Figure S1
Supplementary Information accompanies the paper on www.nature.com/nature.
Hittinger et al.
Table S1. Summary of sequenced strains, sequencing coverage, and spore viability. See attached.
Table S2. Summary of numbers of GAL sites available for various analyses. See attached.
Table S3. Constitutive expression of the GAL network in S. cerevisiae is deleterious under non-inducing conditions. See attached.
Table S4. List of strains used in experiments. See attached.
Figure S1. The S. kudriavzevii GAL loci segregate independently. Histogram of the number of functional GAL+ loci recovered from F2 segregants from a cross of a Gal+ Portuguese strain and a GalJapanese strain. Spore viability was high (82%), and alleles at the GAL loci assort independently (P = 0.36). Blue, observed; red, expected. See attached.
Figure S2. The wild isolates of S. kudriavzevii are not hybrids. Proportion of hits to each Saccharomyces species (y-axis) for each gene (x-axis, sorted by systematic name in S. cerevisiae) based on Solexa short-read data from the Portuguese reference strain (a), a known1 S. cerevisiae/S. kudriavzevii hybrid (b), and another S. cerevisiae/S. kudriavzevii hybrid not isolated from the wild (c). By definition, the sum of the proportion of hits from each species is 1.0 for each gene. Therefore, the closer the value of S. kudriavzevii (blue diamond) is to 1.0 for a given gene, the more confidently introgression can be excluded. In the two hybrid strains, regions are clearly evident where only one species has contributed genetic material, as are likely regions of aneuploidy. Note that Database S1 contains the complete unfiltered data for all 20 strains, as well as analyses with more liberal mismatch criteria, but no convincing evidence of introgression. See attached.
Figure S3. Relaxed molecular clock estimation of coalescence of GAL and non-GAL loci. Estimation of relative timing of coalescence of Saccharomyces GAL genes and pseudogenes (a), an equivalent number of sites selected randomly from non-GAL genes (b), and an equivalent number of sites selected randomly from non-GAL genes using all strains of S. kudriavzevii shown in Fig. 1c. Scales show estimated
2
substitutions per site; branch lengths are printed immediately above or below their respective internodes or above the population whose coalescence they estimate. See attached.
Figure S4. No sequenced strains of Saccharomyces support introgression as the source of the functional S. kudriavzevii GAL genes. Phylogeny built from aligned and concatenated GAL genes and pseudogenes from Fig. 1a with strains added from the imputed Q20 dataset of the Saccharomyces Genome Resequencing Project2, which includes the previously described species of Saccharomyces boulardii and Saccharomyces cariocanus as strains of S. cerevisiae and S. paradoxus, respectively. Values correspond to Bayesian posterior probabilities and bootstrap values obtained with maximum likelihood, respectively. Scales show estimated substitutions per site under the Bayesian framework. Internodes corresponding to recognized species are darker, while Portuguese (blue) and Japanese (red) lineages are colored. See attached.
Figure S5. The divergence of the GAL loci contrasts sharply with the rest of the genome at nearly all sites. Sliding window estimates of pairwise divergence (d) between ZP591 and IFO1802T: GAL80 (a), GAL4 (b), GAL7/GAL10/GAL1 (c), and GAL2 (d). GAL coding regions are red and oriented from left to right (except GAL7 and GAL10), while intergenic regions are black. Tick marks on the x-axis represent 100 aligned bps, and a dashed line shows the genome-wide background of 0.011. Note that nearly all promoters and untranslated regions also possess elevated divergence levels. See attached.
Database S1. Complete supporting data for Figure S2. See Excel file.
Database S2. Divergence of all genes between Japanese (IFO1802T) and Portuguese (ZP591) reference strains. See Excel file.
1
Gonzalez, S. S., Barrio, E., Gafner, J. & Querol, A. Natural hybrids from Saccharomyces
cerevisiae, Saccharomyces bayanus and Saccharomyces kudriavzevii in wine fermentations. FEMS Yeast Res 6, 1221-1234, (2006).
2
Liti, G. et al. Population genomics of domestic and wild yeasts. Nature 458, 337-341, (2009).
2
Table S1. Summary of sequenced strains, sequencing coverage, and spore viability.
Hittinger et al.
Strain
Nationality Locality
Substrate
Isolation date Sequenced strain Coverage10
ZP513
Portugal
Lisbon
Q. pyrenaica
Mar. 2005
FM10691
87% (7807789)
ZP537
Portugal
L. Albufeira
Q. faginea
May 2005
FM10562
82% (3097180)
ZP542
Portugal
Adagoi
Q. pyrenaica
Jun. 2005
FM10572
73% (3269665)
ZP591
Portugal
Cast. Vide
Q. pyrenaica
Aug. 2005
FM10092
94% (12459524)
ZP594
Portugal
A. Dez
Q. faginea
Sep. 2005
FM10781
84% (3138597)
88% (16)
4
ZP595
Portugal
A. Dez
Q. faginea
Sep. 2005
FM10791
79% (5087661)
90% (20)
4
ZP620
Portugal
Arrábida
Q. ilex
Oct. 2005
FM10721
81% (3081430)
100% (14)
4
ZP621
Portugal
Arrábida
Q. ilex (accorn)
Oct. 2005
FM10731
73% (3882742)
94% (16)
4
ZP623
Portugal
Arrábida
Q. ilex (leaf litter)
Oct. 2005
FM10741
76% (5347358)
95% (20)
ZP625
Portugal
Arrábida
Q. faginea
Oct. 2005
FM10622
71% (2786785)
0% (12)
ZP627
Portugal
Arrábida
Q. faginea
Oct. 2005
FM10751
75% (3104399)
100% (16)
4
ZP629
Portugal
L. Albufeira
Q. ilex
Dec. 2005
FM10761
81% (2763826)
100% (16)
4
ZP630
Portugal
L. Albufeira
Q. ilex
Dec. 2005
FM10771
85% (4472512)
88% (16)
ZP634
Portugal
Cast. Vide
Q. pyrenaica
Jan. 2006
FM10662
68% (2864521)
0% (8)
IFO1802
Japan
Mt. Daisen
partially decayed leaf
< 1992
IFO18022
100% (reference)3 IFO18022
IFO10990 Japan
Mt. Daisen
soil
< 2005
IFO109902
IFO10991 Japan
Mt. Daisen
partially decayed leaf
< 2005
IFO1803
Japan
Yakushima Island partially decayed leaf
W27
Switzerland Waedenswil
Lallemand wine strain
CBS679
Other strain Other Coverage Spore viability11 Notes
FM10941
FM10711
67% (2728243)
82% (4544569)
mixture of 25 bp and 36 bp reads
4
11% (28)
No MATa or HMR; ZP537 partially endoreduplicated but not FM1094
4
0% (24)
reason for sterility unknown; 34 bp reads after trimming "GT" barcode
4
94% (16)
Portuguese reference strain; mixture of 32 bp and 36 bp reads (ZP591)
4
4 No MATα or HML; 34 bp reads after trimming "CT" barcode
4 4
type strain and originally sequenced strain
5
89% (3222318)
34 bp reads after trimming "GT" barcode
6
IFO109912
87% (3294821)
34 bp reads after trimming "TT" barcode
6
< 1992
IFO18032
86% (8542642)
very distant subspecies; mixture of 34 bp reads after trimming "CT" barcode and 36 bp reads
5
< 1995
FM1043
NA (635858)
known hybrid of S. cerevisiae and S. kudriavzevii; 34 bp reads after trimming "AT" barcode
7, 8
< 1913
FM1054
NA (755492)
hybrid of S. cerevisiae and S. kudriavzevii; 34 bp reads after trimming "TT" barcode
9
67% (2710233)3
0% (20)
Single spore derivative.
