Additional File 1

Additional file 1

Microbial succession during the transition from active to inactive stages

of deep-sea hydrothermal vent sulfide chimneys

Jialin Hou, Stefan M. Sievert, Yinzhao Wang, Jeff S. Seewald, Vengadesh Perumal Natarajan

Fengping Wang*, Xiang Xiao* a b

100 100

75 75

Taxa

Alphaproteobacteria Taxa Betaproteobacteria Aquificae Deinococcus-Thermus Campylobacteria Deltaproteobacteria 50 Euryarchaeata 50 Euryarchaeata Others Gammaproteobacteria Percentage Percentage Thermodesulfobacterium Muproteobacteria Unclassified Nitrospirae Others Unclassified

25 25

0 0

aclA aclB cdhA cdhC napA napB norB nosZ sat soxA soxC soxY soxZ sqr aprA aprB dsrA dsrB narG nirB PRK rbcL rbcS Gene Gene

Figure S1 Taxonomic classification of key functional genes retrieved from the L- and M-vent chimeny.(a) The key genes enriched in the active L-vent chimney. (b) The key genes enriched in the recently inactive M-vent chimney.

Reductive bacterial type Reductive archaeal type

Chlorobi group Archaeoglobus(3) Oxidative bacterial type Crenarchaeota Alphaproteobacteria(1/16) Nitrospirae(12) Acidobacteria(1/14) Betaproteobacteria Firmicutes groups

Gammaproteobacteria (20)

Tree scale: 1

Deltaproteobacteria(9/10) Reductive bacterial type

Figure S2 Maximum-likelihood phylogeny of dsrA genes retrieved from L- and M-vent chimney. The red branches represent the dsrA genes recovered from active the L-vent chimney, while the blue ones are those from recently inactive M-vent chimney. Numbers of dsrA gene for each sample are displayed in the parenthesis after the clade name.

W W

W W W WP P P

4 P WP

P P P 2 0 0 007601690 1 thiosulfohydrolase SoxB Bradyrhizobium sp WSM1253 P W W

003614695 1 MUL 008967367 1 thiosulfohydrolase SoxB Bradyrhizobium sp STM 3843 6 1 063684315 1 thiosulfohydrolase SoxB Bradyrhizobium stylosanthis W W W 7 0

057197484 1 thiosulfohydrolase SoxB Bradyrhizobium sp Leaf396 5 WP W 0 0 3 W P P 6 1

P P 4 P 9 2 2 06311 012752908 1 thiosulfohydrolase SoxB Methylobacterium extorquens P 2

3 037019901 1 thiosulfohydrolase SoxB RhizobialesP bacterium 015821 1 1 0 041750721 1 thiosulfohydrolase SoxB Bradyrhizobium sp BT N

1 WP 056910972 1 thiosulfohydrolase SoxB Pseudolabrys 0504233 sp Root14621 WP 1 5 056201494 1 thiosulfohydrolase SoxB Methylobacterium sp Leaf123 4 0

W 4 9 7 8

W S

2 8 W 5 4

P 068025945 1 thiosulfohydrolase SoxB Rhodoplanes sp Z2 5 5 069275816 1 thiosulfohydrolase SoxB Bradyrhizobium elkanii 6 6 3 4

4 P 0 02 P 1224 1 thiosulfohydrolase SoxB Methylobacterium radiotolerans Aquificae Gammaproteobacteria 5 9 3 s

1 4

W C 063987252 1 thiosulfohydrolase SoxB Methylobacterium populi 8

3 0

159 1 thiosulfohydrolase SoxB Methylobacterium extorquens 1

1 A t

1 4080312 1 t C P 1 m af07 h h 1

t W 1 3 L u W R V

t 0 i h i i

o h o

t 1 1 1 thiosulfohydrolase SoxB Bradyrhizobium tropiciagri r

i h p P p p

s o 6 P i s W 6 e

o Afipia

s s i s t 850 008389056 1 u e u s

029031962 1 thiosulfohydrolase SoxB Salinarimonas rosea 5 W W s P c l u l a s a

7 TISPECIES thiosulfohydrolase SoxB Methylosinus f a u f

a o 046021260 1 thiosulfohydrolaseP SoxB Magnetospira sp QH 2 o l u

P e e

9 e f W W l

b f 3 h o h l s 0 s 069189482 1 thiosulfohydrolase SoxB s 7 26 o

560 1 th o y h y o o P o P l o

h T W W 28879728 1 t ngens

d 1 Actinobacteria y Campylobacteria d t h y B 047029550 1 t B hiosulfo y 5 02 B a W

