Supplementary Figure 1. Chronogram of 308 Archaeal, Bacterial

Lentisphaerae bacterium Coraliomargarita akajimensis DSM 45221 Methylacidiphilum infernorum V4 Verrucomicrobia bacterium Chlamydia trachomatis D UW−3 CX Omnitrophica bacterium SCGC AAA252−B19 Pirellula staleyi DSM 6068 Planctomycetes MAG Hydrogenedentes bacterium JGI 0000039−J10 Elusimicrobia bacterium 1 Elusimicrobia bacterium 3 Elusimicrobia bacterium 4 Elusimicrobia bacterium 2 Spirochaeta thermophila DSM 6192 Treponema azotonutricium ZAS−9 Treponema primitia ZAS−2 Treponema sp 1 Treponema sp 2 Spirochaeta smaragdinae DSM 11293 Spirochaetes bacterium 2 Spirochaetes bacterium 1 Chlorobium tepidum TLS Chlorobaculum parvum NCIB 8327 Chlorobium luteolum DSM 273 Chlorobium phaeovibrioides DSM 265 Chlorobium chlorochromatii CaD3 Pelodictyon phaeoclathratiforme BU−1 Chlorobium phaeobacteroides DSM 266 Chlorobium limicola DSM 245 Prosthecochloris aestuarii DSM 271 Chloroherpeton thalassium ATCC 35110 Bacteroides fragilis YCH46 Candidatus Azobacteroides pseudotrichonymphae genomovar CFP2 Ignavibacterium album JCM 16511 1 Ignavibacterium album JCM 16511 2 Marinimicrobia bacterium JGI 0000039−D08 Caldithrix abyssi DSM 13497 Latescibacteria bacterium DG 63 candidate division Zixibacteria bacterium Cloacimonetes bacterium JGI 0000039−A21 Fibrobacter succinogenes subsp succinogenes S85 CP0071271 Gemmatirosa kalamazoonesis strain KBS708 Geobacter sulfurreducens PCA Geobacter uraniireducens Rf4 Geobacter daltonii FRC−32 Geobacter bemidjiensis Bem Pelobacter propionicus DSM 2379 Geobacteraceae bacterium 2 Desulfuromonadales bacterium Pelobacteraceae bacterium Geobacteraceae bacterium 1 Geobacteraceae bacterium 3 Geobacteraceae bacterium 4 Geobacteraceae bacterium 5 Geobacter lovleyi SZ Pelobacter carbinolicus DSM 2380 Desulfovibrio magneticus RS−1 Desulfovibrio salexigens DSM 2638 Desulfovibrio desulfuricans ND132 Proteobacterium MAG 4 Desulfovibrionales bacterium Desulfovibrio africanus str Walvis Bay Desulfomicrobium baculatum DSM 4028 Thermodesulfatator indicus DSM 15286 Desulfobacterium autotrophicum HRM2 Desulfobacula sp Proteobacterium MAG 5 Desulfatibacillum alkenivorans AK−01 Desulfurivibrio alkaliphilus AHT2 Deltaproteobacteria bacterium 2 Deltaproteobacteria bacterium 3 Deltaproteobacteria bacterium 4 Deltaproteobacteria bacterium 1 Desulfobulbus propionicus DSM 2032 Desulfarculus baarsii DSM 2075 Syntrophobacter fumaroxidans MPOB Desulfobacca acetoxidans DSM 11109 Anaeromyxobacter sp Fw109−5 Nitrospina gracilis 3/211 Candidatus Tectomicrobia bacterium RIFCSPLOWO2 02 FULL 70 19 Azoarcus sp BH72 Rhodocyclales bacterium Dechloromonas aromatica RCB Candidatus Accumulibacter phosphatis clade IIA str UW−1 Dechlorosoma suillum PS Polaromonas naphthalenivorans CJ2 Leptothrix cholodnii SP−6 Herbaspirillum seropedicae SmR1 Sideroxydans lithotrophicus ES−1 Gallionellales bacterium Hydrogenophilales bacterium Pectobacterium atrosepticum SCRI1043 Dickeya dadantii Ech703 Tolumonas auensis DSM 9187 Azotobacter vinelandii DJ Pseudomonadales bacterium Proteobacterium MAG 3 Proteobacterium MAG 1 Proteobacterium MAG 2 Halorhodospira halophila SL1 Allochromatium vinosum DSM 180 Methylomonas methanica MC09 Acidithiobacillus ferrivorans SS3 Magnetospirillum magneticum AMB−1 Rhodospirillum rubrum F11 Azospirillum sp B510 Azospirillum lipoferum 4B Azospirillum brasilense Sp245 Rhodospirillum centenum SW Gluconacetobacter diazotrophicus PAl 5 Azorhizobium caulinodans ORS 571 Xanthobacter autotrophicus Py2 Beijerinckia indica subsp indica ATCC 9039 Methylocella silvestris BL2 Methylobacterium nodulans ORS 2060 Bradyrhizobium japonicum USDA 6 Bradyrhizobium sp BTAi1 Bradyrhizobium sp ORS 278 Mesorhizobium loti MAFF303099 Mesorhizobium opportunistum WSM2075 Mesorhizobium ciceri biovar biserrulae WSM1271 Rhodomicrobium vannielii ATCC 17100 Rhodobacter capsulatus SB 1003 Magnetococcus marinus MC−1 Mariprofundus ferrooxydans PV−1 Arcobacter sp L Arcobacter nitrofigilis DSM 7299 Sulfuricurvum kujiense DSM 16994 Sulfuricurvum sp 1 Sulfuricurvum sp 3 Sulfuricurvum sp 2 Sulfurimonas sp 1 Sulfurimonas sp 2 Candidatus Lambdaproteobacteria bacterium 1 Candidatus Lambdaproteobacteria bacterium 2 Denitrovibrio acetiphilus DSM 12809 Calditerrivibrio nitroreducens DSM 19672 Desulfurispirillum indicum S5 Thermodesulfovibrio yellowstonii DSM 11347 Candidatus Dadabacteria bacterium CSP1−2 Acidobacterium capsulatum ATCC 51196 Candidatus Rokubacteria bacterium CSP1−6 