2
Wild derivative, presumed diploid.
3
The reference sequence was assumed correct and complete when analyzing other strains, and the Solexa data was used only to estimate the error rate of the assemblies.
4
Sampaio JP, Gonçalves P. 2008. Natural populations of Saccharomyces kudriavzevii in Portugal are associated with oak bark and are sympatric with S. cerevisiae and S. paradoxus. Appl Environ Microbiol 74: 2144-2152.
5
Kaneko Y, Bano I. 1991. Reexamination of Saccharomyces bayanus strains by DNA-DNA hybridization and electrophoretic karyotyping. IFO Res Commun 15: 30-41.
6
Mikata K. Japan National Institute of Technology and Evaluation Biological Resource Center (http://www.nbrc.nite.go.jp).
7
Schuetz M, Gafner J. 1994. Dynamics of the yeast strain population during spontaneous alcoholic fermentation determined by CHEF gel electrophoresis. Lett Appl Microbiol 19: 253-257.
8
González SS, Barrio E, Gafner J, Querol A. 2006. Natural hybrids from Saccharomyces bayanus and Saccharomyces kudriavzevii in wine fermentations. FEMS Yeast Res 6: 1221-1234.
9
Guilliermond A. 1912. Deposited into CBS culture collection.
11
4
No MATa or HMR; 34 bp reads after trimming "AT" barcode
1
10
Reference
62% (16)
Coverage shows the percent of called bases, relative to the IFO1802T reference sequence described in the Methods, while the number of non-adapter (no exact 15-mer hits) sequencing reads obtained is in parentheses.
Spore viability shows the percent of viable spores with the number of spores tested in parentheses.
Table S2. Summary of numbers of GAL sites available for various analyses. 1 2 3 Gene Pseudogene (bp) Codon (bp) S YML053C 573 121 SUR7 909 211 GAL80 1673 1104 254 AIM32 936 206
YPL247C GAL4 GYP5 RPL36B KAP104 GAL7 GAL10 GAL1 FUR4 POA1 SIC1 EMP46 GAL2 SRL2 EMP70
2324
3369 3369 3369
1825
dS4 (0.00, 0.04) (0.04, 0.12) (0.39, 0.62) (0.19, 0.34)
1569 355 (0.01, 0.03) 1560 346 (0.49, 0.71) 2907 558 (0.22, 0.32) 246 58 (0.00, 0.00) 2742 354 561 714 1902 534
586 78 133 164 436 123
(0.03, 0.06) (0.83, 2.48) (0.98, 2.26) (1.06, 2.30) (0.14, 0.22) (0.01, 0.09)
864 1347 645 1179 1995
194 289 153 242 464
(0.01, 0.06) (0.02, 0.07) (1.24, saturated) (0.64, 1.02) (0.01, 0.04)
1
Hittinger et al.
Phylogenetics5 All strains6 Upstream7 Coding8 Downstream9
1068
1017
225
996
199
1659
1339
248
1525
176
360 573 744
352 552 690
0 35 35
271 486 569
135 0 493
456
435
70
559
145
Length of pseudogenes in IFO1802T (entire intergenic region between adjacent functional genes). 2 Aligned bp remaining after including only fully aligned codons between ZP591, IFO1802T, S. bayanus, S. mikatae, S. paradoxus, and S. cerevisiae. 3 Synonymous sites between ZP591 and IFO1802T (Fig. 2). 4 95% confidence interval of average number of synonymous differences between ZP591 and IFO1802T (low, high) (Fig. 2). 5 Number of bp in phylogenetic data matrix with S. castellii and IFO1803 added (Fig. 1a, S3a). 6 Number of bp in phylogenetic data matrix with full strain set (Fig. S4). 7 Number of upstream bp aligned between ZP591 and IFO1802T (Fig. S5). 8 T Number of coding bp aligned between ZP591 and IFO1802 (Fig. S5). 9 Number of downstream bp aligned between ZP591 and IFO1802T (Fig. S5). Missing data was either not calculated or not applicable.
Hittinger et al. Table S3. Constitutive expression of the GAL network in S. cervisiae is deleterious under non-inducing conditions. Genotype 2% Raffinose (non-induced) 2% Glucose (repressed) 2% Galactose (induced) GAL80+ 0.000 ± 0.001 0.000 ± 0.001 0.000 ± 0.001 gal80 -0.116 ± 0.003 -0.008 ± 0.003 +0.069 ± 0.003 Malthusian selection coefficients are reported ± s. d. with N = 12 as described in the Methods. Note that mutants lacking the Gal80 co-repressor are very unfit in non-inducing conditions, 1 as previously observed in 5% glycerol by MacLean in a study of GAL mutants 2 created by the systematic deletion project . 1
Maclean RC. 2007. Pleiotropy and GAL pathway degeneration in yeast. J Evol Biol 20: 1333-1338. Giaever G et al. 2002. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418: 387-391.
