Supplementary Figures

Branch bootstrap support: ≥ 95% endosymbiont of Tevnia jerichonana (WP 005962054.1) endosymbiont of Riftia pachyptila (WP 005962054.1) > 85% endosymbiont of Ridgeia piscesae (WP 060528630.1) > 50% endosymbiont of Escarpia laminata Gammaproteobacterial metagenomic bin, aquifer adjacent to the Colorado River near Rifle, CO, USA (OGT88445.1) Magneto-ovoid bacterium MO-1 (CRH07508.1) Magnetococcus marinus (WP 011715176.1 ) Magnetofaba australis IT-1 (OSM06211.1) 100 Magnetofaba australis (WP 085441418.1) Beggiatoa sp. Orange Guyamas (JGI/IMG Gp0006298) Thioflavicoccus mobilis (WP 041603910.1) Nitrospina gracilis (WP 005009428.1) Deltaproteobacteria metagenomic bin, aquifer adjacent to the Colorado River near Rifle, CO, USA (OGQ25533.1 ) Deltaproteobacteria metagenomic bin, aquifer adjacent to the Colorado River near Rifle, CO, USA (OGP31275.1) metagenomic bin, aquifer adjacent to the Colorado River near Rifle, CO, USA (OIP65732.1) Achromatium sp. WMS2 (KOR30931.1 ) Candidatus Achromatium palustre (WP 048396998.1) Hydrogenophilaceae metagenomic bin, aquifer adjacent to the Colorado River near Rifle, CO, USA (OIO79828.1) Rhodospirillales metagenomic bin, aquifer adjacent to the Colorado River near Rifle, CO, USA (OHC76497.1) Ensifer sp. YIC4027 (WP 069461593.1) Sinorhizobium fredii (WP 037470357.1) Microvirga lotononidis (WP 009493845.1) Paraburkholderia dilworthii (WP 027803596.1) Skermanella aerolata (WP 044430707.1) Thauera butanivorans (WP 068635822.1) Burkholderiales 0.10

Figure S1: Phylogeny of ATP citrate lyase large subunit (AclA). The evolutionary history was inferred by using the Maximum Likelihood method based on the Le Gascuel 2008 model (31). A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.16)). e rate variation model allowed for some sites to be evolutionarily invariable ([+I], 11.2% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. We used 46 amino acid sequences for the analysis including all known aclA sequences from . All positions containing gaps and missing data were eliminated. There were a total of 707 positions in the final dataset. Grey box frames tubeworm sequences, purple color marks Chromatiaceae sequences. endosymbiont of Riftia pachyptila (vent Ph05) (EGV50294.1 ) endosymbiont of Tevnia jerichonana (vent Tica) (WP 006475391.1) endosymbiont of Ridgeia piscesae (KRT56259.1) Branch bootstrap support: endosymbiont of Escarpia laminata ≥ 95% Thioflavicoccus mobilis (WP 015279272.1 ,AGA89122.1 ) > 80% Thiococcus pfennigii (DOE Joint Genome Institute: Gp0139227) > 50% Hydrogenophilaceae bacterium CG1 02 62 390 (OIO79820.1) Gammaproteobacteria bacterium RIFOXYD12 FULL 61 37 (OGT88532.1 ) Gallionellales bacterium RIFCSPLOWO2 02 FULL 57 47 (OGS96735.1) Gallionellales bacterium RIFCSPHIGHO2 02 FULL 57 16 (OGT16595.1 2) Rhodospirillales bacterium RIFCSPLOWO2 02 FULL 58 16 (OHC76583.1) Candidatus Aminicenantes candidate division TA06 bacterium DG 26 (KPJ51039.1) Zixibacteria bacterium SM23 81 (KPL19536.1) candidate division KSB1 bacterium 4484 188 (OPX34974.1) bacterium RIFCSPLOWO2 12 FULL 45 22 (OGW12780.1) candidate division TA06 bacterium DG 24 (KPJ53590.1) candidate division Zixibacteria bacterium SM23 73 (KPK76769.1) candidate division Zixibacteria bacterium SM23 73 2 (KPL03160.1) bacterium RBG 16 68 9 (OFV89107.1) bacterium RBG 16 43 13 (OHB70316.1) Acidobacteria bacterium RBG 13 68 16 (OFV79423.1) Anaeromyxobacter sp. PSR-1 (WP 059438035.1) Syntrophobacter sp. DG 60 (KPJ68541.1) Deltaproteobacteria