r d

r y h

P d 5

P t o YS 1r o W t

r h d

B B 2 B 054309588P 1 r l o 7284263 1 thiosulfohydrola l 0647 e a i o r x a x W x

P o ardiphaga sp l W o

s a l o 0 o s o M T

s 03553062 a

l P e Afipia sp P52 10

s m sp N 404492 e um W a i S P u S osulfohydrolase So s S D4

03 e B Afipia broomeae

l thiosulfohydrolase SoxB Rhodovulum sp PH10 e 043 S 89 hy S Afipia sp Root123D2 m barat

f P e e x e e

o biu

S gil o

s o s

0 S s W o 1 HTCC2 8034880 drolase SoxB Magnetospiril h o

185 S x o a bium marinum x a a

o l S l P 24588 y l m B m 6 B

x o 646 B hiosulfohydr o

x o

d o o u richSK drii u 01 B O e

41 1 thiosulfohydrolase SoxB Hoeflea phototrophica r x r onas coralicida Deinococcus-Thermus Chlorobi r B B r T n H

s B d

1 o d hios d lla pfennigii NS

P ph 7 r 8 a y y 17 y X W l y r 0 l e xa a thi t a s 21 1 H Ad13 r p h h hiosulfohydrol h a e 053 1 thiosulfohyd o d o P 13 s l h r y o o o o ulfohydrolase SoxB Hoeflea sp n bae y s

1 thi f f oxB Rhiz a 0 f Afif zia sp i p l d o l s l bacteri t r t W 557 n h y h s h h u 3 ul m S u a u o sp thi 1 xB Lutibaculu i 0V17 MB20

o s e h i s P 80 o Aurantim m s

o foh z trifican E os thiosu i

b o c o u c o m o se S ale

osulf x o i i f 014515003W 58 1 i ibri r lum olas o l r a So i

b h ulfohy h o h ydr xB i eni t m u h thiosulfoh t c e c P t rolase SoxB Nitratireductor pacificus ov

17 b d u s s t cte r Alphaproteobacteria Betaproteobacteria 038 S d m sp e 1 i 1 i o SoxB 1 op m a o o c

u e So W ol t Magnetospir i olase SoxB Rhizobium sp Root482 u

4 r l b d a b hydro s r r 9 i m 3 W fohy 1 TISPECIES thiosulfohydrolase SoxB 1 h

ase S o f bi a i o onas sp 2 P ase Sox r l W e t sis d e SoxB Polymo seud dr 6 u rol 6 L 7

0 k o b

0241 P t S u s d m atlantic n i

1 P m d 5 5

P 47 e a 1 im olas i su 0 hio xB t i u e

a u 0 e 8 icr o e SoxB Labre 3

01 d y o z oroseobacter sh a B Nisaea ri 09 8 1 1 se SoxB Ho m s

80 lase So 5 z ola n 3 t

rola a 5 yd x o lfohyd n uv h 1 8 s oxB r l R 8 te a i 3 2 um g tim 0 B Rubritepid o thios T T e 46292 0 1945 1 ulf e SoxB t l A 3 1 t n c h 1 W v lum r n 9 erasakiella spe PR1 3 9 r p 1 f Sox oxB 4 i drolase SoxB Labrenzia sp CP4 a

o olase f a 9 B Mes a i o M s f

2 oh rasakiella pusilla 4 i 6 h osu Def P 0 r hydr 1664487 1 thiosulfohydrolase SoxB Rhodopseudomonas palustris y b i c S A KPD29122 1 t e SoxB v c r s 4