Mesoplasma florum L1 Firmicutes bacterium 1 Firmicutes bacterium 2 Paenibacillus terrae HPL−003 Clostridium kluyveri DSM 555 Clostridium ljungdahlii DSM 13528 Clostridium beijerinckii NCIMB 8052 Clostridium cellulovorans 743B Clostridium lentocellum DSM 5427 Coprococcus catus GD 7 Alkaliphilus metalliredigens QYMF Acetobacterium woodii DSM 1030 Caldicellulosiruptor saccharolyticus DSM 8903 Caldicellulosiruptor hydrothermalis 108 Caldicellulosiruptor lactoaceticus 6A Thermoanaerobacterium thermosaccharolyticum DSM 571 Desulfotomaculum reducens MI−1 Desulfotomaculum ruminis DSM 2154 Desulfotomaculum carboxydivorans CO−1−SRB Desulfotomaculum kuznetsovii DSM 6115 Candidatus Desulforudis audaxviator MP104C Desulfotomaculum acetoxidans DSM 771 Thermincola potens JR Heliobacterium modesticaldum Ice1 Syntrophobotulus glycolicus DSM 8271 Desulfosporosinus orientis DSM 765 Syntrophothermus lipocalidus DSM 12680 Actinobacteria bacterium 1 Atribacteria bacterium SCGC AAA252−M02 Actinobacteria bacterium 2 Candidatus Margulisbacteria bacterium 1 Candidatus Margulisbacteria bacterium 2 Nostoc sp PCC 7120 Nostoc azollae 0708 Cyanothece sp PCC 7425 Trichodesmium erythraeum IMS101 Cyanothece sp PCC 7424 Cyanothece sp PCC 7822 Cyanothece sp PCC 8802 Candidatus Atelocyanobacterium thalassa isolate ALOHA Candidatus Acetylcyanobacterium MAG Synechococcus sp JA−3−3Ab Candidatus Melainabacteria bacterium Frankia symbiont of Datisca glomerata Fimbriimonas ginsengisoli Gsoil 348 Roseiflexus castenholzii DSM 13941 Chloroflexi bacterium Candidatus Beckwithbacteria bacterium GW2011 GWC1 49 16 Candidatus Gottesmanbacteria bacterium Candidatus Levybacteria bacterium GW2011 GWB1 36 18 US07 C0001 Candidatus Roizmanbacteria bacterium GW2011 GWC2 37 13 Candidatus Daviesbacteria bacterium GW2011 GWA1 36 8 Candidatus Curtissbacteria bacterium GW2011 GWA1 41 11 Candidate division WS6 bacterium GW2011 GWF2 39 15 Candidate division WWE3 bacterium RIFOXYD2 FULL 43 10 Candidatus Falkowbacteria bacterium GW2011 GWF2 38 1205 Candidatus Jorgensenbacteria bacterium GW2011 GWA1 48 13 Candidatus Kaiserbacteria bacterium GW2011 GWB1 50 17 Candidatus Wirthbacteria bacterium CG2 30 54 11 Deinococcus radiodurans R1 Fusobacterium nucleatum subsp nucleatum ATCC 25586 Thermanaerovibrio acidaminovorans DSM 6589 Dictyoglomus turgidum DSM 6724 Caldisericum sp CG2 30 36 11 Thermotoga maritima MSB8 Hydrogenobacter thermophilus TK−6 Thermocrinis albus DSM 14484 Aquifex aeolicus VF5 Calescamantes bacterium JGI 0000106−G12 Methanothermobacter thermautotrophicus str Delta H Methanothermobacter marburgensis str Marburg Methanococcus vannielii SB Methanococcus aeolicus Nankai−3 Methanothermococcus okinawensis IH1 Methanotorris igneus Kol 5 Methanocaldococcus vulcanius M7 Methanocaldococcus sp FS406−22 Methanocaldococcus jannaschii DSM 2661 Methanocaldococcus infernus ME Methanosarcina mazei Go1 Methanosarcina acetivorans C2A Methanosarcina barkeri str Fusaro Methanohalophilus mahii DSM 5219

Methanosaeta concilii GP6 Methanocella arvoryzae MRE50 Methanocella conradii HZ254 Methanospirillum hungatei JF−1 Methanoregula boonei 6A8 Methanosphaerula palustris E1−9c Methanocorpusculum labreanum Z Halorubrum lacusprofundi ATCC 49239 Geoglobus ahangari strain 234 Candidatus Iainarchaeum andersonii SCGC AAA011−E11 Candidatus Nanopusillus acidilobi strain 7A Candidatus Pacearchaeota archaeon RBG 13 36 9 Candidatus Woesearchaeota archaeon RBG 13 36 6 Nanohaloarchaea archaeon SG9 Candidatus Micrarchaeota archaeon RBG 16 49 10 Candidatus Aenigmarchaeota archaeon CG1 02 38 14 Sulfolobus solfataricus P2 Candidatus Korarchaeum cryptofilum OPF8 Nitrososphaera viennensis EN76 1 Nitrososphaera viennensis EN76 2 Candidatus Bathyarchaeota archaeon RBG 16 57 9 Candidatus Thorarchaeota archaeon SMTZ1−83 1 Candidatus Thorarchaeota archaeon SMTZ1−83 2 Candidatus Lokiarchaeota archaeon CR 4 1 Candidatus Lokiarchaeota archaeon CR 4 2 Candidatus Odinarchaeota archaeon LCB 4 Candidatus Heimdallarchaeota archaeon AB 125 Picrophilus torridus DSM 9790 Oryza sativa Sorghum bicolor Zea mays mays cv B73 Glycine max cultivar Williams 82 Populus trichocarpa Ricinus communis Arabidopsis thaliana Volvox carteri f nagariensis 69 1b Chlorella sp NC64A Micromonas pusilla NOUM17 PCC 299 Theileria annulata strain Ankara clone C9 Phaeodactylum tricornutum CCAP 10551 Thalassiosira pseudonana CCMP1335 Pavlova lutheri Allomyces macrogynus ATCC 38327 Blastocystis hominis Coemansia reversa NRRL 