JCVI Role Category_1 JCVI Role Category_2. histidinol phosphatase-related protein Unknown function NT01ST0303 AAL19211 STM0248

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1 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 JCVI Locus (LT2) Genbank Locus (LT2) Primary Annotation No. (LT2) histidinol phosphatase-related protein Unknown function NT01ST0303 AAL19211 STM0248 conserved hypothetical protein Hypothetical proteins NT01ST1594 AAL20205 STM1280 multidrug resistance protein norm Cellular processes NT01ST1758 AAL20347 STM1425 acyl carrier protein acpd Fatty acid and phospholipid NT01ST2039 AAL20560 STM1642 phosphodiesterase metabolism outer membrane protein c precursor ompc Cell Envelope NT01ST2829 AAL21169 STM2267 CorE b2611 Viral functions NT01ST3328 AAL21567 STM2678 4Fe-4S binding domain protein Unknown function Cell Envelope NT01ST3543 AAL21728 STM2848 transcriptional regulator, DeoR family glpr Regulatory functions NT01ST4096 AAL22134 STM3262 conserved hypothetical protein Hypothetical proteins NT01ST4169 AAL22198 STM3329 TIGR01212 conserved hypothetical protein Hypothetical proteins NT01ST4329 AAL22325 STM3463 potential nitrite transporter Energy metabolism NT01ST4346 AAL22338 STM3476 acetyltransferase, GNAT family family Unknown function NT01ST4455 AAL22425 STM3565 PTS system, mannitol-specific IIC Transport and binding NT01ST4604 AAL22544 STM3685 component subfamily, putative proteins DNA polymerase III, beta subunit dnan DNA metabolism NT01ST4790 AAL22696 STM3837 vgrg protein, putative vgrg Unclassified NT01ST0349 AAL19246 STM0289 glycosyl hydrolase, family 13 malz Energy metabolism NT01ST0499 AAL19355 STM0401 catalase/peroxidase HPI katg Cellular processes NT01ST5107 AAL22946 STM4106 hypothetical protein Hypothetical proteins NT01ST5613 AAL23340 STM oxoglutarate dehydrogenase, E1 suca Energy metabolism NT01ST0884 AAL19680 STM0736 component carbamoyl-phosphate synthase, small subunit cara Purines, pyrimidines, nucleosides, and nucleotides NT01ST0081 AAL19030 STM0066

2 conserved hypothetical protein Hypothetical proteins NT01ST0185 AAL19112 STM0148 conserved hypothetical protein Hypothetical proteins NT01ST0198 AAL19121 STM0157 salmonella atypical fimbria outer safc Cell Envelope NT01ST0369 AAL19258 STM0301 membrane usher prpe protein prpe Unknown function NT01ST0460 AAL19325 STM0371 phosphoribosylaminoimidazole pure Purines, pyrimidines, NT01ST0653 AAL19488 STM0534 carboxylase, catalytic subunit nucleosides, and nucleotides aminotransferase, class I arat Central intermediary NT01ST0731 AAL19554 STM0603 metabolism phosphoglucomutase, alpha-dglucose pgm Energy metabolism NT01ST0841 AAL19642 STM0698 phosphate-specific lipoprotein, putative Cell Envelope NT01ST0899 AAL19687 STM0743 conserved hypothetical protein Hypothetical proteins NT01ST1873 AAL20434 STM1515 TIGR00156 domain protein GDP-mannose mannosyl hydrolase Purines, pyrimidines, nucleosides, and nucleotides NT01ST2623 AAL21011 STM2107 cysteine synthase B cysm Amino acid biosynthesis NT01ST3032 AAL21334 STM2440 nrdi protein nrdi Purines, pyrimidines, nucleosides, and nucleotides NT01ST3491 AAL21691 STM2806 PTS system IIA component, putative Transport and binding proteins NT01ST4092 AAL22130 STM3258 oligopeptide ABC transporter, ATPbinding oppf Transport and binding NT01ST4531 AAL22487 STM3626 protein proteins aldehyde dehydrogenase B aldb Energy metabolism NT01ST4599 AAL22539 STM3680 xanthine/uracil permease family Transport and binding NT01ST4808 AAL22709 STM3851 protein proteins rard protein rard Unknown function NT01ST4920 AAL22799 STM3955 thih protein thih Biosynthesis of cofactors, NT01ST5159 AAL22987 STM4159 prosthetic groups, and carriers acetate operon repressor. iclr Energy metabolism NT01ST5191 AAL23011 STM4187 trehalose-6-phosphate hydrolase trec Energy metabolism NT01ST5527 AAL23273 STM4453 surface antigen b0177 Unknown function NT01ST0277 AAL19188 STM0224