0 ovu 6 T 0 luen d 0 01 i ase SoxB Candidatus Filomicr ti DI z ydrola i W ul

e 1 thiosulfohy hi

c l B gr c 1504631 1 thiosulfohydrolase SoxB Rhodopseudomonas palustris h illum m a yd e d ubr W 7 1 t 5 T 056714039 1 thiosulfohydrolase SoxB Bosea sp Leaf344 lfo c Acidithiobacillia Deltaproteobacteria h 057191334 1 thiosulfohydrolase SoxB Bosea sp Root483D1 055728613 1 thiosulfohydrolase SoxB Bosea thiooxidans ia philum YIM 77505 i h nf r N xB Cae u 047574680 1 thiosulfohydrolase SoxB Methylobacterium sp ZNC0032 x W l 1 OL 65 1 thiosulfohy h fohydrolaseiosu So P e n se 5 n P 01 drolase SoxB Labrenzlase P n P l u al Ai1 027279129 1 thiosulfohydrolase SoxB Rhodopseudomonas palustris A o D

P 84 a 044409978 1 thiosulfohydrolase SoxB Rhodopseudomonas palustris 1 t o A rola s i y ho a 1 047308425 1 thiosulfohydrolase SoxB Rhodopseudomonas palustris 083 1 thiosulfohydrolase SoxB Stappia stellulata y i s M s P YC6860 s la ch SoxB Ma i 0 lfo ra 027 Thermopet assospira pro i ro W Co W 041798973 1 thiosulfohydrolase SoxB Rhodopseudomonas palustris or phiswaldense W 0 O 7 os h WP 068732006 1 thiosulfohydrolase SoxB o rm 02 61 t WP i u d K WP 034463065 1 thiosulfohydrolase SoxB 38 lfoh WP 8664 oh WP e ios h efl 063676819 1 thiosulfohydrolase SoxB Bradyrhizobium neotropicale 63 hydro se hizobium sp 1M WP m ZK WP 006019993 1 thiosulfohydrolase SoxB lf h A WP o 876 38 iosul 056301239 1 thiosulfohydrolase SoxB hy nticu t 1 u 2 0 o WP th ydro xB R aschi s sp t s M scaffold21824 WP li ea sp BAL378 a f 062314242 1 thiosulfohydrolase SoxB Bradyrhizobium sp CCH10 C7 lf ase S SoxB oxB a sp 3 isop 2 7 ulfoh S n WP 67333538 1 thiosulfohydrolase SoxB Stappia indica 1 o u a 91 1 6 ydrolase s WP 024919724 1 MU su i W 8 i IMCC20628 859 33 1 thiosulfohydrolase SoxB Fulvimarina pelagi u S m M L 9 0505 1 thiosu is hoef WP o oh ol x C0 g 6 878 coviens WP P s ell 8 1 th ox locculans 0 02905 6 i f hydrolase SoxB Rho So Jannas u 4 WP l i n M P fo p o o ase rib 55 hi WP P 138 1 thiosulfohydrolase h va o s 4 M g 00 63404 f hydrolase SoxB Di a Eh l ydrolas B M i W l xB tan B u 027881 1 1 t o h rill 052639129 1 thiosulfohydrola 67 1 o vi 1 ne M scaffold31746 gene9 u rol lase k i p e um H e 7 s y scaffold10 r l W 037153841 1 thiosulfohydrolase SoxB Rhizobium0 sp M vax atla C i Th scaffo ea sp 2 05 20 1 thios hiosulf lase crob s 1 thio os d obact WP P ohydr d i n t ulf xB um 53436 7430 1 thiosulfoh6 vi m C i drolase SoxB M toco WP P y oxB Jann hiosulfoh r M 023430402 1 thiosulfohydrola 1 thi hiosu Lo u andonensig h u ola S fund W P 07 6 8 1 t xB ose i cus o ermu s 026379258 1 thiosulfohydrolase SoxB dro h lim p i e W ydro o l hia t W io hy lfoh T sca 0 1 t R s