1564 Cryptococcus neoformans var neoformans B 3501A Botryobasidium botryosum Phanerochaete chrysosporium RP 78 Serpula lacrymans lacrymans S79 Hypholoma sublateritium Gymnopus luxurians Laccaria bicolor S238N H82 LACBI Schizophyllum commune H4 8 Postia placenta Mad 698 R POSPL Coprinopsis cinerea strain okayama7130 Malassezia globosa CBS Beauveria bassiana CH Ministeria vibrans Andalucia incarcerata Roombia truncata Dictyostelium discoideum AX4 Oikopleura dioica Collodictyon triciliatum Euglena gracilis Leishmania Leishmania Leishmania infantum JPCM5 MCANES98LLM 877 Trypanosoma brucei TREU927 Reclinomonas americana Jakoba libera Babesia bovis T2Bo Guttulinopsis vulgaris Bigelowiella natans Paramecium tetraurelia strain Stock d4−2 macronuclear 1 Paramecium tetraurelia strain Stock d4−2 macronuclear 2 Tetrahymena pyriformis Tetrahymena thermophila SB210 macronuclear Entamoeba histolytica HM−1 IMSS 1 Entamoeba histolytica HM−1 IMSS 2 Sawyeria marylandensis Naegleria gruberi strain NEG−M Giardia lamblia ATCC 50803 strain WB C6 Trichomonas vaginalis G3 Guillardia theta

4450 3560 2670 1780 890 0

Supplementary Figure 1. Chronogram of 308 archaeal, bacterial, and eukaryotic genomes generated using PhyloBayes with a CIR clock model and conservative fossil calibration points. Chronogram was built based on an alignment of concatenated universal, single-copy genes. Branch lengths indicate time in thousands of years. Error bars represent the 95% confidence intervals for each node. Lentisphaera araneosa HTCC2155 Lentisphaerae bacterium Coraliomargarita akajimensis DSM 45221 Methylacidiphilum infernorum V4 Verrucomicrobia bacterium Chlamydia trachomatis D UW−3 CX Omnitrophica bacterium SCGC AAA252−B19 Pirellula staleyi DSM 6068 Planctomycetes MAG Hydrogenedentes bacterium JGI 0000039−J10 Elusimicrobia bacterium 1 Elusimicrobia bacterium 3 Elusimicrobia bacterium 4 Elusimicrobia bacterium 2 Spirochaeta thermophila DSM 6192 Treponema azotonutricium ZAS−9 Treponema primitia ZAS−2 Treponema sp 1 Treponema sp 2 Spirochaeta smaragdinae DSM 11293 Spirochaetes bacterium 2 Spirochaetes bacterium 1 Chlorobium tepidum TLS Chlorobaculum parvum NCIB 8327 Chlorobium luteolum DSM 273 Chlorobium phaeovibrioides DSM 265 Chlorobium chlorochromatii CaD3 Pelodictyon phaeoclathratiforme BU−1 Chlorobium phaeobacteroides DSM 266 Chlorobium limicola DSM 245 Prosthecochloris aestuarii DSM 271 Chloroherpeton thalassium ATCC 35110 Bacteroides fragilis YCH46 Candidatus Azobacteroides pseudotrichonymphae genomovar CFP2 Ignavibacterium album JCM 16511 1 Ignavibacterium album JCM 16511 2 Marinimicrobia bacterium JGI 0000039−D08 Caldithrix abyssi DSM 13497 Latescibacteria bacterium DG 63 candidate division Zixibacteria bacterium Cloacimonetes bacterium JGI 0000039−A21 Fibrobacter succinogenes subsp succinogenes S85 CP0071271 Gemmatirosa kalamazoonesis strain KBS708 Geobacter sulfurreducens PCA Geobacter uraniireducens Rf4 Geobacter daltonii FRC−32 Geobacter bemidjiensis Bem Pelobacter propionicus DSM 2379 Geobacteraceae bacterium 2 Desulfuromonadales bacterium Pelobacteraceae bacterium Geobacteraceae bacterium 1 Geobacteraceae bacterium 3 Geobacteraceae bacterium 4 Geobacteraceae bacterium 5 Geobacter lovleyi SZ Pelobacter carbinolicus DSM 2380 Desulfovibrio magneticus RS−1 Desulfovibrio salexigens DSM 2638 Desulfovibrio desulfuricans ND132 Proteobacterium MAG 4 Desulfovibrionales bacterium Desulfovibrio africanus str Walvis Bay Desulfomicrobium baculatum DSM 4028 Thermodesulfatator indicus DSM 15286 Desulfobacterium autotrophicum HRM2 Desulfobacula sp Proteobacterium MAG 5 Desulfatibacillum alkenivorans AK−01 Desulfurivibrio alkaliphilus AHT2 Deltaproteobacteria bacterium 2 Deltaproteobacteria bacterium 3 Deltaproteobacteria bacterium 4 Deltaproteobacteria bacterium 1 Desulfobulbus propionicus DSM 2032 Desulfarculus baarsii DSM 2075 Syntrophobacter fumaroxidans MPOB Desulfobacca acetoxidans DSM 11109 Anaeromyxobacter sp Fw109−5 Nitrospina gracilis 3/211 Candidatus Tectomicrobia bacterium RIFCSPLOWO2 02 FULL 70 19 Azoarcus sp BH72 Rhodocyclales bacterium Dechloromonas aromatica RCB Candidatus Accumulibacter phosphatis clade IIA str UW−1 Dechlorosoma suillum PS Polaromonas naphthalenivorans CJ2 Leptothrix cholodnii SP−6 Herbaspirillum seropedicae SmR1 Sideroxydans lithotrophicus ES−1 Gallionellales bacterium Hydrogenophilales bacterium Pectobacterium atrosepticum SCRI1043 Dickeya dadantii Ech703 Tolumonas auensis DSM 9187 