJ, USA(individual ID number NG07921)
Macacathibetana
Coriell Institute, IPBIR, Camden, NJ, USA(individual ID number PR00711)
Macacanigra
Coriell Institute, IPBIR, Camden, NJ, USA(individual ID number NG07101)
Mandrillus sphinx
San Diego Zoo/CRES, CA, USA (Drs. Oliver Ryder and Leona Chemnick)
M.leucophaeus
San Diego Zoo/CRES, CA, USA (Drs. Oliver Ryder and Leona Chemnick)
Lophocebusaterrimus
Tulane Regional Primate Research Center, LA, USA (Dr. Bobby Gormus)
Cercocebustorquatuslunatus
Tulane Regional Primate Research Center, LA, USA (Dr. Bobby Gormus)
C.galerituschrysogaster
Charles Paddock Zoo, Atascadero, CA, (Dr. Cathi Lehn, Texas A&M, TX, USA)
Cercopithecusmitis
Animal Research Center, The University of Texas at Austin, USA (Jim Letchworth)
* See Material and Methods for information about geographic origin of samples.
VI-EvoNC hypothesized positively selected sites and their posterior probability values
Site
Site
Site
Site
Site
Site
Site
5: 0.989037
154: 0.932526
296: 0.926272
486: 0.926413
581: 0.919703
663: 0.925781
762: 0.933935
Site
Site
Site
Site
Site
Site
Site
6: 0.930001
247: 0.989254
302: 0.942589
513: 0.933935
588: 0.929422
674: 0.927571
765: 0.925887
Site
Site
Site
Site
Site
Site
Site
3
77: 0.920158
263: 0.920457
329: 0.937435
517: 0.914334
605: 0.915384
704: 0.996492
792: 0.925783
Site
Site
Site
Site
Site
Site
Site
132: 0.923882
278: 0.934672
466: 0.920173
577: 0.914334
644: 0.987727
729: 0.929422
843: 0.922005
Figure 1.-Scheme showing the strategy used for primers design.
4
VII ─ Supplementary
Tables
Table 1. Likelihood values, parameter estimates and likelihood ratio statistics (2ΔlnL)
under branch-sites model A for hominoid clade of phylogenetic tree.
2ΔlnL
Model
ℓ
estimate of parameters
Positively Selected Sites
A ()
A ( free)
-2393.314268
-2392.498033
0=0.0000
1=1.0000
2=1.0000
p0=0.41717
p1=0.32803
p2a+p2b=0.2548
0=0.0000
p0=0.5709
1=1.0000
2=4.90174
p1=0.39258
p2a+p2b=0.10025
−
0.254058
None with P > 70%
Note: The parameters x(x = 0,1 and 2)are thedN/dS ratio included in each model.
Table 2. Likelihood values, parameter estimates and likelihood ratio statistics (2ΔlnL)
under models of variable  ratios among sites (M1a, M2a, M0, M3, M7 and M8).
2ΔlnL
Model
p
ℓ
Positively Selected Sites
Distribution
M1a: neutral
2
-2393.9340
M2a: selection
3
-2392.0471
M0: one ratio
M3: discrete
1
5
-2403.2928
-2392.0471
M7: beta
M8: beta&
2
4
-2393.9880
-2392.0471
M8a: beta&
4
-2394.3872
0=0.0000
1=1.0000
0=0.2315
1=1.0000
2=2.4734
p0=0.52041
p1=0.4796
p0=0.8484
p1=0.0000
p2=0.1516
0=0.5261
0=0.2313
1=0.2315
2=2.4734
p0=0.2023
p1=0.6461
p2=0.15.16
p=0.0050
p0= 0.8505
p=30.3189
q=99.0000
p0= 0.5204
p=0.0050
q=1.3173
Not Allowed
3.7739
22.4915*
q=0.0050
p1=0.1495
=2.4908
7. 22. 25. 28. 31. 34. 39. 211. 332
(0.5 < P < 0.87)
7. 22. 25. 28. 39. 332 (P > 0.95)
Not Allowed
3.8817
p1=0.4796
=1
7. 22. 25. 28. 31. 34. 39. 122. 124.
211. 329. 332 (0.53 < P< 0.93)
Not Allowed
4.6801
Note:p is the number of parameters of each model. The parameters x(x = 0,1 and 2)are thedN/dS ratio included in each model.
* Significant values (P < 1%; 2 = 13.48).
Table 3. Likelihood values, parameter estimates and likelihood ratio statistics (2ΔlnLTa)
under model for partitioned data.
ℓ
r1
r2
r3
r4


A: (≠ rs)
-2396.7621
1
0.5013
0.5264
0.6745
4.6886
0.5166
B: (≠ rs , S)
-2373.4453
1
0.6084
0.5693
0.7165
4.6405
C: (≠ rs, , )
-2388.4290
1
0.5084
0.5405
0.7004
Model
0.5412
1=3.1194
3=5.0850
=1.0329
=0.6967
2=4.3222
4=16.2266
=0.3152
=0.6633
Cnull: (≠ rs, )
-2389.9189
1
0.5084
0.5405
0.7264
-
-
=1
D: (≠ rs, , , S)
-2364.4738
1
0.6107
0.5717
0.7167
1=3.216
3=5.2231
=1.1190
=0.5138
2=4.3324
4=16.0279
=0.3432
=0.4950
-
-
=1
Dnull: (≠ rs, , , S)
-2366.3254
1
0.6107
0.5717
0.7536
2ΔlnL
2.9799
3.7032
Note:Parameters xand x (x = 1, 2, 3 and 4) are, respectively, dN/dS ratio and transition/transversion ratio for each of four defined
regions. Parameters r1, r2, r3 and r4 are the codon frequencies for each region.
5
VIII─ References
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Ewing B, Hillier L, Wendl MC, Green P (1998) Base-calling of automated sequencer traces using
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Ewing B, Green P (1998)Base-calling of automated sequencer traces using phred. II. Error
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Gordon D,Abajian C, Green P (1998) Consed: a graphical tool for sequence finishing. Genome
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Gordon D,Desmarais C, Green P (2001) Automated finishing with autofinish. Genome Res
11(4):614–25.
Guindon S,Gascuel OA (2003) simple, fast, and accurate algorithm to estimate largephylogenies
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Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F,
Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and
Clustal X version 2.0. Bioinformatics 23(21):2947–2948.
Nickerson DA,Tobe VO, Taylor SL (1997) PolyPhred: automating the detection and genotyping
of single nucleotide substitutions using fluorescence-basedresequencing. Nucleic Acids Res
25(14):2745–51.
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