3 tola protein tola Cellular processes NT01ST0904 AAL19691 STM0747 conserved hypothetical protein Hypothetical proteins NT01ST1047 AAL19797 STM0861 SseC ssec Unclassified NT01ST1735 AAL20324 STM1400 SseF ssef Unclassified NT01ST1739 AAL20328 STM1404 OmpS2 protein omps2 Cell Envelope NT01ST1814 AAL20393 STM1473 hypothetical protein Hypothetical proteins NT01ST1963 AAL20501 STM1583 outer membrane protein S1 (omps1) omps1 Cell Envelope NT01ST2489 AAL20905 STM1995 PTS system, IIC component, putative Transport and binding proteins Amino acid biosynthesis NT01ST2916 AAL21243 STM2342 sulfate ABC transporter, ATP-binding cysa Transport and binding NT01ST3033 AAL21335 STM2441 protein proteins glycine dehydrogenase gcvp Energy metabolism NT01ST3817 AAL21928 STM3053 ubih protein ubih Biosynthesis of cofactors, NT01ST3824 AAL21932 STM3057 prosthetic groups, and carriers 99 pct identical to TraF of plasmid Viral functions NT01ST5263 AAL23066 STM4242 R64 lipoate-protein ligase A lpla Protein fate Biosynthesis of cofactors, prosthetic groups, and carriers NT01ST5673 AAL23391 STM amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase folk-2 Biosynthesis of cofactors, prosthetic groups, and carriers NT01ST0225 AAL19147 STM0183 exonuclease SbcC, putative DNA metabolism NT01ST0490 AAL19349 STM0395 PipB pipb Unclassified NT01ST1346 AAL20020 STM1088 membrane protein, putative Cell Envelope NT01ST1778 AAL20363 STM1441 pyridoxal kinase Biosynthesis of cofactors, NT01ST1789 AAL20372 STM1450 prosthetic groups, and carriers Anaerobic dimethyl sulfoxide b1590 Energy metabolism NT01ST1847 AAL20415 STM1496 reductase chain C (DMSO reductase anchor subunit). hypothetical protein Hypothetical proteins NT01ST1895 AAL20451 STM1532 hypothetical protein Hypothetical proteins NT01ST2070 AAL20585 STM1668

4 oligopeptide ABC transporter, periplasmic oligopeptide-binding protein oppa Transport and binding proteins NT01ST2082 AAL20596 STM1679 conserved hypothetical protein Hypothetical proteins NT01ST2197 AAL20683 STM1768 conserved hypothetical protein Hypothetical proteins NT01ST2854 AAL21188 STM2287 thiosulfate-binding protein precursor Transport and binding NT01ST3036 AAL21338 STM2444 proteins glucarate dehydratase b2787 Energy metabolism NT01ST3694 AAL21840 STM2960 proton/peptide symporter family Unclassified NT01ST4488 AAL22452 STM3592 protein glycyl-trna synthetase, beta subunit glys Protein synthesis NT01ST4569 AAL22514 STM3655 glucosamine--fructose-6-phosphate glms Central intermediary NT01ST4821 AAL22719 STM3861 aminotransferase (isomerizing) metabolism hypothetical protein Hypothetical proteins NT01ST4989 AAL22854 STM hydroxyisobutyrate dehydrogenase, Energy metabolism NT01ST4997 AAL22862 STM4023 putative conserved hypothetical protein Hypothetical proteins NT01ST2037 AAL20558 STM1640 malt regulatory protein. malt Energy metabolism NT01ST4393 AAL22377 STM3515 conserved hypothetical protein Hypothetical proteins NT01ST5283 AAL23082 STM4258 membrane protein, putative Cell Envelope NT01ST5539 AAL23283 STM4464 hypothetical protein Hypothetical proteins NT01ST2302 AAL20769 STM1854 similar to Cell Envelope NT01ST3484 AAL21686 STM2801 conserved hypothetical protein Hypothetical proteins NT01ST4820 AAL22718 STM3860 lipoprotein, putative Cell Envelope NT01ST5016 AAL22878 STM4039 cell division protein FtsK ftsk Cellular processes NT01ST1184 AAL19895 STM0960

5 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 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 Baye indicates the posterior probability; ";" separates the aa sites; aa sites are based on site location in the alignment used for positive selection analyses (pro method is used to calculate posterior probabilities for site classes under alternative models (Model 2a and Model A if the LRT suggests presence of cod 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, T 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 codon is exclusively used for each amino acid) to 61 (which indicates that the use of alternative synonymous codons is equally likely (Wright Gen

6 n which all genes, including genes with recombination, were used JCVI Locus (CT18) a d N b d S Alignment Length, bp 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 NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST E NT03ST E E NT03ST E NT03ST NT03ST E NT03ST NT03ST NT03ST

7 NT03ST NT03ST NT03ST E NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST E

8 NT03ST NT03ST NT03ST NT03ST NT03ST E NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST E NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST

9 NT03ST NT03ST E E NT03ST E NT03ST NT03ST NT03ST E NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST NT03ST E E NT03ST NT03ST E