Azotobacter vinelandii DJ Pseudomonadales bacterium Proteobacterium MAG 3 Proteobacterium MAG 1 Proteobacterium MAG 2 Halorhodospira halophila SL1 Allochromatium vinosum DSM 180 Methylomonas methanica MC09 Acidithiobacillus ferrivorans SS3 Magnetospirillum magneticum AMB−1 Rhodospirillum rubrum F11 Azospirillum sp B510 Azospirillum lipoferum 4B Azospirillum brasilense Sp245 Rhodospirillum centenum SW Gluconacetobacter diazotrophicus PAl 5 Azorhizobium caulinodans ORS 571 Xanthobacter autotrophicus Py2 Beijerinckia indica subsp indica ATCC 9039 Methylocella silvestris BL2 Methylobacterium nodulans ORS 2060 Bradyrhizobium japonicum USDA 6 Bradyrhizobium sp BTAi1 Bradyrhizobium sp ORS 278 Mesorhizobium loti MAFF303099 Mesorhizobium opportunistum WSM2075 Mesorhizobium ciceri biovar biserrulae WSM1271 Rhodomicrobium vannielii ATCC 17100 Rhodobacter capsulatus SB 1003 Magnetococcus marinus MC−1 Mariprofundus ferrooxydans PV−1 Arcobacter sp L Arcobacter nitrofigilis DSM 7299 Sulfuricurvum kujiense DSM 16994 Sulfuricurvum sp 1 Sulfuricurvum sp 3 Sulfuricurvum sp 2 Sulfurimonas sp 1 Sulfurimonas sp 2 Candidatus Lambdaproteobacteria bacterium 1 Candidatus Lambdaproteobacteria bacterium 2 Denitrovibrio acetiphilus DSM 12809 Calditerrivibrio nitroreducens DSM 19672 Desulfurispirillum indicum S5 Thermodesulfovibrio yellowstonii DSM 11347 Candidatus Dadabacteria bacterium CSP1−2 Acidobacterium capsulatum ATCC 51196 Candidatus Rokubacteria bacterium CSP1−6 Mesoplasma florum L1 Firmicutes bacterium 1 Firmicutes bacterium 2 Paenibacillus terrae HPL−003 Clostridium kluyveri DSM 555 Clostridium ljungdahlii DSM 13528 Clostridium beijerinckii NCIMB 8052 Clostridium cellulovorans 743B Clostridium lentocellum DSM 5427 Coprococcus catus GD 7 Alkaliphilus metalliredigens QYMF Acetobacterium woodii DSM 1030 Caldicellulosiruptor saccharolyticus DSM 8903 Caldicellulosiruptor hydrothermalis 108 Caldicellulosiruptor lactoaceticus 6A Thermoanaerobacterium thermosaccharolyticum DSM 571 Desulfotomaculum reducens MI−1 Desulfotomaculum ruminis DSM 2154 Desulfotomaculum carboxydivorans CO−1−SRB Desulfotomaculum kuznetsovii DSM 6115 Candidatus Desulforudis audaxviator MP104C Desulfotomaculum acetoxidans DSM 771 Thermincola potens JR Heliobacterium modesticaldum Ice1 Syntrophobotulus glycolicus DSM 8271 Desulfosporosinus orientis DSM 765 Syntrophothermus lipocalidus DSM 12680 Actinobacteria bacterium 1 Atribacteria bacterium SCGC AAA252−M02 Actinobacteria bacterium 2 Candidatus Margulisbacteria bacterium 1 Candidatus Margulisbacteria bacterium 2 Nostoc sp PCC 7120 Nostoc azollae 0708 Cyanothece sp PCC 7425 Trichodesmium erythraeum IMS101 Cyanothece sp PCC 7424 Cyanothece sp PCC 7822 Cyanothece sp PCC 8802 Candidatus Atelocyanobacterium thalassa isolate ALOHA Candidatus Acetylcyanobacterium MAG Synechococcus sp JA−3−3Ab Candidatus Melainabacteria bacterium Frankia symbiont of Datisca glomerata Fimbriimonas ginsengisoli Gsoil 348 Roseiflexus castenholzii DSM 13941 Chloroflexi bacterium Candidatus Beckwithbacteria bacterium GW2011 GWC1 49 16 Candidatus Gottesmanbacteria bacterium Candidatus Levybacteria bacterium GW2011 GWB1 36 18 US07 C0001 Candidatus Roizmanbacteria bacterium GW2011 GWC2 37 13 Candidatus Daviesbacteria bacterium GW2011 GWA1 36 8 Candidatus Curtissbacteria bacterium GW2011 GWA1 41 11 Candidate division WS6 bacterium GW2011 GWF2 39 15 Candidate division WWE3 bacterium RIFOXYD2 FULL 43 10 Candidatus Falkowbacteria bacterium GW2011 GWF2 38 1205 Candidatus Jorgensenbacteria bacterium GW2011 GWA1 48 13 Candidatus Kaiserbacteria bacterium GW2011 GWB1 50 17 Candidatus Wirthbacteria bacterium CG2 30 54 11 Deinococcus radiodurans R1 Fusobacterium nucleatum subsp nucleatum ATCC 25586 Thermanaerovibrio acidaminovorans DSM 6589 Dictyoglomus turgidum DSM 6724 Caldisericum sp CG2 30 36 11 Thermotoga maritima MSB8 Hydrogenobacter thermophilus TK−6 Thermocrinis albus DSM 14484 Aquifex aeolicus VF5 Calescamantes bacterium JGI 0000106−G12 Methanothermobacter thermautotrophicus str Delta H Methanothermobacter marburgensis str Marburg Methanococcus vannielii SB Methanococcus aeolicus Nankai−3 Methanothermococcus okinawensis IH1 Methanotorris igneus Kol 5 Methanocaldococcus vulcanius M7 Methanocaldococcus sp FS406−22 Methanocaldococcus jannaschii DSM 2661 Methanocaldococcus infernus ME Methanosarcina mazei Go1 Methanosarcina acetivorans C2A Methanosarcina barkeri str Fusaro Methanohalophilus mahii DSM 5219

4940 3950 2960 1970 987 0

