<p> Supplementary Information</p><p>Inversions and adaptation to the plant toxin ouabain shape DNA sequence variation within and between chromosomal inversions of Drosophila subobscura. </p><p>Cinta Pegueroles, Albert Ferrés-Coy, Maria Martí-Solano, Charles F Aquadro, Marta Pascual and Francesc Mestres Supplementary Information</p><p>Supplementary Figure 1: Genetic differentiation (FST) between chromosomal arrange- ments 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</p><p>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</p><p>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</p><p>Pif1A O3+4+1 0.172 0.055*</p><p>O3+4+1 - O3+4 0.066 0.055*</p><p>O3+4+7 0.178 0.030</p><p>O3+4+7 - O3+4 0.094 0.055*</p><p>Abi O3+4+7 0.327 0.008</p><p>O3+4+7 - O3+4 0.144 0.013</p><p>Sqd O3+4+1 0.714 0.000</p><p>O3+4+1 - O3+4 0.149 0.030</p><p>O3+4+7 0.206 0.021</p><p>O3+4+7 - O3+4 0.165 0.070*</p><p>Yrt O3+4+1 0.195 0.046</p><p>O3+4+1 - O3+4 0.090 0.110*</p><p>O3+4+7 0.182 0.067</p><p>O3+4+7 - O3+4 0.078 0.110*</p><p>Atpα O3+4+1 n.a. 0.009</p><p>O3+4+1 - O3+4 0.335 0.007</p><p>O3+4+7 0.200 0.000</p><p>O3+4+7 - O3+4 0.262 0.003</p><p>Fmr1 O3+4+1 0.089 0.004</p><p>O3+4+1 - O3+4 0.089 0.033</p><p>O3+4+7 0.365 0.006</p><p>O3+4+7 - O3+4 0.114 0.012 * Rho per gene > 100 Supplementary Information</p><p>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.</p><p> a b O3+4 - O3+4+1 Converted Informative sites ψ Length (bp) Pif1A 1 0.00017 Sqd 3 0.00041</p><p>Yrt O3+4 4 0.00063 111</p><p>Atpα O3+4 17 0.00541 1573</p><p>Atpα O3+4+1 17 0.00541 52 Fmr1 0 0</p><p>O3+4 - O3+4+7</p><p>Pif1A O3+4 32 0.00464 466</p><p>Pif1A O3+4+7 32 0.00464 103</p><p>Pif1A O3+4+7 32 0.00464 208 Abi 1 0.00023</p><p>Sqd O3+4+7 3 0.00043 8</p><p>Yrt O3+4+7 2 0.00037 7</p><p>Atpα O3+4 18 0.00614 1422</p><p>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</p><p>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.</p><p>* * * * * * * * * * 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</p><p>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</p><p>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</p><p>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</p><p>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.</p><p>Tajima's D P-value Fu and Li's D a P-value</p><p>Pif1A O3+4+1 -0.654 0.262 -0.972 0.223</p><p>O3+4+7 -0.581 0.277 -0.990 0.211</p><p>Abi O3+4+7 -0.899 0.194 -0.794 0.248</p><p>Sqd O3+4+1 -0.731 0.257 -1.598 0.083</p><p>O3+4+7 -1.331 0.101 -1.561 0.085</p><p>Yrt O3+4+1 -0.684 0.253 0.118 0.550</p><p>O3+4+7 -0.888 0.219 -0.789 0.275</p><p>Atpα O3+4+1 -1.728 0.011 -2.236 0.008</p><p>O3+4+7 -1.347 0.104 -0.309 0.421</p><p>Fmr1 O3+4+1 -1.620 0.050 -1.956 0.050</p><p>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</p><p>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.</p><p>Positions of the Atpα Chromosomal Tajima’s D Fu and Li’s D gene arrangement</p><p> all positions O3+4+1 -1.728* -2.236*</p><p> all positions O3+4+7 -1.347 -0.309</p><p> silent sites O3+4+1 -1 -2,236*</p><p> 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</p><p>Table S6: Synonymous and nonsynonymous polymorphism and divergence (Ps, Pn, Ds,</p><p>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.</p><p>Ps Pn Ds Dn DoS</p><p>Pif1A O3+4+1 0 1 15 0 -1.000 Pif1A O3+4+7 0 2 15 0 -1.000</p><p>Abi O3+4+7 16 0 54 0 0.000</p><p>Sqd O3+4+1 1 0 23 4 0.148 Sqd O3+4+7 4 0 23 4 0.148</p><p>Yrt O3+4+1 24 3 57 9 0.025 Yrt O3+4+7 28 1 57 9 0.102</p><p>Atpα O3+4+1 2 0 51 10 0.164 Atpα O3+4+7 8 10 47 6 -0.442</p><p>Fmr1 O3+4+1 10 2 37 1 -0.140 Fmr1 O3+4+7 7 1 37 1 -0.099 Supplementary Information</p><p>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.</p><p>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</p>