Supplementary Table 1: Polymorphic Sites Among the 42 D

Supplementary Information

Inversions and adaptation to the plant toxin ouabain shape DNA sequence variation within and between chromosomal inversions of Drosophila subobscura.

Cinta Pegueroles, Albert Ferrés-Coy, Maria Martí-Solano, Charles F Aquadro, Marta Pascual and Francesc Mestres Supplementary Information

Supplementary Figure 1: Genetic differentiation (FST) between chromosomal arrangements for each gene vs their distance to the nearest inversion breakpoint. Squares designate O3+4+1 - O3+4 comparisons, diamonds O3+4+7 - O3+4 and triangles O3+4+1 - O3+4+7. Grey symbols correspond to genes located inside inversions. Supplementary Information

Supplementary Figure 2: Recombination networks constructed using SplitsTree4 program 67 for Sqd (a gene located within the inversions) and Atpα and Fmr1 genes (both located outside the studied inversions) for individuals with O3+4 (O3+4), O3+4+1 (O1) and O3+4+7 (O7) arrangements. Supplementary Information

Table S1: ZnS statistic values and the recombination parameter (Rho) per bp. Shadowed genes are located inside inversions. ZnS is not available for the Atpα gene in the O3+4+1 arrangement due to the lack of parsimony informative sites. Arrangement ZnS Rho/length

Pif1A O3+4+1 0.172 0.055*

O3+4+1 - O3+4 0.066 0.055*

O3+4+7 0.178 0.030

O3+4+7 - O3+4 0.094 0.055*

Abi O3+4+7 0.327 0.008

O3+4+7 - O3+4 0.144 0.013

Sqd O3+4+1 0.714 0.000

O3+4+1 - O3+4 0.149 0.030

O3+4+7 0.206 0.021

O3+4+7 - O3+4 0.165 0.070*

Yrt O3+4+1 0.195 0.046

O3+4+1 - O3+4 0.090 0.110*

O3+4+7 0.182 0.067

O3+4+7 - O3+4 0.078 0.110*

Atpα O3+4+1 n.a. 0.009

O3+4+1 - O3+4 0.335 0.007

O3+4+7 0.200 0.000

O3+4+7 - O3+4 0.262 0.003

Fmr1 O3+4+1 0.089 0.004

O3+4+1 - O3+4 0.089 0.033

O3+4+7 0.365 0.006

O3+4+7 - O3+4 0.114 0.012 * Rho per gene > 100 Supplementary Information

Table S2: Gene conversion tracts detected for each of the studied gene regions. The Ψ parameter is defined as the probability of a site to be informative for a gene conversion event. Shadowed genes are located inside inversions.

a b O3+4 - O3+4+1 Converted Informative sites ψ Length (bp) Pif1A 1 0.00017 Sqd 3 0.00041

Yrt O3+4 4 0.00063 111

Atpα O3+4 17 0.00541 1573

Atpα O3+4+1 17 0.00541 52 Fmr1 0 0

O3+4 - O3+4+7

Pif1A O3+4 32 0.00464 466

Pif1A O3+4+7 32 0.00464 103

Pif1A O3+4+7 32 0.00464 208 Abi 1 0.00023

Sqd O3+4+7 3 0.00043 8

Yrt O3+4+7 2 0.00037 7

Atpα O3+4 18 0.00614 1422

Atpα O3+4+7 18 0.00614 1573 Fmr1 0 0 a converted arrangement when identification was possible. b probability of a site being informative of a gene conversion event. Supplementary Information

Table S3: Polymorphic sites among the 42 D. subobscura isochromosomal lines for the Atpα gene. Sequences are grouped according to their gene arrangement. Exonic regions appear shaded and boxes identify gene conversion tracts. The position number of each polymorphic site is in relation to the nucleotide alignment of D. subobscura sequences.