Supplementary Figure 2. Chronogram of 308 archaeal, bacterial, and eukaryotic genomes generated using PhyloBayes with a CIR clock model and liberal fossil calibration points. Chronogram was built based on an alignment of concatenated universal, single-copy genes. Branch lengths indicate time in thousands of years. Error bars represent the 95% confidence intervals for each node. Lentisphaera araneosa HTCC2155 Lentisphaerae bacterium Coraliomargarita akajimensis DSM 45221 Methylacidiphilum infernorum V4 Verrucomicrobia bacterium Chlamydia trachomatis D UW−3 CX Omnitrophica bacterium SCGC AAA252−B19 Pirellula staleyi DSM 6068 Planctomycetes MAG Hydrogenedentes bacterium JGI 0000039−J10 Elusimicrobia bacterium 1 Elusimicrobia bacterium 3 Elusimicrobia bacterium 4 Elusimicrobia bacterium 2 Spirochaeta thermophila DSM 6192 Treponema azotonutricium ZAS−9 Treponema primitia ZAS−2 Treponema sp 1 Treponema sp 2 Spirochaeta smaragdinae DSM 11293 Spirochaetes bacterium 2 Spirochaetes bacterium 1 Chlorobium tepidum TLS Chlorobaculum parvum NCIB 8327 Chlorobium luteolum DSM 273 Chlorobium phaeovibrioides DSM 265 Chlorobium chlorochromatii CaD3 Pelodictyon phaeoclathratiforme BU−1 Chlorobium phaeobacteroides DSM 266 Chlorobium limicola DSM 245 Prosthecochloris aestuarii DSM 271 Chloroherpeton thalassium ATCC 35110 Bacteroides fragilis YCH46 Candidatus Azobacteroides pseudotrichonymphae genomovar CFP2 Ignavibacterium album JCM 16511 1 Ignavibacterium album JCM 16511 2 Marinimicrobia bacterium JGI 0000039−D08 Caldithrix abyssi DSM 13497 Latescibacteria bacterium DG 63 candidate division Zixibacteria bacterium Cloacimonetes bacterium JGI 0000039−A21 Fibrobacter succinogenes subsp succinogenes S85 CP0071271 Gemmatirosa kalamazoonesis strain KBS708 Geobacter sulfurreducens PCA Geobacter uraniireducens Rf4 Geobacter daltonii FRC−32 Geobacter bemidjiensis Bem Pelobacter propionicus DSM 2379 Geobacteraceae bacterium 2 Desulfuromonadales bacterium Pelobacteraceae bacterium Geobacteraceae bacterium 1 Geobacteraceae bacterium 3 Geobacteraceae bacterium 4 Geobacteraceae bacterium 5 Geobacter lovleyi SZ Pelobacter carbinolicus DSM 2380 Desulfovibrio magneticus RS−1 Desulfovibrio salexigens DSM 2638 Desulfovibrio desulfuricans ND132 Proteobacterium MAG 4 Desulfovibrionales bacterium Desulfovibrio africanus str Walvis Bay Desulfomicrobium baculatum DSM 4028 Thermodesulfatator indicus DSM 15286 Desulfobacterium autotrophicum HRM2 Desulfobacula sp Proteobacterium MAG 5 Desulfatibacillum alkenivorans AK−01 Desulfurivibrio alkaliphilus AHT2 Deltaproteobacteria bacterium 2 Deltaproteobacteria bacterium 3 Deltaproteobacteria bacterium 4 Deltaproteobacteria bacterium 1 Desulfobulbus propionicus DSM 2032 Desulfarculus baarsii DSM 2075 Syntrophobacter fumaroxidans MPOB Desulfobacca acetoxidans DSM 11109 Anaeromyxobacter sp Fw109−5 Nitrospina gracilis 3/211 Candidatus Tectomicrobia bacterium RIFCSPLOWO2 02 FULL 70 19 Azoarcus sp BH72 Rhodocyclales bacterium Dechloromonas aromatica RCB Candidatus Accumulibacter phosphatis clade IIA str UW−1 Dechlorosoma suillum PS Polaromonas naphthalenivorans CJ2 Leptothrix cholodnii SP−6 Herbaspirillum seropedicae SmR1 Sideroxydans lithotrophicus ES−1 Gallionellales bacterium Hydrogenophilales bacterium Pectobacterium atrosepticum SCRI1043 Dickeya dadantii Ech703 Tolumonas auensis DSM 9187 Azotobacter vinelandii DJ Pseudomonadales bacterium Proteobacterium MAG 3 Proteobacterium MAG 1 Proteobacterium MAG 2 Halorhodospira halophila SL1 Allochromatium vinosum DSM 180 Methylomonas methanica MC09 Acidithiobacillus ferrivorans SS3 Magnetospirillum magneticum AMB−1 Rhodospirillum rubrum F11 Azospirillum sp B510 Azospirillum lipoferum 4B Azospirillum brasilense Sp245 Rhodospirillum centenum SW Gluconacetobacter diazotrophicus PAl 5 Azorhizobium caulinodans ORS 571 Xanthobacter autotrophicus Py2 Beijerinckia indica subsp indica ATCC 9039 Methylocella silvestris BL2 Methylobacterium nodulans ORS 2060 Bradyrhizobium japonicum USDA 6 Bradyrhizobium sp BTAi1 Bradyrhizobium sp ORS 278 Mesorhizobium loti MAFF303099 Mesorhizobium opportunistum WSM2075 Mesorhizobium ciceri biovar biserrulae WSM1271 Rhodomicrobium vannielii ATCC 17100 Rhodobacter capsulatus SB 1003 Magnetococcus marinus MC−1 Mariprofundus ferrooxydans PV−1 Arcobacter sp L Arcobacter nitrofigilis DSM 7299 Sulfuricurvum kujiense DSM 16994 Sulfuricurvum sp 1 Sulfuricurvum sp 3 Sulfuricurvum sp 2 Sulfurimonas sp 1 Sulfurimonas sp 2 