2
Table S4. List of strains used in experiments. Strain FM1009 FM1071 FM1097
Species S. kudriavzevii S. kudriavzevii S. kudriavzevii
Population Portuguese Portuguese Japanese
Derived from ZP591 ZP591 IFO1802T
FM1098 FM1109 FM1110 FM1111 FM1112 FM1123 FM1131
S. S. S. S. S. S. S.
Japanese Portuguese Portuguese hybrid hybrid Portuguese Portuguese
IFO1802 FM1071 FM1071 FM1097/FM1109 FM1098/FM1110 FM1109 FM1110
kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii
T
FM1142 S. kudriavzevii Portuguese FM1146 S. kudriavzevii Portuguese
FM1123 FM1142
FM1153 S. kudriavzevii Portuguese
FM1071/FM1146
FM1157 S. kudriavzevii Portuguese FM1159 S. kudriavzevii Portuguese
FM1123/FM1131 FM1153
FM1160 S. kudriavzevii Portuguese FM1161 S. kudriavzevii Portuguese
FM1153 FM1153
FM1162 S. kudriavzevii Portuguese
FM1153
FM1163 S. kudriavzevii Portuguese FM1164 S. kudriavzevii Portuguese
FM1153 FM1153
FM1165 FM1166 FM1183 FM1184 FM1185 FM1186 FM1187 FM1188 FM1189 FM1190 FM1282 FM1283
FM1153 FM1153 FM1157 FM1157 FM1157 FM1157 FM1157 FM1157 FM1157 FM1157 BY4724 BY4724
S. S. S. S. S. S. S. S. S. S. S. S.
kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii cerevisiae cerevisiae
Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese laboratory laboratory
Hittinger et al. Genotype MATa/MAT MATa/MAT
Use Table S1 Table S1
Notes 1 2
MAT ho ::natMX
3
MATa ho ::natMX MATa ho ::kanMX MAT ho ::kanMX MATa/MAT ho ::kanMX/ho ::natMX MATa/MAT ho ::kanMX/ho ::natMX MATa ho ::kanMX ura3- MAT ho ::kanMX trp1- + MATa ho ::kanMX ura3- trp1 ::ScerURA3
3 3 3 4 4 5 6 7
Fig. S1 Fig. S1
MATa ho :kanMX ura3- trp1 ::ScerGAL3+ +
8
+
+
MATa/MAT ho ::kanMX/HO ura3- /URA3 trp1 ::ScerGAL3 /TRP1 + + MATa/MAT ho ::kanMX/ho ::kanMX ura3- /URA3 trp1- /TRP1
+
9 9
MATa ho ::kanMX ura3- trp1 ::ScerGAL3+ + MATa ho ::kanMX ura3- trp1 ::ScerGAL3
Fig. 3c Fig. 3c
MATa ho ::kanMX ura3- trp1 ::ScerGAL3+
Fig. 3c
MATa ho ::kanMX ura3- trp1 ::ScerGAL3 + MATa ho ::kanMX ura3- trp1 ::ScerGAL3
+
Fig. 3c Fig. 3c
MATa ho ::kanMX ura3- trp1 ::ScerGAL3+ + MATa ho ::kanMX ura3- trp1 ::ScerGAL3
Fig. 3c Fig. 3c
MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa
+
ho ::kanMX ura3- trp1 ::ScerGAL3 ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ura3- lys2- PTDH3-yEGFP-TCYC1 ura3- lys2- PTDH3-yBGFP-TCYC1
Fig. 3c Fig. 3c Fig. 3c Fig. 3c Fig. 3c Fig. 3c Fig. 3c Fig. 3c Fig. 3c Table S3 Table S3
10 11
+
FM1284 S. cerevisiae
laboratory
FM1282
MATa ura3- lys2- PTDH3-yEGFP-TCYC1 GAL80
Table S3
12
FM1285 S. cerevisiae
laboratory
FM1282
+ MATa ura3- lys2- PTDH3-yEGFP-TCYC1 GAL80
Table S3
12
FM1286 S. cerevisiae FM1287 S. cerevisiae
laboratory laboratory
FM1282 FM1282
lys2- PTDH3-yEGFP-TCYC1 GAL80+
Table S3
12
FM1288 S. cerevisiae FM1289 S. cerevisiae
laboratory laboratory
FM1282 FM1282
MATa ura3- MATa ura3- MATa ura3- MATa ura3-
lys2- PTDH3-yEGFP-TCYC1 gal80- lys2- PTDH3-yEGFP-TCYC1 gal80- lys2- PTDH3-yEGFP-TCYC1 gal80-
Table S3 Table S3 Table S3
13 13 13
FM1290 S. cerevisiae
laboratory
FM1282
MATa ura3- lys2- PTDH3-yEGFP-TCYC1 GAL3+
Fig. 3c
14
FM1291 S. cerevisiae
laboratory
FM1282
MATa ura3- lys2- PTDH3-yEGFP-TCYC1 GAL3
+
Fig. 3c
14
+
lys2- PTDH3-yEGFP-TCYC1 GAL3 lys2- PTDH3-yEGFP-TCYC1 gal3- lys2- PTDH3-yEGFP-TCYC1 gal3-
Fig. 3c Fig. 3c Fig. 3c
14 15 15
lys2- PTDH3-yEGFP-TCYC1 gal3-
Fig. 3c
FM1292 FM1293 FM1294 FM1295
S. S. S. S.
cerevisiae cerevisiae cerevisiae cerevisiae
laboratory laboratory laboratory laboratory
FM1282 FM1282 FM1282 FM1282
MATa MATa MATa MATa
ura3- ura3- ura3- ura3-
FM1332 FM1334 FM1335 FM1336 FM1337
S. S. S. S. S.
kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii
Portuguese Portuguese Portuguese Portuguese Portuguese
FM1123 FM1123 FM1123 FM1123 FM1123
MATa MATa MATa MATa MATa
ho ::kanMX ho ::kanMX ho ::kanMX ho ::kanMX ho ::kanMX
FM1338 FM1340 FM1343 FM1345 FM1346 FM1348 FM1351 FM1352 FM1353 FM1354 FM1355 FM1356 FM1357 FM1358 FM1359 FM1360 FM1361 FM1362
S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.
kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii
Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese
FM1123 FM1123 FM1123 FM1123 FM1123 FM1123 FM1132 FM1132 FM1132 FM1134 FM1135 FM1135 FM1136 FM1137 FM1137 FM1137 FM1138 FM1138
MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa
+
ura3- ura3- ura3- ura3-
gal80 ::ScerURA3 gal80 ::ScerURA3+ gal80 ::ScerURA3+ gal80 ::ScerURA3+
ura3- ho ::kanMX ura3- ho ::natMX ura3- ho ::natMX ura3- ho ::natMX ura3- ho ::natMX ura3- ho ::natMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3-
gal80 ::ScerURA3 + gal80 ::ScerURA3
+
gal80 gal80 gal80 gal80 gal80 gal80 gal80 gal80 gal80 gal80 gal80 gal80
15 16 16 16 16
Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.
3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b
16 16 17, 18 17 17 17 17 19 19 19 19 19 19 19 19 19 19 19 19
1
Lab stock of wild ZP591 isolate; Portuguese reference strain20. Homozygote of ZP591 derived from a single spore. 3 Heterothallic single spore isolate after deletion of HO. 4 F1 Japanese/Portuguese strain sporulated to generate F2 segregants. 2
5
Start codon to stop codon deletion made transforming with PCR product and selecting against URA3+ . Start codon to stop codon deletion made transforming with PCR product and selecting against TRP1+ . + Start codon to stop codon replacement made transforming with PCR product and selecting for URA3 plus its intergenic sequences. 8 Start codon to stop codon replacement made transforming with PCR product containing ScerGAL3+ plus its intergenic sequences and selecting against URA3+ . 9 Cross used to generate panel of experimental strains. 10 GFP competition strain21. 11 BFP competition strain21. 12 + + + GAL80 re-introduced in triplicate strains to MATa ura3- lys2- PTDH3-yEGFP-TCYC1 gal80 ::URA3 by transforming with PCR product and selecting against URA3 . 6 7
13
URA3+ removed from MATa ura3- lys2- PTDH3-yEGFP-TCYC1 gal80 ::URA3+ by transforming with engineered oligonucleotides and selecting against URA3+.
14
GAL3+ re-introduced in triplicate strains to MATa ura3- lys2- PTDH3-yEGFP-TCYC1 gal3 ::URA3+ by transforming with PCR product and selecting against URA3+21.
15
+
+
+21
URA3 removed from MATa ura3- lys2- PTDH3-yEGFP-TCYC1 gal3 ::URA3 by transforming with engineered oligonucleotides and selecting against URA3
16
Start codon to stop codon replacement made transforming with PCR product and selecting for ScerURA3+ plus its intergenic sequences. 17 Drug marker changed by selecting for natMX, control competition strains competed against parental FM1123. 18 Competition strain with no detectable defect. 19 Start codon to stop codon deletion made transforming with PCR product and selecting against URA3+ , competed against FM1340. 20 Sampaio JP, Gonçalves P. 2008. Natural populations of Saccharomyces kudriavzevii in Portugal are associated with oak bark and are sympatric with S. cerevisiae and S. paradoxus. Appl Environ Microbiol. 74: 2144-2152. 21 Hittinger CT, Carroll SB. 2007. Gene duplication and the adaptive evolution of a classic genetic switch. Nature 449: 677-681.
.
Hittinger et al.
Figure S1 50
Number of F2 segregants
40
30 Observed Expected 20
10
0 0
1
2
Number of functional GAL+ loci
3
4
Figure S2
a
Hittinger et al.
Proportion of hits
1.0 0.9
S. kudriavzevii
0.8
S. cerevisiae
0.7
S. paradoxus
0.6
S. mikatae
0.5
S. bayanus
0.4 0.3 0.2 0.1
Genes
ZP591 (Portuguese S. kudriavzevii)
Genes
W27 (known hybrid)
Genes
CBS679 (hybrid)
0.0
b
1.0 0.9
Proportion of hits
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
c
1.0 0.9
Proportion of hits
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
Hittinger et al.
Figure S3 0.0644
a
S. cerevisiae
0.0514 0.0644
S. paradoxus
0.0828
0.0500
0.1158
S. mikatae
0.0603 0.1711
0.0581
0.0276
0.1883
S. kudriavzevii ZP591 S. kudriavzevii IFO1802T
0.1130 0.0581
S. kudriavzevii IFO1803
0.2589
S. bayanus
0.4472
S. castellii
b
0.0455
S. cerevisiae
0.0455
S. paradoxus
0.0395 0.0362
0.0300
0.0850
S. mikatae
0.0442
0.0042
S. kudriavzevii ZP591
0.0166
S. kudriavzevii IFO1802T
0.1004
0.0727
0.0042 0.0209
0.1654
S. kudriavzevii IFO1803 S. bayanus
0.2382
S. castellii
c 0.0010 0.0020
ZP591 Portuguese Population
0.0037
0.0094 0.0008 0.0039
0.0141
IFO1802T Japanese Population IFO1803
Hittinger et al.
Figure S4 S. bayanus
98/91
S. kudriavzevii
100/100
S. mikatae
99/84
99/100
99/100
S. paradoxus
99/100
99/100
S. cerevisiae
S. castellii
0.08
Hittinger et al.
Figure S5 a
b
0.4
0.4
GAL80
0.3
0.3
d
GAL4
d 0.2
0.2
0.1
0.1
0
0
c
d
0.6
0.6
0.5 0.4
d
GAL7
GAL10
0.5
GAL2
GAL1
0.4
d 0.3
0.3
0.2
0.2
0.1
0.1
0
0
Hittinger et al.
