JCVI Locus Protein Name. JCVI Role Category JCVI Locus (LT2) Annotation name

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1 Additional File 4. Detailed information on all 41 genes that showed evidence for positive selection from positive selection analysis, in which gene Alignment Primary Gene Genbank Locus JCVI Locus Protein Name JCVI Role Category JCVI Locus (LT2) Annotation name (LT2) (CT18) Length, No. (LT2) bp pyridoxal kinase phosphoglucomutase, alpha-dglucose phosphate-specific PTS system IIA component, putative oligopeptide ABC transporter, ATPbinding protein conserved hypothetical protein TIGR00156 domain protein phosphoribosylaminoimidazole carboxylase, catalytic subunit Biosynthesis of cofactors, prosthetic groups, and carriers NT01ST1789 AAL20372 STM1450 NT03ST pgm Energy metabolism NT01ST0841 AAL19642 STM0698 NT03ST oppf pure Transport and binding proteins Transport and binding proteins NT01ST4092 AAL22130 STM3258 NT03ST NT01ST4531 AAL22487 STM3626 NT03ST Hypothetical proteins NT01ST1873 AAL20434 STM1515 NT03ST Purines, pyrimidines, nucleosides, and nucleotides NT01ST0653 AAL19488 STM0534 NT03ST rard protein rard Unknown function NT01ST4920 AAL22799 STM3955 NT03ST malt regulatory protein. malt Energy metabolism NT01ST4393 AAL22377 STM3515 NT03ST glycyl-trna synthetase, beta subunit glys Protein synthesis NT01ST4569 AAL22514 STM3655 NT03ST nrdi protein nrdi Purines, pyrimidines, nucleosides, and NT01ST3491 AAL21691 STM2806 NT03ST nucleotides hypothetical protein Hypothetical proteins NT01ST4989 AAL22854 STM4015 NT03ST acetate operon repressor. iclr Energy metabolism NT01ST5191 AAL23011 STM4187 NT03ST conserved hypothetical protein Hypothetical proteins NT01ST5283 AAL23082 STM4258 NT03ST exonuclease SbcC, putative DNA metabolism NT01ST0490 AAL19349 STM0395 NT03ST hypothetical protein Hypothetical proteins NT01ST1895 AAL20451 STM1532 NT03ST aminotransferase, class I arat Central intermediary metabolism NT01ST0731 AAL19554 STM0603 NT03ST salmonella atypical fimbria outer membrane usher safc Cell Envelope NT01ST0369 AAL19258 STM0301 NT03ST lipoprotein, putative Cell Envelope NT01ST0899 AAL19687 STM0743 NT03ST PipB pipb Unclassified NT01ST1346 AAL20020 STM1088 NT03ST

2 membrane protein, putative Cell Envelope NT01ST1778 AAL20363 STM1441 NT03ST oligopeptide ABC transporter, Transport and binding periplasmic oligopeptide-binding oppa NT01ST2082 AAL20596 STM1679 NT03ST proteins protein GDP-mannose mannosyl hydrolase Purines, pyrimidines, nucleosides, and nucleotides NT01ST2623 AAL21011 STM2107 NT03ST proton/peptide symporter family protein Unclassified NT01ST4488 AAL22452 STM3592 NT03ST aldehyde dehydrogenase B aldb Energy metabolism NT01ST4599 AAL22539 STM3680 NT03ST hydroxyisobutyrate dehydrogenase, putative Energy metabolism NT01ST4997 AAL22862 STM4023 NT03ST histidinol phosphatase-related protein Unknown function NT01ST0303 AAL19211 STM0248 NT03ST glycosyl hydrolase, family 13 malz Energy metabolism NT01ST0499 AAL19355 STM0401 NT03ST conserved hypothetical protein Hypothetical proteins NT01ST1594 AAL20205 STM1280 NT03ST multidrug resistance protein norm Cellular processes NT01ST1758 AAL20347 STM1425 NT03ST outer membrane protein c precursor ompc Cell Envelope NT01ST2829 AAL21169 STM2267 NT03ST CorE b2611 Viral functions NT01ST3328 AAL21567 STM2678 NT03ST similar to Cell Envelope NT01ST3484 AAL21686 STM2801 NT03ST ubih protein ubih Biosynthesis of cofactors, prosthetic groups, and NT01ST3824 AAL21932 STM3057 NT03ST carriers conserved hypothetical protein TIGR01212 Hypothetical proteins NT01ST4169 AAL22198 STM3329 NT03ST conserved hypothetical protein Hypothetical proteins NT01ST4329 AAL22325 STM3463 NT03ST acetyltransferase, GNAT family family Unknown function NT01ST4455 AAL22425 STM3565 NT03ST PTS system, mannitol-specific IIC Transport and binding component subfamily, putative proteins NT01ST4604 AAL22544 STM3685 NT03ST catalase/peroxidase HPI katg Cellular processes NT01ST5107 AAL22946 STM4106 NT03ST pct identical to TraF of plasmid R64 Viral functions NT01ST5263 AAL23066 STM4242 NT03ST conserved hypothetical protein Hypothetical proteins NT01ST1047 AAL19797 STM0861 NT03ST hypothetical protein Hypothetical proteins NT01ST2302 AAL20769 STM1854 NT03ST