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Figure S2: Phylogeny of the alpha subunit (KorA) of the 4-subunit 2-oxoglutarate:ferredoxin oxidoreductase (OGOR, 33 amino acid sequences, 362 positions). The evolutionary history was inferred using the Maximum Likelihood method based on the Le Gascuel 2008 model (31). A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.98)). e rate variation model allowed for some sites to be evolutionarily invariable ([+I], 13.0% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Grey box frames tubeworm sequences, purple color marks Chromatiaceae sequences. Branch bootstrap support: endosymbiont of Ridgeia piscesae (WP 060528622.1) ≥ 95% endosymbiont of Ri ia pachyptila and Tevnia jerichonana (WP 005965519.1) > 85% endosymbiont of Escarpia laminata (this study) > 50% iococcus pfennigii (DOE Joint Genome Institute: Gp0139227) ioavicoccus mobilis (WP 015279268.1) Gammaproteobacteria bacterium RIFOXYD12 FULL 61 37 (OGT88433.1) Gallionellales bacterium RIFCSPLOWO2 02 FULL 59 110 (OGT00635.1) Gallionellales bacterium RIFCSPHIGHO2 02 FULL 57 16 (OGT08275.1) Betaproteobacteria bacterium RBG 16 56 24 (OFZ67799.1) Gallionellales bacterium RIFCSPLOWO2 02 FULL 57 47 (OGS96740.1) Hydrogenophilaceae bacterium CG1 02 62 390 (OIO79816.1) Hydrogenophilales bacterium CG18 big l WC 8 21 14 2 50 58 12 (PIQ11058.1) Rhodospirillales bacterium RIFCSPLOWO2 12 FULL 58 28 (OHC78560.1 ) Candidatus Aminicenantes bacterium RBG 13 59 9 (OGD16533.1) Candidatus Atribacteria bacterium CG17 big l post rev 8 21 14 2 50 34 11 (PIW12242.1) Candidatus Atribacteria bacterium HGW-Atribacteria-1 (PKP59757.1) Deltaproteobacteria

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Figure S3: Phylogeny of the alpha subunit (TfrA) of the thiol:fumarate reductase (21 amino acid sequences, 483 positions). The evolutionary history was inferred using the Maximum Likelihood method based on the Le Gascuel 2008 model (31). A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.22)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 17% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Grey box frames tubeworm sequences, purple color marks Chromatiaceae sequences. a) RNA-seq transcript levels in E. laminata symbiont 4 3 Expression [log(TPM)] 2 1 0

sbcC sbcD tfrB tfrA hdrB korA korB hdrA xC xA aclB aclA idh rbr acn pntA pntB mdh capsular polysaccharide synthesis cluster

hdrC korD korC xD xB rbr 4 3 2 1 0

gltA mdh2 fdx* frd*nuoF* korA korB por-like rnfB por sucC sucD *** poorly de ned p rnfCDGEA

pyruvate

b) FDox Por CO2 FDred RnfB RnfA NAD+ NADH acetyl-CoA RnfD RnfG Na+/H+ Mdh NAD+ OAA RnfE malate ATP RnfC Mdh NADH FumB AclB AclA ADP+Pi dsrC? fumarate GltA citrate SH SH TfrA TfrB HdrB S S Acn HdrC succinate ATP NADH dsrC? HdrA SucC SucD - FlxB isocitrate NADH e FlxD ADP+Pi NAD(P)+ PntB Succinyl-CoA FlxA FlxC H+ CO + 2 Idh PntA NAD FDred KorD