Table S1. Summary of sequenced strains, sequencing coverage, and spore viability. See attached.
Table S2. Summary of numbers of GAL sites available for various analyses. See attached.
Table S3. Constitutive expression of the GAL network in S. cerevisiae is deleterious under non-inducing conditions. See attached.
Table S4. List of strains used in experiments. See attached.
Figure S1. The S. kudriavzevii GAL loci segregate independently. Histogram of the number of functional GAL+ loci recovered from F2 segregants from a cross of a Gal+ Portuguese strain and a GalJapanese strain. Spore viability was high (82%), and alleles at the GAL loci assort independently (P = 0.36). Blue, observed; red, expected. See attached.
Figure S2. The wild isolates of S. kudriavzevii are not hybrids. Proportion of hits to each Saccharomyces species (y-axis) for each gene (x-axis, sorted by systematic name in S. cerevisiae) based on Solexa short-read data from the Portuguese reference strain (a), a known1 S. cerevisiae/S. kudriavzevii hybrid (b), and another S. cerevisiae/S. kudriavzevii hybrid not isolated from the wild (c). By definition, the sum of the proportion of hits from each species is 1.0 for each gene. Therefore, the closer the value of S. kudriavzevii (blue diamond) is to 1.0 for a given gene, the more confidently introgression can be excluded. In the two hybrid strains, regions are clearly evident where only one species has contributed genetic material, as are likely regions of aneuploidy. Note that Database S1 contains the complete unfiltered data for all 20 strains, as well as analyses with more liberal mismatch criteria, but no convincing evidence of introgression. See attached.
Figure S3. Relaxed molecular clock estimation of coalescence of GAL and non-GAL loci. Estimation of relative timing of coalescence of Saccharomyces GAL genes and pseudogenes (a), an equivalent number of sites selected randomly from non-GAL genes (b), and an equivalent number of sites selected randomly from non-GAL genes using all strains of S. kudriavzevii shown in Fig. 1c. Scales show estimated
2
substitutions per site; branch lengths are printed immediately above or below their respective internodes or above the population whose coalescence they estimate. See attached.
Figure S4. No sequenced strains of Saccharomyces support introgression as the source of the functional S. kudriavzevii GAL genes. Phylogeny built from aligned and concatenated GAL genes and pseudogenes from Fig. 1a with strains added from the imputed Q20 dataset of the Saccharomyces Genome Resequencing Project2, which includes the previously described species of Saccharomyces boulardii and Saccharomyces cariocanus as strains of S. cerevisiae and S. paradoxus, respectively. Values correspond to Bayesian posterior probabilities and bootstrap values obtained with maximum likelihood, respectively. Scales show estimated substitutions per site under the Bayesian framework. Internodes corresponding to recognized species are darker, while Portuguese (blue) and Japanese (red) lineages are colored. See attached.
Figure S5. The divergence of the GAL loci contrasts sharply with the rest of the genome at nearly all sites. Sliding window estimates of pairwise divergence (d) between ZP591 and IFO1802T: GAL80 (a), GAL4 (b), GAL7/GAL10/GAL1 (c), and GAL2 (d). GAL coding regions are red and oriented from left to right (except GAL7 and GAL10), while intergenic regions are black. Tick marks on the x-axis represent 100 aligned bps, and a dashed line shows the genome-wide background of 0.011. Note that nearly all promoters and untranslated regions also possess elevated divergence levels. See attached.
Database S1. Complete supporting data for Figure S2. See Excel file.
Database S2. Divergence of all genes between Japanese (IFO1802T) and Portuguese (ZP591) reference strains. See Excel file.
1
Gonzalez, S. S., Barrio, E., Gafner, J. & Querol, A. Natural hybrids from Saccharomyces
cerevisiae, Saccharomyces bayanus and Saccharomyces kudriavzevii in wine fermentations. FEMS Yeast Res 6, 1221-1234, (2006).
2
Liti, G. et al. Population genomics of domestic and wild yeasts. Nature 458, 337-341, (2009).
2
Table S1. Summary of sequenced strains, sequencing coverage, and spore viability.
Hittinger et al.
Strain
Nationality Locality
Substrate
Isolation date Sequenced strain Coverage10
ZP513
Portugal
Lisbon
Q. pyrenaica
Mar. 2005
FM10691
87% (7807789)
ZP537
Portugal
L. Albufeira
Q. faginea
May 2005
FM10562
82% (3097180)
ZP542
Portugal
Adagoi
Q. pyrenaica
Jun. 2005
FM10572
73% (3269665)
ZP591
Portugal
Cast. Vide
Q. pyrenaica
Aug. 2005
FM10092
94% (12459524)
ZP594
Portugal
A. Dez
Q. faginea
Sep. 2005
FM10781
84% (3138597)
88% (16)
4
ZP595
Portugal
A. Dez
Q. faginea
Sep. 2005
FM10791
79% (5087661)
90% (20)
4
ZP620
Portugal
Arrábida
Q. ilex
Oct. 2005
FM10721
81% (3081430)
100% (14)
4
ZP621
Portugal
Arrábida
Q. ilex (accorn)
Oct. 2005
FM10731
73% (3882742)
94% (16)
4
ZP623
Portugal
Arrábida
Q. ilex (leaf litter)
Oct. 2005
FM10741
76% (5347358)
95% (20)
ZP625
Portugal
Arrábida
Q. faginea
Oct. 2005
FM10622
71% (2786785)
0% (12)
ZP627
Portugal
Arrábida
Q. faginea
Oct. 2005
FM10751
75% (3104399)
100% (16)
4
ZP629
Portugal
L. Albufeira
Q. ilex
Dec. 2005
FM10761
81% (2763826)
100% (16)
4
ZP630
Portugal
L. Albufeira
Q. ilex
Dec. 2005
FM10771
85% (4472512)
88% (16)
ZP634
Portugal
Cast. Vide
Q. pyrenaica
Jan. 2006
FM10662
68% (2864521)
0% (8)
IFO1802
Japan
Mt. Daisen
partially decayed leaf
< 1992
IFO18022
100% (reference)3 IFO18022
IFO10990 Japan
Mt. Daisen
soil
< 2005
IFO109902
IFO10991 Japan
Mt. Daisen
partially decayed leaf
< 2005
IFO1803
Japan
Yakushima Island partially decayed leaf
W27
Switzerland Waedenswil
Lallemand wine strain
CBS679
Other strain Other Coverage Spore viability11 Notes
FM10941
FM10711
67% (2728243)
82% (4544569)
mixture of 25 bp and 36 bp reads
4
11% (28)
No MATa or HMR; ZP537 partially endoreduplicated but not FM1094
4
0% (24)
reason for sterility unknown; 34 bp reads after trimming "GT" barcode
4
94% (16)
Portuguese reference strain; mixture of 32 bp and 36 bp reads (ZP591)
4
4 No MATα or HML; 34 bp reads after trimming "CT" barcode
4 4
type strain and originally sequenced strain
5
89% (3222318)
34 bp reads after trimming "GT" barcode
6
IFO109912
87% (3294821)
34 bp reads after trimming "TT" barcode
6
< 1992
IFO18032
86% (8542642)
very distant subspecies; mixture of 34 bp reads after trimming "CT" barcode and 36 bp reads
5
< 1995
FM1043
NA (635858)
known hybrid of S. cerevisiae and S. kudriavzevii; 34 bp reads after trimming "AT" barcode
7, 8
< 1913
FM1054
NA (755492)
hybrid of S. cerevisiae and S. kudriavzevii; 34 bp reads after trimming "TT" barcode
9
67% (2710233)3
0% (20)
Single spore derivative.