Candidatus Lambdaproteobacteria bacterium 1 Candidatus Lambdaproteobacteria bacterium 2 Denitrovibrio acetiphilus DSM 12809 Calditerrivibrio nitroreducens DSM 19672 Desulfurispirillum indicum S5 Thermodesulfovibrio yellowstonii DSM 11347 Candidatus Dadabacteria bacterium CSP1−2 Acidobacterium capsulatum ATCC 51196 Candidatus Rokubacteria bacterium CSP1−6 Mesoplasma florum L1 Firmicutes bacterium 1 Firmicutes bacterium 2 Paenibacillus terrae HPL−003 Clostridium kluyveri DSM 555 Clostridium ljungdahlii DSM 13528 Clostridium beijerinckii NCIMB 8052 Clostridium cellulovorans 743B Clostridium lentocellum DSM 5427 Coprococcus catus GD 7 Alkaliphilus metalliredigens QYMF Acetobacterium woodii DSM 1030 Caldicellulosiruptor saccharolyticus DSM 8903 Caldicellulosiruptor hydrothermalis 108 Caldicellulosiruptor lactoaceticus 6A Thermoanaerobacterium thermosaccharolyticum DSM 571 Desulfotomaculum reducens MI−1 Desulfotomaculum ruminis DSM 2154 Desulfotomaculum carboxydivorans CO−1−SRB Desulfotomaculum kuznetsovii DSM 6115 Candidatus Desulforudis audaxviator MP104C Desulfotomaculum acetoxidans DSM 771 Thermincola potens JR Heliobacterium modesticaldum Ice1 Syntrophobotulus glycolicus DSM 8271 Desulfosporosinus orientis DSM 765 Syntrophothermus lipocalidus DSM 12680 Actinobacteria bacterium 1 Atribacteria bacterium SCGC AAA252−M02 Actinobacteria bacterium 2 Candidatus Margulisbacteria bacterium 1 Candidatus Margulisbacteria bacterium 2 Nostoc sp PCC 7120 Nostoc azollae 0708 Cyanothece sp PCC 7425 Trichodesmium erythraeum IMS101 Cyanothece sp PCC 7424 Cyanothece sp PCC 7822 Cyanothece sp PCC 8802 Candidatus Atelocyanobacterium thalassa isolate ALOHA Candidatus Acetylcyanobacterium MAG Synechococcus sp JA−3−3Ab Candidatus Melainabacteria bacterium Frankia symbiont of Datisca glomerata Fimbriimonas ginsengisoli Gsoil 348 Roseiflexus castenholzii DSM 13941 Chloroflexi bacterium Candidatus Beckwithbacteria bacterium GW2011 GWC1 49 16 Candidatus Gottesmanbacteria bacterium Candidatus Levybacteria bacterium GW2011 GWB1 36 18 US07 C0001 Candidatus Roizmanbacteria bacterium GW2011 GWC2 37 13 Candidatus Daviesbacteria bacterium GW2011 GWA1 36 8 Candidatus Curtissbacteria bacterium GW2011 GWA1 41 11 Candidate division WS6 bacterium GW2011 GWF2 39 15 Candidate division WWE3 bacterium RIFOXYD2 FULL 43 10 Candidatus Falkowbacteria bacterium GW2011 GWF2 38 1205 Candidatus Jorgensenbacteria bacterium GW2011 GWA1 48 13 Candidatus Kaiserbacteria bacterium GW2011 GWB1 50 17 Candidatus Wirthbacteria bacterium CG2 30 54 11 Deinococcus radiodurans R1 Fusobacterium nucleatum subsp nucleatum ATCC 25586 Thermanaerovibrio acidaminovorans DSM 6589 Dictyoglomus turgidum DSM 6724 Caldisericum sp CG2 30 36 11 Thermotoga maritima MSB8 Hydrogenobacter thermophilus TK−6 Thermocrinis albus DSM 14484 Aquifex aeolicus VF5 Calescamantes bacterium JGI 0000106−G12 Methanothermobacter thermautotrophicus str Delta H Methanothermobacter marburgensis str Marburg Methanococcus vannielii SB Methanococcus aeolicus Nankai−3 Methanothermococcus okinawensis IH1 Methanotorris igneus Kol 5 Methanocaldococcus vulcanius M7 Methanocaldococcus sp FS406−22 Methanocaldococcus jannaschii DSM 2661 Methanocaldococcus infernus ME Methanosarcina mazei Go1 Methanosarcina acetivorans C2A Methanosarcina barkeri str Fusaro Methanohalophilus mahii DSM 5219

4550 3640 2730 1820 909 0

Supplementary Figure 3. Chronogram of 308 archaeal, bacterial, and eukaryotic genomes generated using PhyloBayes with a lognormal clock model and conservative fossil calibration points. Chronogram was built based on an alignment of concatenated universal, single-copy genes. Branch lengths indicate time in thousands of years. Error bars represent the 95% confidence intervals for each node. Lentisphaera araneosa HTCC2155 Lentisphaerae bacterium Coraliomargarita akajimensis DSM 45221 Methylacidiphilum infernorum V4 Verrucomicrobia bacterium Chlamydia trachomatis D UW−3 CX Omnitrophica bacterium SCGC AAA252−B19 Pirellula staleyi DSM 6068 Planctomycetes MAG Hydrogenedentes bacterium JGI 0000039−J10 Elusimicrobia bacterium 1 Elusimicrobia bacterium 3 Elusimicrobia bacterium 4 Elusimicrobia bacterium 2 Spirochaeta thermophila DSM 6192 Treponema azotonutricium ZAS−9 Treponema primitia ZAS−2 Treponema sp 1 Treponema sp 2 Spirochaeta smaragdinae DSM 11293 Spirochaetes bacterium 2 Spirochaetes bacterium 1 Chlorobium tepidum TLS Chlorobaculum parvum NCIB 8327 Chlorobium luteolum DSM 273 Chlorobium