rru s oxB salinarum W thios fo h osei c r w 193 1 s e S se caf 13 415 0744 1 t l s e rc P he c WP P 02435 48 iosulf oxB cter f a ul sulfoh drol l er sp 624 8 1 u a ter 008 y d5787 cus m i WP 0 h lfo cte r l So s mar WW 67 51 t lfohy s lase S se S S f f drola oxB rm fo ffold454 1 W P 0 79364 u ae bacteria o oh T a 06 e Rube nn ac 1 WP 4 1 thiosulf1 s ro a b h yd as he P a cte r ant 285 thiosul us mylo ld544 W 55 64 982 6 o d oxB R o a s yd yd xB gen P 009469448 1 thiosulfohydrolase3177 SoxB Roseibium sp J ob ace d te W ydro 63 TC1 i W 0 4 6 thio 1 partial y xB b aquimarie W r e Sox s P 4669 29 hi se SoxB o r o s T r ar hiosu 1 lfohydrolase So o d P 94 o rolase s m ge W 94 t t ydrol a e S Roseibaod ter ac i P l e S hermusThe thermophil 4 P 0 7 ne u oh So h rh d ter l ase Thermus thermophilusp inu W gene8 f h ol o b ro 0 01 a u liqu gene8 e5 P 063 1 1 s r as o o W 1 9 se la CC s 0 g W ul o SoxB xB d e fohydrol g n P 028 21 7 9 io f rh d P 2 1 S s B o r cal W ge s o u o o 9 W 0 e x m e9 s P 00803 h o fohydrolas obac 0125 6 t 88 SoxB Meio e ene1 en W 25 sul l hyd S d hi oxB S T B B P 70 se rh a 4 P S u fa W 01 9 157 hi o o rolase u o c osulfoh o herm idi 0358441 253205 t i lf d SoxB Rhodobac r spha i 1 1 oxB Th s ig US3 UF1 cie e9 P 1 olase se P d hod t W 4 0 17 xB W 53 54 8 1 t h y r a r e n 1 ter P d t fohydrol l e u o a 14 9 ase M e W B38122 5 h d xB e ct la P 29 4 0 ol 1 hiosu osu o y o o 1 Th r nite ns W 035 f i f las a ific 50 029551971 9 1 th eiot The us mus r N f 6 h l b th a A P 054 128 1 1 t t osul oh ydr oxB R S Ps eud o 7 1 t 18 y SoxB e e fold4 1 1 su B ac io d sc r P f 2 h dro d va at p 0 r i t he rm r r W o ulf o y se SoxB Psex Ps a ica sae su olase SoxBos h m a sca 3 83 i lf e l a y 2 L e otoduco W ca 7 1 1 1 thi h se S la B v alba hi lf u L rm us islandicus s e s 3 t oh s So ind s os o 1 s scaff Meiothermusrmus u M 0550869 1 su lf ro ioc h lfoh L fi himai s 6 1 o e h cla roph th c s u M P 40 i u yd s xB RhoT o va ul yd L s 055683 7 1 h a i s 1 iosu a c lifo P 0 60380 2 os h l e Sox o sanguinis u fohy ro y sc ffold L cerbereus W i ydrola S cla mona t drol affo old t tus 37 9 3 1 thios lfo ro as xB Th ii hil hios l sc a rmis W P 05 8410897 1 t th h d o a lf a i 4 9 29 4 lfohydrola o y se S B uv cus op drol se o Hy ffo w W P 0 1 su lf h Thio l W 1 06 W hyd as 3 a l 6441 gen a 0 4 6 io u drol la us sphae L s ulf 7 ffol d us W P 043 1 3 fo se Sox B c ha p AC ilu P a S d e ld 6 176 n 2 36 2 32 osu ro a x acoc c s h W 06 se ohy o rolase S r So e W 1 th ios l o r s R xB Hy oge 32 4 n P 0 h fohy yd B Defl u ip P g d 68 3 73 1 h ro So c e p r 73 S drolas 21 38 4 rufus si W P 0 5 5 ul d lase ct s a s 00 o x e s 2 29 1 