* * * * * * * * * * 0 8 3 7 3 9 3 9 1 5 1 7 0 4 7 7 9 5 4 4 7 2 3 6 9 1 7 2 4 8 9 6 2 6 8 0 3 7 9 7 4 4 8 3 5 7 8 8 2 4 8 4 4 5 6 9 2 2 3 8 9 1 1 2 7 7 8 8 9 0 1 2 3 1 3 4 4 6 4 0 0 0 1 2 2 2 3 3 3 3 3 4 4 4 5 5 5 5 5

Atpα 2 5 5 5 5 7 9 1 1 1 1 1 1 2 2 2 2 3 4 7 7 8 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 FMP2 G G C C A G C A C A A C T A C C T T C C C G A G C T G C G A C C C C G G C C G C T C T 3+4 FMP3 A T . . . . A . . G . . C . A G ...... G ...... A T C . A 3+4 FMP4 A T . . . . A . . G . . C C ...... T . . A T . . A 3+4 FMP6 A T . . . . A . T G C A C . A G C ...... T . . A T C . A 3+4 FMP10 A T . . . . A ...... T . . T T . A T . . A 3+4 FMP16 A T . . . . A . . G . . C . . G ...... T . . A T C . A 3+4 MP1 A T . . . . A ...... C . . . . G ...... A T . . A 3+4 MP2 A T . . . . A . T G C A C . A G C C . . . C ...... A T C . A 3+4 MP3 A T . . . . A . . G . . C . A G . C ...... T . . A T . . A 3+4 MP4 A T . . . . A . . G . . C . A G ...... A T . . . 3+4 MP6 A T . . . . A . T G C A C . A G C . . . . . G ...... T . . A T C . A 3+4 MP10 A T . . . . A . T G C A C . A G . C . . . . G ...... A T . . A 3+4 FBC5 A T . . . . A . . G . . C . A G . C ...... C ...... T . . A T . . A 3+4 FBC12 A T . . . . A . . G . . C . . G ...... G . . . A ...... T . . . . A T C . A 3+4 FBC15 A T . . . . A . . G . . C . A G ...... G ...... T . . A T C . A 3+4 FBC29 A T . . . . A . T G C A C . A G C . . T ...... A ...... T . . A T . . A 3+4 FBC37 A T . . . . A . T G C A C . A G . . . . . C ...... A T . . A 3+4 FBC48 A T . . . . A . . G . . C . A G . . T . T ...... T . T . . . . A T C T A 3+4 FBC49 A T . . . . A ...... A . . . . 3+4 BC23 A T . . . . A . . G . . C C . G ...... G ...... A T C . A 3+4 BC31 A T . . . . A . . G . . C . A ...... T . T A T . . A 3+4 BC34 A T . . . . A . . G . . C . A ...... A . . . . T . . A T . . . 3+4 BC45 A T . . . . A . T G C A C . A G C . . . . . G ...... A T . . A 3+4 BC56 A T . . . . A . T G C A C . A G C . . . . . G ...... A T . . A 3+4

FMP25 ...... 3+4+1 MP9 ...... 3+4+1 MP36 ...... G . . C C . G ...... 3+4+1 MP44 ...... 3+4+1 MP50 ...... 3+4+1 MP63 ...... C ...... 3+4+1 MP78 ...... A ...... 3+4+1 MP90 ...... 3+4+1 MP104 ...... 3+4+1

FBC28 . . G G T ...... A ...... A . . . . 3+4+7 FBC46 . . . G T . . G ...... A . . . . 3+4+7 FBC76 A T . . . . A . . G . . C . A G . C . . T . G ...... A T C . A 3+4+7 FBC94 . . G ...... A . . . . 3+4+7 FBC96 . . . G T C ...... A . . . . 3+4+7 FBC101 . . . G T ...... C ...... A . . . . 3+4+7 FBC103 . . G G T ...... A ...... A . . . . 3+4+7 FBC106 . . G G T ...... A ...... A . . . . 3+4+7 BC21 . . . G T C ...... 3+4+7 * nonsynonymous changes Supplementary Information

Table S4: Tajima’s D and Fu and Li’s D neutrality tests for O3+4+1 and O3+4+7 chromosomal arrangements. Shadowed genes are located inside inversions.