phaeovibrioides DSM 265 Chlorobium chlorochromatii CaD3 Pelodictyon phaeoclathratiforme BU−1 Chlorobium phaeobacteroides DSM 266 Chlorobium limicola DSM 245 Prosthecochloris aestuarii DSM 271 Chloroherpeton thalassium ATCC 35110 Bacteroides fragilis YCH46 Candidatus Azobacteroides pseudotrichonymphae genomovar CFP2 Ignavibacterium album JCM 16511 1 Ignavibacterium album JCM 16511 2 Marinimicrobia bacterium JGI 0000039−D08 Caldithrix abyssi DSM 13497 Latescibacteria bacterium DG 63 candidate division Zixibacteria bacterium Cloacimonetes bacterium JGI 0000039−A21 Fibrobacter succinogenes subsp succinogenes S85 CP0071271 Gemmatirosa kalamazoonesis strain KBS708 Geobacter sulfurreducens PCA Geobacter uraniireducens Rf4 Geobacter daltonii FRC−32 Geobacter bemidjiensis Bem Pelobacter propionicus DSM 2379 Geobacteraceae bacterium 2 Desulfuromonadales bacterium Pelobacteraceae bacterium Geobacteraceae bacterium 1 Geobacteraceae bacterium 3 Geobacteraceae bacterium 4 Geobacteraceae bacterium 5 Geobacter lovleyi SZ Pelobacter carbinolicus DSM 2380 Desulfovibrio magneticus RS−1 Desulfovibrio salexigens DSM 2638 Desulfovibrio desulfuricans ND132 Proteobacterium MAG 4 Desulfovibrionales bacterium Desulfovibrio africanus str Walvis Bay Desulfomicrobium baculatum DSM 4028 Thermodesulfatator indicus DSM 15286 Desulfobacterium autotrophicum HRM2 Desulfobacula sp Proteobacterium MAG 5 Desulfatibacillum alkenivorans AK−01 Desulfurivibrio alkaliphilus AHT2 Deltaproteobacteria bacterium 2 Deltaproteobacteria bacterium 3 Deltaproteobacteria bacterium 4 Deltaproteobacteria bacterium 1 Desulfobulbus propionicus DSM 2032 Desulfarculus baarsii DSM 2075 Syntrophobacter fumaroxidans MPOB Desulfobacca acetoxidans DSM 11109 Anaeromyxobacter sp Fw109−5 Nitrospina gracilis 3/211 Candidatus Tectomicrobia bacterium RIFCSPLOWO2 02 FULL 70 19 Azoarcus sp BH72 Rhodocyclales bacterium Dechloromonas aromatica RCB Candidatus Accumulibacter phosphatis clade IIA str UW−1 Dechlorosoma suillum PS Polaromonas naphthalenivorans CJ2 Leptothrix cholodnii SP−6 Herbaspirillum seropedicae SmR1 Sideroxydans lithotrophicus ES−1 Gallionellales bacterium Hydrogenophilales bacterium Pectobacterium atrosepticum SCRI1043 Dickeya dadantii Ech703 Tolumonas auensis DSM 9187 Azotobacter vinelandii DJ Pseudomonadales bacterium Proteobacterium MAG 3 Proteobacterium MAG 1 Proteobacterium MAG 2 Halorhodospira halophila SL1 Allochromatium vinosum DSM 180 Methylomonas methanica MC09 Acidithiobacillus ferrivorans SS3 Magnetospirillum magneticum AMB−1 Rhodospirillum rubrum F11 Azospirillum sp B510 Azospirillum lipoferum 4B Azospirillum brasilense Sp245 Rhodospirillum centenum SW Gluconacetobacter diazotrophicus PAl 5 Azorhizobium caulinodans ORS 571 Xanthobacter autotrophicus Py2 Beijerinckia indica subsp indica ATCC 9039 Methylocella silvestris BL2 Methylobacterium nodulans ORS 2060 Bradyrhizobium japonicum USDA 6 Bradyrhizobium sp BTAi1 Bradyrhizobium sp ORS 278 Mesorhizobium loti MAFF303099 Mesorhizobium opportunistum WSM2075 Mesorhizobium ciceri biovar biserrulae WSM1271 Rhodomicrobium vannielii ATCC 17100 Rhodobacter capsulatus SB 1003 Magnetococcus marinus MC−1 Mariprofundus ferrooxydans PV−1 Arcobacter sp L Arcobacter nitrofigilis DSM 7299 Sulfuricurvum kujiense DSM 16994 Sulfuricurvum sp 1 Sulfuricurvum sp 3 Sulfuricurvum sp 2 Sulfurimonas sp 1 Sulfurimonas sp 2 Candidatus Lambdaproteobacteria bacterium 1 Candidatus Lambdaproteobacteria bacterium 2 Denitrovibrio acetiphilus DSM 12809 Calditerrivibrio nitroreducens DSM 19672 Desulfurispirillum indicum S5 Thermodesulfovibrio yellowstonii DSM 11347 Candidatus Dadabacteria bacterium CSP1−2 Acidobacterium capsulatum ATCC 51196 Candidatus Rokubacteria bacterium CSP1−6 Mesoplasma florum L1 Firmicutes bacterium 1 Firmicutes bacterium 2 Paenibacillus terrae HPL−003 Clostridium kluyveri DSM 555 Clostridium ljungdahlii DSM 13528 Clostridium beijerinckii NCIMB 8052 Clostridium cellulovorans 743B Clostridium lentocellum DSM 5427 Coprococcus catus GD 7 Alkaliphilus metalliredigens QYMF Acetobacterium woodii DSM 1030 Caldicellulosiruptor saccharolyticus DSM 8903 Caldicellulosiruptor hydrothermalis 108 Caldicellulosiruptor lactoaceticus 6A Thermoanaerobacterium thermosaccharolyticum DSM 571 Desulfotomaculum reducens MI−1 Desulfotomaculum ruminis DSM 2154 Desulfotomaculum carboxydivorans CO−1−SRB Desulfotomaculum kuznetsovii DSM 6115 Candidatus Desulforudis audaxviator MP104C Desulfotomaculum acetoxidans DSM 