2
Wild derivative, presumed diploid.
3
The reference sequence was assumed correct and complete when analyzing other strains, and the Solexa data was used only to estimate the error rate of the assemblies.
4
Sampaio JP, Gonçalves P. 2008. Natural populations of Saccharomyces kudriavzevii in Portugal are associated with oak bark and are sympatric with S. cerevisiae and S. paradoxus. Appl Environ Microbiol 74: 2144-2152.
5
Kaneko Y, Bano I. 1991. Reexamination of Saccharomyces bayanus strains by DNA-DNA hybridization and electrophoretic karyotyping. IFO Res Commun 15: 30-41.
6
Mikata K. Japan National Institute of Technology and Evaluation Biological Resource Center (http://www.nbrc.nite.go.jp).
7
Schuetz M, Gafner J. 1994. Dynamics of the yeast strain population during spontaneous alcoholic fermentation determined by CHEF gel electrophoresis. Lett Appl Microbiol 19: 253-257.
8
González SS, Barrio E, Gafner J, Querol A. 2006. Natural hybrids from Saccharomyces bayanus and Saccharomyces kudriavzevii in wine fermentations. FEMS Yeast Res 6: 1221-1234.
9
Guilliermond A. 1912. Deposited into CBS culture collection.
11
4
No MATa or HMR; 34 bp reads after trimming "AT" barcode
1
10
Reference
62% (16)
Coverage shows the percent of called bases, relative to the IFO1802T reference sequence described in the Methods, while the number of non-adapter (no exact 15-mer hits) sequencing reads obtained is in parentheses.
Spore viability shows the percent of viable spores with the number of spores tested in parentheses.
Table S2. Summary of numbers of GAL sites available for various analyses. 1 2 3 Gene Pseudogene (bp) Codon (bp) S YML053C 573 121 SUR7 909 211 GAL80 1673 1104 254 AIM32 936 206
YPL247C GAL4 GYP5 RPL36B KAP104 GAL7 GAL10 GAL1 FUR4 POA1 SIC1 EMP46 GAL2 SRL2 EMP70
2324
3369 3369 3369
1825
dS4 (0.00, 0.04) (0.04, 0.12) (0.39, 0.62) (0.19, 0.34)
1569 355 (0.01, 0.03) 1560 346 (0.49, 0.71) 2907 558 (0.22, 0.32) 246 58 (0.00, 0.00) 2742 354 561 714 1902 534
586 78 133 164 436 123
(0.03, 0.06) (0.83, 2.48) (0.98, 2.26) (1.06, 2.30) (0.14, 0.22) (0.01, 0.09)
864 1347 645 1179 1995
194 289 153 242 464
(0.01, 0.06) (0.02, 0.07) (1.24, saturated) (0.64, 1.02) (0.01, 0.04)
1
Hittinger et al.
Phylogenetics5 All strains6 Upstream7 Coding8 Downstream9
1068
1017
225
996
199
1659
1339
248
1525
176
360 573 744
352 552 690
0 35 35
271 486 569
135 0 493
456
435
70
559
145
Length of pseudogenes in IFO1802T (entire intergenic region between adjacent functional genes). 2 Aligned bp remaining after including only fully aligned codons between ZP591, IFO1802T, S. bayanus, S. mikatae, S. paradoxus, and S. cerevisiae. 3 Synonymous sites between ZP591 and IFO1802T (Fig. 2). 4 95% confidence interval of average number of synonymous differences between ZP591 and IFO1802T (low, high) (Fig. 2). 5 Number of bp in phylogenetic data matrix with S. castellii and IFO1803 added (Fig. 1a, S3a). 6 Number of bp in phylogenetic data matrix with full strain set (Fig. S4). 7 Number of upstream bp aligned between ZP591 and IFO1802T (Fig. S5). 8 T Number of coding bp aligned between ZP591 and IFO1802 (Fig. S5). 9 Number of downstream bp aligned between ZP591 and IFO1802T (Fig. S5). Missing data was either not calculated or not applicable.
Hittinger et al. Table S3. Constitutive expression of the GAL network in S. cervisiae is deleterious under non-inducing conditions. Genotype 2% Raffinose (non-induced) 2% Glucose (repressed) 2% Galactose (induced) GAL80+ 0.000 ± 0.001 0.000 ± 0.001 0.000 ± 0.001 gal80 -0.116 ± 0.003 -0.008 ± 0.003 +0.069 ± 0.003 Malthusian selection coefficients are reported ± s. d. with N = 12 as described in the Methods. Note that mutants lacking the Gal80 co-repressor are very unfit in non-inducing conditions, 1 as previously observed in 5% glycerol by MacLean in a study of GAL mutants 2 created by the systematic deletion project . 1
Maclean RC. 2007. Pleiotropy and GAL pathway degeneration in yeast. J Evol Biol 20: 1333-1338. Giaever G et al. 2002. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418: 387-391.