10 m branch specific test es (BEB), the number in bracket ovided as Additional file 5); BEB dons under positive selection on the Ty2= Typhi Ty2, LT2= Typhimurium LT2 0 (in case of extreme bias where one ne 87:23-29)

11 BEB (TO) f p-value for Ch# c q-value for Ch# Prop. of sites under pos sel. (Ch#) d ω(ch#) e BEB (Ch#) f p-value for Tym# c q-value for Tym# Prop. of sites under pos sel. (Tym#) d 175 (0.985); 184 (0.959) 3.36E E (0.999);184 (0.997); (0.992); 191 (0.982) (0.970) (0.992) (0.964) (1.000) (0.990); 45 (0.990); 129 (0.959); 597(0.957); 622 (0.959); 631 (0.973)

12 (0.974) (0.951); 111 (0.955); (0.954); 692 (0.950) 95 (0.985) (0.976) (0.984) (0.969) (0.985); 679 (0.989); 691 (0.986) (0.963) (0.998); 691(0.998)

13 (0.953) (0.954); 198 (0.970); (0.965); 282 (0.982) 189 (0.984) (0.977) E (0.970) (0.983) (0.986); 324 (0.986) 3.52E (0.997); 146 (0.996); 147 (0.962); 150 (0.986); 151 (0.965); 152 (0.994); 155 (0.998) (0.997) (0.991); 178 (0.973); 261 (0.973) 2.85E (0.996); 264 (0.984)

14 1.19E (1.000) (0.994);189 (0.998) E E (0.955) (0.983) (0.955) (0.976); 7 (0.962); 13 (0.983);15 (0.988); 19 (0.973); 22 (0.973); 24 (0.979); 41 (0.979); 51 (0.976); 60 (0.971); 68 (0.975) (0.988) 1.54E E (0.966); 272 (0.975); 278 (0.975); 279 (0.997)

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16 ω(tym#) e BEB (Tym#) f p-value q-value Ty# Prop. of sites Ty# c 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 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 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

17 (0.984); 396 (0.986); (0.983); 401 (0.989); 405 (0.997) (0.956); 31(0.955) (0.967); 139 (0.996); (0.984); 144 (0.970); 147 (0.977) ND ND ND ND ND

18 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 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND E E (0.977); 146 (0.999); 147 (0.988); 149 (0.975); 150 (0.991); 151 (0.986); 152 (0.997); 155 (0.999) (0.982); 41 (0.993)

19 (0.966) (0.979) 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

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21 ω (Pty#) e BEB (Pty#) f Tree group no g 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 (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); ND ND ND ND ND (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (((ATCC9150,ch),CT18),LT2,Ty2); (0.977); 45 (0.958); 2 (((ATCC9150,ch),LT2),CT18,Ty2); (0.959); 83 (0.989); 84 (0.978); 85 (0.985); 97 (0.961); 114 (0.984); 124 (0.982); 129 (0.988) (((ATCC9150,ch),LT2),CT18,Ty2); (((ATCC9150,ch),LT2),CT18,Ty2); (((ATCC9150,ch),LT2),CT18,Ty2); (((ATCC9150,CT18),ch),LT2,Ty2); ((ATCC9150,ch),(CT18,Ty2),LT2);

22 (0.958); 235(0.992) 5 ((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,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,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,LT2)),CT18,Ty2);

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

24 ((ATCC9150,(CT18,Ty2)),ch,LT2); ((ATCC9150,(CT18,Ty2)),ch,LT2); ((ATCC9150,(CT18,Ty2)),ch,LT2); ((ATCC9150,(CT18,Ty2)),ch,LT2); (0.973) 7 ((ATCC9150,(CT18,Ty2)),ch,LT2); ((ATCC9150,(CT18,Ty2)),ch,LT2); (0.568) 7 ((ATCC9150,(CT18,Ty2)),ch,LT2); ((ATCC9150,(CT18,Ty2)),ch,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,(ch,(CT18,Ty2)),LT2); (0.992); 40 (0.992); 9 (((ATCC9150,LT2),ch),CT18,Ty2); ND ND ND ND ND 44 (0.958); 45 (0.977) (((ATCC9150,LT2),ch),CT18,Ty2); ND ND ND ND ND (0.999); 15 (0.999); 9 (((ATCC9150,LT2),ch),CT18,Ty2); (0.998); 21 (0.990); 22 (0.997); 24 (0.999); 25 (0.989); 39 (0.995); 41 (0.998); 44 (0.991); 45 (0.988); 48 (0.964); 51 (0.998); 60 (0.998); 66 (0.992); 67 (0.993); 68 (0.999); 71 (0.991) (((ATCC9150,LT2),ch),CT18,Ty2); ((ATCC9150,CT18),(ch,LT2),Ty2);

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26 q-value for PHI GENE- CONV inner p-value q-value for GENE-CONV Nc value for Paratyphi A i 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 ND

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28 ND

29 ND ND