t fo y o se oc a ct n 2 32 n B n 0 g W P 0 8 thiosulos l h r a b m e 8 xB L ivi gen en e13 51 5 o d l e Sox Parac ac n ca C0 24 d Aq e 4 W 4 4 1 su y o riu fi a h 62 H rogen o s r 1 P 06 2 6 6 thi o lf h r e r ri 1 e xB ca ga uifex 9 g e10 5 3 1 i u o ct e it E 52 3 yd e1 p e W P 91635 1 thi 8 th yd olas e SoxBoxB Par Par mmo ct n 1 So ne 0 0 0 ios ulf r B e a e 56 m 72 rog T ff s a W P 5 7 1 h oh d S G ba b d utus iu th x o ol p p r P 2 941 lf e o s r W 1 B h 128 ae t 9 0 2 0 t ios B s TG1 io en b e d ar ial K P 0 9 1 h su as x cte P thios s Therma rmocri 4 0518 7 7 t lfohy e Sox Agro mm ccu C a 012083 u oba ct oli ti W P 5 526 u s B e b lf e 2 al 2 1 hio ohydrolas a So e o gene15 c 0 50 2 5 t os f ol e G co cus ver p M s ul hydrolase SoxB r R1 1 us W P 3 1 s ra c a u 8 f L c th 0 1910 4 hi ohydroldr la Sox a o ce e 0 oh ul oc n W 1 1 70 4 y xB P c ola s a n 195 L s um e is P 0 2 ro o ra r a s CA W y s caffo rinis rm 8 7 32 hiosul S rr u P dr c a 48 W P 3 osulf foh yd lase xB nic cte te r L o l 0 80 1 t i ul h se o Pa a i o UC 0 1 o scaffoa s p b W 3 27 972 1 t th o dro a S B ea b ed v 1 t las ff ld p s h u P 0 1 lf y l x c o no 3 hios ol Y p i s 8 14 1 u ro O d m ti 3 e S 519 04 lu W P 745 s oh se So o um HL 26 d2 G s 03 f yd la ius la ri 8 ulf BS W ul h o e oxB Rh b e W 0 ohy o ld229089 8 genA P o r s o g cte P 0 x Su 68 A 0668 8593 os lf yd la S s s a 1 L B S W P 1 thiohi u h xB s iu W 02 dr sc Sul lf 1 1 043132 1058 10 thios t s o dro o la e b 1 th olas L u ge W 6 1 io lf y lase S a sob a P 2 io aff rovum e5 P 3671 2 h h s e 049 W 0083403088 s s fur n W 0 1 5 t su foh dro se T la c 0 s P 66 u e c o gene e P 0 1 io la B a ra I lu 8 lfo L a ld ov 4 3 12 5 h ro x te hi 01 7 h S ffold1 1 um W 036744 2 t Th c p W 1371 1 y ox sc 66 r P 43 2 1 osul yd So B a sp H cus li W d B L affo ift 6 W 05 90 0 3 thi ulfohy h e b ka P th ro s 27 sp ia P 3 8 s do ius di al 6 s 0 P 900 io la Nit c 5 e 06 0 3 1 hios lfo la Sox o in r 64 tu 29 0134 1 su s a l 1 g A W 8 0 9 t su ro e e 3 ra 52 thi lfo e rat ff d 0 en R P 2 5 o d ct L e 1 os S L ol 14 0 ge W 0 7 77 1 4 y as B Rh arius a J p 29 59313 t h o s irupt d5 e P 1 7 thi e h rol x ib p m 09 83 hi ulf yd x c 1 3 01 75 094 lfo d o Roseovarov m s te AR W osulf ohy rol B L a 1 g n W 6 4 1 u y S se iti la r M P 1 S s ffo or 9 en e4 P 60 4 gen s oh e o ar co te 0 thi 1 ohyd a u c sp 3 W 03 6 2 io lf s oxB R hi ac ATI W 1 o t dr se lf a ld gen e6 P 0 th u la S M g b P 26 s hi olase So urimo ff 7 S 6 9 s ro xB n a 5R 0 7 u osulf r old1 6 B W 036582 1 io d oxB o ec p 35 28 69 lfo o xB Su 7 155 e P 6 7 th y ase S S Do d s L 3b s W 9 2 hydr ohyd lase SoxB L scaffold184n 1 9 W 0 old218027 h ol