2
Table S4. List of strains used in experiments. Strain FM1009 FM1071 FM1097
Species S. kudriavzevii S. kudriavzevii S. kudriavzevii
Population Portuguese Portuguese Japanese
Derived from ZP591 ZP591 IFO1802T
FM1098 FM1109 FM1110 FM1111 FM1112 FM1123 FM1131
S. S. S. S. S. S. S.
Japanese Portuguese Portuguese hybrid hybrid Portuguese Portuguese
IFO1802 FM1071 FM1071 FM1097/FM1109 FM1098/FM1110 FM1109 FM1110
kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii
T
FM1142 S. kudriavzevii Portuguese FM1146 S. kudriavzevii Portuguese
FM1123 FM1142
FM1153 S. kudriavzevii Portuguese
FM1071/FM1146
FM1157 S. kudriavzevii Portuguese FM1159 S. kudriavzevii Portuguese
FM1123/FM1131 FM1153
FM1160 S. kudriavzevii Portuguese FM1161 S. kudriavzevii Portuguese
FM1153 FM1153
FM1162 S. kudriavzevii Portuguese
FM1153
FM1163 S. kudriavzevii Portuguese FM1164 S. kudriavzevii Portuguese
FM1153 FM1153
FM1165 FM1166 FM1183 FM1184 FM1185 FM1186 FM1187 FM1188 FM1189 FM1190 FM1282 FM1283
FM1153 FM1153 FM1157 FM1157 FM1157 FM1157 FM1157 FM1157 FM1157 FM1157 BY4724 BY4724
S. S. S. S. S. S. S. S. S. S. S. S.
kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii cerevisiae cerevisiae
Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese laboratory laboratory
Hittinger et al. Genotype MATa/MAT MATa/MAT
Use Table S1 Table S1
Notes 1 2
MAT ho ::natMX
3
MATa ho ::natMX MATa ho ::kanMX MAT ho ::kanMX MATa/MAT ho ::kanMX/ho ::natMX MATa/MAT ho ::kanMX/ho ::natMX MATa ho ::kanMX ura3- MAT ho ::kanMX trp1- + MATa ho ::kanMX ura3- trp1 ::ScerURA3
3 3 3 4 4 5 6 7
Fig. S1 Fig. S1
MATa ho :kanMX ura3- trp1 ::ScerGAL3+ +
8
+
+
MATa/MAT ho ::kanMX/HO ura3- /URA3 trp1 ::ScerGAL3 /TRP1 + + MATa/MAT ho ::kanMX/ho ::kanMX ura3- /URA3 trp1- /TRP1
+
9 9
MATa ho ::kanMX ura3- trp1 ::ScerGAL3+ + MATa ho ::kanMX ura3- trp1 ::ScerGAL3
Fig. 3c Fig. 3c
MATa ho ::kanMX ura3- trp1 ::ScerGAL3+
Fig. 3c
MATa ho ::kanMX ura3- trp1 ::ScerGAL3 + MATa ho ::kanMX ura3- trp1 ::ScerGAL3
+
Fig. 3c Fig. 3c
MATa ho ::kanMX ura3- trp1 ::ScerGAL3+ + MATa ho ::kanMX ura3- trp1 ::ScerGAL3
Fig. 3c Fig. 3c
MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa
+
ho ::kanMX ura3- trp1 ::ScerGAL3 ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ho ::kanMX ura3- trp1- ura3- lys2- PTDH3-yEGFP-TCYC1 ura3- lys2- PTDH3-yBGFP-TCYC1
Fig. 3c Fig. 3c Fig. 3c Fig. 3c Fig. 3c Fig. 3c Fig. 3c Fig. 3c Fig. 3c Table S3 Table S3
10 11
+
FM1284 S. cerevisiae
laboratory
FM1282
MATa ura3- lys2- PTDH3-yEGFP-TCYC1 GAL80
Table S3
12
FM1285 S. cerevisiae
laboratory
FM1282
+ MATa ura3- lys2- PTDH3-yEGFP-TCYC1 GAL80
Table S3
12
FM1286 S. cerevisiae FM1287 S. cerevisiae
laboratory laboratory
FM1282 FM1282
lys2- PTDH3-yEGFP-TCYC1 GAL80+
Table S3
12
FM1288 S. cerevisiae FM1289 S. cerevisiae
laboratory laboratory
FM1282 FM1282
MATa ura3- MATa ura3- MATa ura3- MATa ura3-
lys2- PTDH3-yEGFP-TCYC1 gal80- lys2- PTDH3-yEGFP-TCYC1 gal80- lys2- PTDH3-yEGFP-TCYC1 gal80-
Table S3 Table S3 Table S3
13 13 13
FM1290 S. cerevisiae
laboratory
FM1282
MATa ura3- lys2- PTDH3-yEGFP-TCYC1 GAL3+
Fig. 3c
14
FM1291 S. cerevisiae
laboratory
FM1282
MATa ura3- lys2- PTDH3-yEGFP-TCYC1 GAL3
+
Fig. 3c
14
+
lys2- PTDH3-yEGFP-TCYC1 GAL3 lys2- PTDH3-yEGFP-TCYC1 gal3- lys2- PTDH3-yEGFP-TCYC1 gal3-
Fig. 3c Fig. 3c Fig. 3c
14 15 15
lys2- PTDH3-yEGFP-TCYC1 gal3-
Fig. 3c
FM1292 FM1293 FM1294 FM1295
S. S. S. S.
cerevisiae cerevisiae cerevisiae cerevisiae
laboratory laboratory laboratory laboratory
FM1282 FM1282 FM1282 FM1282
MATa MATa MATa MATa
ura3- ura3- ura3- ura3-
FM1332 FM1334 FM1335 FM1336 FM1337
S. S. S. S. S.
kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii
Portuguese Portuguese Portuguese Portuguese Portuguese
FM1123 FM1123 FM1123 FM1123 FM1123
MATa MATa MATa MATa MATa
ho ::kanMX ho ::kanMX ho ::kanMX ho ::kanMX ho ::kanMX
FM1338 FM1340 FM1343 FM1345 FM1346 FM1348 FM1351 FM1352 FM1353 FM1354 FM1355 FM1356 FM1357 FM1358 FM1359 FM1360 FM1361 FM1362
S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.
kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii kudriavzevii
Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese Portuguese
FM1123 FM1123 FM1123 FM1123 FM1123 FM1123 FM1132 FM1132 FM1132 FM1134 FM1135 FM1135 FM1136 FM1137 FM1137 FM1137 FM1138 FM1138
MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa
+
ura3- ura3- ura3- ura3-
gal80 ::ScerURA3 gal80 ::ScerURA3+ gal80 ::ScerURA3+ gal80 ::ScerURA3+
ura3- ho ::kanMX ura3- ho ::natMX ura3- ho ::natMX ura3- ho ::natMX ura3- ho ::natMX ura3- ho ::natMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3- ho ::kanMX ura3-
gal80 ::ScerURA3 + gal80 ::ScerURA3
+
gal80 gal80 gal80 gal80 gal80 gal80 gal80 gal80 gal80 gal80 gal80 gal80
15 16 16 16 16
Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.
3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b
16 16 17, 18 17 17 17 17 19 19 19 19 19 19 19 19 19 19 19 19
1
Lab stock of wild ZP591 isolate; Portuguese reference strain20. Homozygote of ZP591 derived from a single spore. 3 Heterothallic single spore isolate after deletion of HO. 4 F1 Japanese/Portuguese strain sporulated to generate F2 segregants. 2
5
Start codon to stop codon deletion made transforming with PCR product and selecting against URA3+ . Start codon to stop codon deletion made transforming with PCR product and selecting against TRP1+ . + Start codon to stop codon replacement made transforming with PCR product and selecting for URA3 plus its intergenic sequences. 8 Start codon to stop codon replacement made transforming with PCR product containing ScerGAL3+ plus its intergenic sequences and selecting against URA3+ . 9 Cross used to generate panel of experimental strains. 10 GFP competition strain21. 11 BFP competition strain21. 12 + + + GAL80 re-introduced in triplicate strains to MATa ura3- lys2- PTDH3-yEGFP-TCYC1 gal80 ::URA3 by transforming with PCR product and selecting against URA3 . 6 7
13
URA3+ removed from MATa ura3- lys2- PTDH3-yEGFP-TCYC1 gal80 ::URA3+ by transforming with engineered oligonucleotides and selecting against URA3+.
14
GAL3+ re-introduced in triplicate strains to MATa ura3- lys2- PTDH3-yEGFP-TCYC1 gal3 ::URA3+ by transforming with PCR product and selecting against URA3+21.
15
+
+
+21
URA3 removed from MATa ura3- lys2- PTDH3-yEGFP-TCYC1 gal3 ::URA3 by transforming with engineered oligonucleotides and selecting against URA3
16
Start codon to stop codon replacement made transforming with PCR product and selecting for ScerURA3+ plus its intergenic sequences. 17 Drug marker changed by selecting for natMX, control competition strains competed against parental FM1123. 18 Competition strain with no detectable defect. 19 Start codon to stop codon deletion made transforming with PCR product and selecting against URA3+ , competed against FM1340. 20 Sampaio JP, Gonçalves P. 2008. Natural populations of Saccharomyces kudriavzevii in Portugal are associated with oak bark and are sympatric with S. cerevisiae and S. paradoxus. Appl Environ Microbiol. 74: 2144-2152. 21 Hittinger CT, Carroll SB. 2007. Gene duplication and the adaptive evolution of a classic genetic switch. Nature 449: 677-681.
.
Hittinger et al.
Figure S1 50
Number of F2 segregants
40
30 Observed Expected 20
10
0 0
1
2
Number of functional GAL+ loci
3
4
Figure S2
a
Hittinger et al.
Proportion of hits
1.0 0.9
S. kudriavzevii
0.8
S. cerevisiae
0.7
S. paradoxus
0.6
S. mikatae
0.5
S. bayanus
0.4 0.3 0.2 0.1
Genes
ZP591 (Portuguese S. kudriavzevii)
Genes
W27 (known hybrid)
Genes
CBS679 (hybrid)
0.0
b
1.0 0.9
Proportion of hits
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
c
1.0 0.9
Proportion of hits
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
Hittinger et al.
Figure S3 0.0644
a
S. cerevisiae
0.0514 0.0644
S. paradoxus
0.0828
0.0500
0.1158
S. mikatae
0.0603 0.1711
0.0581
0.0276
0.1883
S. kudriavzevii ZP591 S. kudriavzevii IFO1802T
0.1130 0.0581
S. kudriavzevii IFO1803
0.2589
S. bayanus
0.4472
S. castellii
b
0.0455
S. cerevisiae
0.0455
S. paradoxus
0.0395 0.0362
0.0300
0.0850
S. mikatae
0.0442
0.0042
S. kudriavzevii ZP591
0.0166
S. kudriavzevii IFO1802T
0.1004
0.0727
0.0042 0.0209
0.1654
S. kudriavzevii IFO1803 S. bayanus
0.2382
S. castellii
c 0.0010 0.0020
ZP591 Portuguese Population
0.0037
0.0094 0.0008 0.0039
0.0141
IFO1802T Japanese Population IFO1803
Hittinger et al.
Figure S4 S. bayanus
98/91
S. kudriavzevii
100/100
S. mikatae
99/84
99/100
99/100
S. paradoxus
99/100
99/100
S. cerevisiae
S. castellii
0.08
Hittinger et al.
Figure S5 a
b
0.4
0.4
GAL80
0.3
0.3
d
GAL4
d 0.2
0.2
0.1
0.1
0
0
c
d
0.6
0.6
0.5 0.4
d
GAL7
GAL10
0.5
GAL2
GAL1
0.4
d 0.3
0.3
0.2
0.2
0.1
0.1
0
0