771 Thermincola potens JR Heliobacterium modesticaldum Ice1 Syntrophobotulus glycolicus DSM 8271 Desulfosporosinus orientis DSM 765 Syntrophothermus lipocalidus DSM 12680 Actinobacteria bacterium 1 Atribacteria bacterium SCGC AAA252−M02 Actinobacteria bacterium 2 Candidatus Margulisbacteria bacterium 1 Candidatus Margulisbacteria bacterium 2 Nostoc sp PCC 7120 Nostoc azollae 0708 Cyanothece sp PCC 7425 Trichodesmium erythraeum IMS101 Cyanothece sp PCC 7424 Cyanothece sp PCC 7822 Cyanothece sp PCC 8802 Candidatus Atelocyanobacterium thalassa isolate ALOHA Candidatus Acetylcyanobacterium MAG Synechococcus sp JA−3−3Ab Candidatus Melainabacteria bacterium Frankia symbiont of Datisca glomerata Fimbriimonas ginsengisoli Gsoil 348 Roseiflexus castenholzii DSM 13941 Chloroflexi bacterium Candidatus Beckwithbacteria bacterium GW2011 GWC1 49 16 Candidatus Gottesmanbacteria bacterium Candidatus Levybacteria bacterium GW2011 GWB1 36 18 US07 C0001 Candidatus Roizmanbacteria bacterium GW2011 GWC2 37 13 Candidatus Daviesbacteria bacterium GW2011 GWA1 36 8 Candidatus Curtissbacteria bacterium GW2011 GWA1 41 11 Candidate division WS6 bacterium GW2011 GWF2 39 15 Candidate division WWE3 bacterium RIFOXYD2 FULL 43 10 Candidatus Falkowbacteria bacterium GW2011 GWF2 38 1205 Candidatus Jorgensenbacteria bacterium GW2011 GWA1 48 13 Candidatus Kaiserbacteria bacterium GW2011 GWB1 50 17 Candidatus Wirthbacteria bacterium CG2 30 54 11 Deinococcus radiodurans R1 Fusobacterium nucleatum subsp nucleatum ATCC 25586 Thermanaerovibrio acidaminovorans DSM 6589 Dictyoglomus turgidum DSM 6724 Caldisericum sp CG2 30 36 11 Thermotoga maritima MSB8 Hydrogenobacter thermophilus TK−6 Thermocrinis albus DSM 14484 Aquifex aeolicus VF5 Calescamantes bacterium JGI 0000106−G12 Methanothermobacter thermautotrophicus str Delta H Methanothermobacter marburgensis str Marburg Methanococcus vannielii SB Methanococcus aeolicus Nankai−3 Methanothermococcus okinawensis IH1 Methanotorris igneus Kol 5 Methanocaldococcus vulcanius M7 Methanocaldococcus sp FS406−22 Methanocaldococcus jannaschii DSM 2661 Methanocaldococcus infernus ME Methanosarcina mazei Go1 Methanosarcina acetivorans C2A Methanosarcina barkeri str Fusaro Methanohalophilus mahii DSM 5219

5000 4000 3000 2000 1000 0

Supplementary Figure 4. Chronogram of 308 archaeal, bacterial, and eukaryotic genomes generated using PhyloBayes with a lognormal clock model and liberal fossil calibration points. Chronogram was built based on an alignment of concatenated universal, single-copy genes. Branch lengths indicate time in thousands of years. Error bars represent the 95% confidence intervals for each node. anfD anfK

vnfD vnfK

Supplementary Figure 5. Histograms and density plots of all speciation, loss, duplication, and HGT events (blue) and just horizontal gene transfer events (green) for anf and vnf genes for the CIR clock model using conservative calibration points. nitrogen fxation denitrifcation from nitrate denitrifcation from nitrite nitrifcation DNRA Figure S6. Histograms and density plots of all speciation, loss, duplication and HGT events (blue) and just horizontal gene transfer events (green) over time using the lognormal model with conservative calibration points. Borders around graphs indicate the metabolic process that the gene is involved in. Histograms are ordered according to the earliest time point at which a speciation, loss, duplication, or HGT event nitrogen fxation denitrifcation from nitrate denitrifcation from nitrite nitrifcation DNRA Figure S7. Histograms and density plots of all speciation, loss, duplication and HGT events (blue) and just horizontal gene transfer events (green) over time using the lognormal model with liberal calibration points. Borders around graphs indicate the metabolic process that the gene is involved in. Histograms are ordered according to the earliest time point at which a speciation, loss, duplication, or HGT event occurred. nitrogen fxation denitrifcation from nitrate denitrifcation from nitrite nitrifcation DNRA

Figure S8. Histograms and density plots of all speciation, loss, duplication and HGT events (blue) and just horizontal gene transfer events (green) over time using the lognormal model with liberal calibration points. Borders around graphs indicate the metabolic process that the gene is involved in. Histograms are ordered according to the earliest time point at which a speciation, loss, duplication, or HGT event occurred.