Tajima's D P-value Fu and Li's D a P-value

Pif1A O3+4+1 -0.654 0.262 -0.972 0.223

O3+4+7 -0.581 0.277 -0.990 0.211

Abi O3+4+7 -0.899 0.194 -0.794 0.248

Sqd O3+4+1 -0.731 0.257 -1.598 0.083

O3+4+7 -1.331 0.101 -1.561 0.085

Yrt O3+4+1 -0.684 0.253 0.118 0.550

O3+4+7 -0.888 0.219 -0.789 0.275

Atpα O3+4+1 -1.728 0.011 -2.236 0.008

O3+4+7 -1.347 0.104 -0.309 0.421

Fmr1 O3+4+1 -1.620 0.050 -1.956 0.050

O3+4+7 -0.637 0.276 -1.432 0.068 a Fu and Li's D using D. pseudoobscura as an outgroup. * P-values assessed by 1000 coalescent simulations Supplementary Information

Table S5: Tajima’s D and Fu and Li’s D neutrality tests for the Atpα gene in the O3+4+1 and O3+4+7 chromosomal arrangements. Tests were performed including all positions, only silent sites, and including and excluding recombinant individuals.

Positions of the Atpα Chromosomal Tajima’s D Fu and Li’s D gene arrangement

all positions O3+4+1 -1.728* -2.236*

all positions O3+4+7 -1.347 -0.309

silent sites O3+4+1 -1 -2,236*

silent sites O3+4+7 -1.668 -0.081 all positions, O3+4+1 -1,310 -1,677 without recombinants all positions, O3+4+7 -2.126* -0.252 without recombinants silent sites, O3+4+1 -1.310 -1.677 without recombinants silent sites, O3+4+7 -0.448 -0.312 without recombinants * P-value < 0.05 Supplementary Information

Table S6: Synonymous and nonsynonymous polymorphism and divergence (Ps, Pn, Ds,

Dn respectively) and DoS calculated as Dn/(Dn+Ds) - Pn/(Pn+Ps) for O3+4+1 and O3+4+7 chromosomal arrangements. Divergence was calculated using D. pseudoobscura as outgroup. Shadowed genes are located inside inversions.

Ps Pn Ds Dn DoS

Pif1A O3+4+1 0 1 15 0 -1.000 Pif1A O3+4+7 0 2 15 0 -1.000

Abi O3+4+7 16 0 54 0 0.000

Sqd O3+4+1 1 0 23 4 0.148 Sqd O3+4+7 4 0 23 4 0.148

Yrt O3+4+1 24 3 57 9 0.025 Yrt O3+4+7 28 1 57 9 0.102

Atpα O3+4+1 2 0 51 10 0.164 Atpα O3+4+7 8 10 47 6 -0.442

Fmr1 O3+4+1 10 2 37 1 -0.140 Fmr1 O3+4+7 7 1 37 1 -0.099 Supplementary Information

Table S7: Synonymous site and branch-site tests implemented in CodeML of the PAML v4 package20. Site tests were performed comparing the neutral model M1a (model=0; NSsites=1) with alternative M2a (model=0; NSsites=2), and the neutral model M7 (model=0; NSsites=7, ncatG=10) with the alternative M8 (model=0; NSsites=8, ncatG=10). For the branch-site test 2, in the neutral model we used the parameters model=2; NSsites=2; fix_omega = 1; omega=1 and, for the alternative, model=2; NSsites=2; fix_omega = 0; omega=1.5.

Site test model aa in position 109 LRT pval M1a vs M2a A 0 1 M7 vs M8 A -0.001 1 M1a vs M2a G 0.125 0.939 M7 vs M8 G 0.216 0.897 branch-site test 2 branch aa in position 109 LRT pval O3+4+7 A 11.428 0.0007 O3+4+7 G 11.773 0.0006 O3+4+1 A 0.426 0.514 O3+4 S 1.20e-05 0.997