KorB NAD(P)H + KorC 2-oxoglutarate NAD FDox KorA KorA KorB

Figure S4: Genome-based reconstruction of the TCA cycle in tubeworm symbionts: A. Expression pro le of both gene clusters that encode the TCA cycle in Escarpia laminata tubeworm symbionts. B. The TCA cycle reconstruction. Each box is named after the respective protein. Frame color corresponds to a gene cluster and box color corresponds to the gene color code in panel A. FumB is not colored as the standalone fumB gene located elsewhere in the genome. OAA is oxaloacetate. fumarate succinate

FAD TfrA TfrB HdrB HdrC NADH FlxB FlxD HdrA - e FAD KorD FlxA FlxC

NAD+ KorC KorA KorB

succinyl-CoA 2-oxoglutarate

CO2

Figure S5: Proposed function of the putative HdrABC-FlxABCD-KorABCD-TfrAB complex. Electrons from NADH oxidation are transferred to HdrA, which may bifurcate them to KorABCD (alternatively mediate electron transfer via ferredoxin) and to TfrAB (alternatively mediate electron transfer via the thiol/disul de pair of DsrC). Squares and hexagons mark the estimated presence of [4Fe-4S]2+/1+ and [2Fe-2S]2+/1 centers, respectively. endosymbiont of Ridgeia piscesae (WP 057955770.1) endosymbiont of Ridgeia piscesae (KRT54083.1) Branch bootstrap support: ≥ 95% endosymbiont of Tevnia jerichonana / Riftia pachyptila (WP 005958508.1) endosymbiont of Tevnia jerichonana (vent Tica) (EGW53450.1) > 80% endosymbiont of Escarpia laminata > 50% endosymbiont of Solemya velesiana (WP 078484252.1) Gammaproteobacteria bacterium RIFOXYD12 FULL 61 37 (OGT89358.1) Gammaproteobacteria bacterium LUC14 002 19 P1 (OQX32180.1) Candidatus Thiomargarita nelsonii (KHD08044.1) Beggiatoa Orange Guyamas Beggiatoa sp. PS (EDN69037.1) Candidatus Achromatium palustre (WP 048400259.1) uncultured Thiohalocapsa sp. PB-PSB1 (ESQ15560.1) Candidatus Thiosymbion oneisti (WP 089726309.1) Candidatus Thiodiazotropha endolucinida (ODJ87695.1) Candidatus Thiodiazotropha endolucinida (WP 083220676.1) Flavobacteriales bacterium (PCJ27237.1) Marivirga sericea (WP 085516022.1) Muricauda sp. TMED12 (OUT56390.1) Deltaproteobacteria bacterium CG2 30 63 29 (OIP35220.1) Gammaproteobacteria bacterium RIFCSPLOWO2 12 FULL 52 10 (OGT86260.1) Gammaproteobacteria bacterium RIFCSPLOWO2 02 FULL 56 15 (OGT70055.1) Betaproteobacteria bacterium RIFCSPLOWO2 12 FULL 62 58 (OGA47866.1) Tropicimonas sediminicola (SNT09822.1) Tropicimonas sediminicola (WP 089234212.1) Pseudoruegeria sabulilitoris (WP 068312701.1) Pseudoruegeria lutimaris (SDK73065.1) Pseudoruegeria lutimaris (WP 093161153.1) Candidatus Rokubacteria bacterium RIFCSPLOWO2 02 FULL 72 37 (OGL10744.1) Candidatus Rokubacteria bacterium RIFCSPHIGHO2 02 FULL 73 26 (OGK99289.1) magneto-ovoid bacterium MO-1 (CRH05823.1) bacterium CG1 02 55 237 (OIO69204.1) Hydrogenobacter thermophilus (WP 012963731.1) Thermocrinis albus (WP 012991705.1) Hydrogenobaculum sp. (WP 015419993.1) Hydrogenobaculum sp. Y04AAS1 (WP 012514477.1) Nitrospirae bacterium GWA2 42 11 (OGW14346.1) Nitrospirae bacterium RBG 16 64 22 (OGW59366.1) Persephonella hydrogeniphila (SNZ08054.1) Sulfurihydrogenibium sp. YO3AOP1 (WP 012459334.1) Sulfurihydrogenibium subterraneum (WP 028950037.1) tuberculosis (WP 079090607.1) Mycobacterium marinum (WP 012395405.1)