3 a d N = number of non-synonymous changes per non-synonymous sites b d S = number of synonymous changes per synonymous sites c TO = overall test; Ch#= Choleraesuis branch specific test; Pty#= Paratyphi A branch specific test ; Ty# = Typhi branch specific test; Tym#= Typhimurium br d Proportion of sites under positive selection e w =omega (i.e., d N /d S ) f only sites with a probability >95% as being under positive seletion are shown; the first number indicates the aa sites identified by Bayes Empirical Bayes (B bracket indicates the posterior probability; ";" separates the aa sites; aa sites are based on site location in the alignment used for positive selection analyses File 5); BEB method is used to calculate posterior probabilities for site classes under alternative models (Model 2a and Model A if the LRT suggests presenc positive selection on the foreground branch) g Each distinct gene tree (n=18) was designated with a unique gene tree number h Gene trees used for positive seletion analyses are shown in Newick format; ATCC9150 = S. Paratyphi A, ch= S. Choleraesuis, CT18= S. Typhi CT18, Ty2= i Nc= effective number of codons used in a gene sequence was calculated using the program chips from EMBOSS package; Nc can take values from 20 (in where one codon is exclusively used for each amino acid) to 61 (which indicates that the use of alternative synonymous codons is equally likely (Wright. 199

5 es without recombination were used d N a d S b Estimated diversity No. of informative sites p-value for TO c q-value for TO Prop. of sites under pos sel. (TO) d ω (TO) e BEB (TO) f p-value for Ch# c q-value for Ch# (0.984) (0.976) (0.969) E (0.951); 111 (0.955); 405 (0.954); 692 (0.950) (0.997)

6 E E E E (0.985); 184 (0.959) 3.36E (0.955) (0.983) (0.964) E E (0.983)

7 ranch specific test BEB), the number in s (provided as Additional ce of codons under = Typhi Ty2, LT2= Typhimurium LT2 case of extreme bias 90. Gene 87:23-29)

9 Prop. of sites under pos sel. ω(ch#) e p-value BEB (Ch#) f (Ch#) d for Tym# c q-value for Tym# Prop. of sites under pos sel. (Tym#) d ω(tym#) e BEB (Tym#) f p-value Ty# c q-value Ty# (0.991); 178 (0.973); 261 (0.973)

10 (0.999);184 (0.997); 185 (0.992); 191 (0.982) ND ND ND ND ND ND ND ND (0.970) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

13 Prop. of sites under pos sel. (Ty#) d ω (Ty#) e BEB (Ty#) f p-value for Pty# c q-value for Pty# Prop. of sites under pos sel. (Pty#) d ω (Pty#) e BEB (Pty#) f Tree group no g (0.967); 139 (0.996); 143 (0.984); 144 (0.970); 147 (0.977) (0.979) (0.966) (0.568) (0.956); 31(0.955)

14 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND (0.992); 40 (0.992); 9 44 (0.958); 45 (0.977)

17 Gene Tree h p value for NSS q-value for NSS p-value Max Chi q-value for Max Chi Permutationbased p-value for PHI q-value for PHI GENE- CONV inner p-value q-value for GENE-CONV Nc value for Paratyphi A i ((ATCC9150,(CT18,Ty2)),ch,LT2); ((ATCC9150,ch),(CT18,Ty2),LT2); ((ATCC9150,ch),(CT18,Ty2),LT2); ((ATCC9150,ch),(CT18,Ty2),LT2); ((ATCC9150,ch),(CT18,Ty2),LT2); ((ATCC9150,ch),(CT18,Ty2),LT2); ((ATCC9150,ch),(CT18,Ty2),LT2); (ATCC9150,(ch,(CT18,Ty2)),LT2); ((ATCC9150,(CT18,Ty2)),ch,LT2); ((ATCC9150,ch),(CT18,Ty2),LT2); ((ATCC9150,(CT18,Ty2)),ch,LT2); ((ATCC9150,ch),(CT18,Ty2),LT2); (ATCC9150,(ch,(CT18,Ty2)),LT2); ((ATCC9150,(CT18,Ty2)),ch,LT2); ((ATCC9150,(CT18,Ty2)),ch,LT2); ((ATCC9150,ch),(CT18,Ty2),LT2); ((ATCC9150,ch),(CT18,Ty2),LT2); ((ATCC9150,ch),(CT18,Ty2),LT2); ((ATCC9150,(CT18,Ty2)),ch,LT2);

18 ((ATCC9150,(CT18,Ty2)),ch,LT2); ((ATCC9150,(CT18,Ty2)),ch,LT2); ((ATCC9150,ch),(CT18,Ty2),LT2); ((ATCC9150,(CT18,Ty2)),ch,LT2); ((ATCC9150,ch),(CT18,Ty2),LT2); ((ATCC9150,(CT18,Ty2)),ch,LT2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),LT2),CT18,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,LT2),ch),CT18,Ty2); ND ND ND ND ND ND ((ATCC9150,(ch,LT2)),CT18,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); ND ND ND ND ND ND (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),LT2),CT18,Ty2); ((ATCC9150,(ch,LT2)),CT18,Ty2); ((ATCC9150,(ch,LT2)),CT18,Ty2); ND ND ND ND ND ND (((ATCC9150,LT2),ch),CT18,Ty2); ND ND ND ND ND ND

21 Nc value for Choleraesuis i Nc value for Typhi CT18 i Nc value for Typhimurium LT2 i Nc value for Typhi Ty2 i Average Nc i

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Additional Table 3. Detailed information on all 81 genes that showed evidence for positive selection from initial positive selection analysis, in Protein Name Gene name JCVI Role Category_1 JCVI Role Category_2