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Figure S6: Phylogeny of the alpha subunit (KorA) from the 2-subunit 2-oxoglutarate ferredoxin oxidoreductase (OGOR, 44 amino acid sequences, 541 positions). The evolutionary history was inferred by using the Maximum Likelihood method based on the Le Gascuel 2008 model (31). A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.2)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 6.0% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Grey box frames tubeworm sequences, purple color marks Chromatiaceae sequences. endosymbiont of Ridgeia piscesae (WP 057957090.1) endosymbiont of Tevnia jerichonana / Riftia pachyptila (WP 005958507.1) Branch bootstrap support: Endosymbiont of Escarpia laminata ≥ 95% Thiococcus pfennigii (DOE Joint Genome Institute: Gp0139227)) > 80% Thioflavicoccus mobilis (WP 015279275) > 50% Gammaproteobacteria bacterium RIFOXYD12 FULL 61 37 (OGT88440.1) Hydrogenophilaceae bacterium CG1 02 62 390 (OIO79822.1) Gallionellales bacterium RIFCSPLOWO2 02 FULL 59 110 (OGS99002.1) Gallionellales bacterium RIFCSPLOWO2 02 FULL 57 47 (OGS96733.1) Gallionellales bacterium RIFCSPHIGHO2 02 FULL 57 16 (OGT16593.1) Rhodospirillales bacterium RIFCSPLOWO2 02 FULL 58 16(OHC76502.1) Candidatus Aminicenantes bacterium RBG 13 59 9 (OGD16537.1) Candidatus Aminicenantes bacterium RBG 13 59 9 (OGD22118.1) Candidatus Aminicenantes bacterium RBG 19FT COMBO 58 17 (OGD37786.1) Acetothermia bacterium 64 32 (KUK27013.1) Syntrophobacter sp. DG 60 (KPJ68543.1) archaeon ex4484 74 (OYT34320.1) Desulfurococcales archaeon ex4484 58 (OYT40487.1 ) Thermoplasmatales archaeon ex4484 30 (OYT61384.1) Arc I group archaeon U1lsi0528 Bin055 (KYC53320.1) Arc I group archaeon B03fssc0709 Meth Bin005 (KYC45110.1) Desulfuromonas sp. SDB (KQC12678.1) Candidatus Edwardsbacteria bacterium RifOxyC12 full 54 24 (OGF05880.1) Candidatus Aegiribacteria bacterium MLS C (OPL19372.1) candidate division WOR-3 bacterium JGI Cruoil 03 44 89 (OYD16623.1 ) Candidatus Atribacteria bacterium RBG 16 35 8 (OGD31325.1) Candidatus Cloacimonas sp. 4484 209 (OQX51896.1 Tindallia californiensis (SDY33670.1) Tindallia magadiensis (WP 093370007.1) Maledivibacter halophilus (WP 079492716.1) Natranaerobius thermophilus (WP 012449054.1) Chlorobium phaeovibrioides (WP 011890083.1) Deltaproteobacteria

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Figure S7: Phylogeny of the alpha subunit (HdrA) from the NADH dehydrogenase/heterodisulde reductase (HdrABC-FlxABCD ) electron bifurcating complex, co-localized with the 4-subunit OGOR (38 amino acid sequences, 576 positions). The evolutionary history was inferred by using the Maximum Likelihood method based on the Le Gascuel 2008 model (31). A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.95)). e rate variation model allowed for some sites to be evolutionarily invariable ([+I], 9.3% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Grey box frames tubeworm sequences, purple color marks Chromatiaceae sequences.