se B ta H K si P 0 50 1 a 35 g P ff 4 1 lfo la se ox sp w n 1 2 1 ol S s 9 en 2 4 56 su ydr o la S Oc anella Az ue 2 65 th ase So rol oxB lfu a 1 e W ca 82 1 io foh dr ro e xB kt la p o 45 1 io Sulf r ut g 1 s 5 63 y yd s o ico s oh is 9 t s ase im o e 5 M 0 2 th h la S Lo n ia 23 hi ulfo Sul ur o tro n P 1 osul foh fo ro e B ter iens 4 osu xB Pe S n p e2 58 8 hi ul l yd cea as q 1 hyd oxB fu im as sp 9 gene9h W 0 0 t os su h las Sox O bac th lfo ri onas ic P 37 1 io fo ro e eo on anha io hy ro monas a W 8 thi th ul yd xB s im su d la rseph Sul AST P 989 1 s h So Ro ea n lf ro se f got P 4 io lfo rolas B imin tal oh la So uri K 37 76 1 9 th u d se ox ed ti C1 yd se on curvumden landi 1 ZY 73 80 1 os ola S p J ans ro S xB P el 0 K 2 9 hi lfohy r e S B imen s yi vor la ox la it O0 3 39 t u yd ox ed ra ro idi se B e sp K rif ca 08 1 1 las S ge e l S Su rs icans OA 0 55 23 thios lfoh dro B S in om oha o l L e kuj P 7 2 1 su y ase is p yl xB fu scaffold9207ph M iense W 6 34 4 o Le ria th S rih L o 09 0 8 thi ulfoh e Sox B ge u yd s ne Lau P 49 704 1 s hydrol as x e a me emixtae lfu ro ca ll W 0 3 o fo ol So er ua rih g ff a m P 27 thi ul dr ng q ns yd en ol 8 W 9 1 os lase xB Ru i a a is ro ib d1 ar 0556880 hi fohy ro er g iu 5 g in P 4 256 t ul d So B Leis g inhibe iens en m 60 ene9 a W 02 8 1 os hy se ter ib s ge P 8 lfo ola Sox eisin aec 5382 iu ub 781 739 thi r e L gall T ns m te ne W 00 5 iosu hyd as xB ter ora 11 sp rra 3 P 67 48 1 th lfo rol So haeobac iv IMB Y n 5 W 02 1 su d e B P sp CEC en H O3 eu P 2614 68 io ohy las ox ter um A m W 8 th ulf ro S haeobacac hthal eri OP 027 68 1 yd e B P nap ct P 52 45 hios oh as ox er ba 1 W 06 67 1 t ulf drol S haeob ct e 35 P 04 ios e B P iba 77 W 11 58 th fohy pic eracea T 0 897 1 ul drolas Sox Tro act CEC P 40 07 hios ob sp W 72 t ohy rolase oxB er P 02 28 1 d S Rhod ct inus W 58 hiosulf ohy ase ba ar sis 0 1 t ulf ol eo m en P os ydr SoxB a don W 061049250566 thi foh lase xB Ph ibacter aek P sul ro So r b s W hio se Celer cte osu 065270 2397 1 1 t fohyd la iba uc P 5 ro ler s m W 05833 16 thiosul hyd Ce riu ns P 47 1 lfo va era W 82 61 su olase SoxBSoxB seo tol cus 05 16 thio ydr e Ro lanti P 57 1 as B arius la at W 22 drol Sox eov co W 022 02 thiosulfohohy se os ani P WP 831 1 lf la B R ooce 0194 05 70 iosu ydro ox osus WP 3 P th oh e S Pseud nsis 06 0127 W 22158 1 1 ulf olas xB muc de 240 1 06 53 hios dr o ipiger rmu WP 299 thios WP 571 1 t be 0 4 1 ul 009 63 ulfohy rolase S B Sal ca 553 thio foh P 762 os iba 57 601 sulf ydro W 72 1 thi fohyd ase Sox Pelag 3 W 90 1 oh las 03 625 osul l xB lla sp P th ydro e S WP thi hydro So ice 45 057 iosul las oxB 662 1 ulfo ase itre p SE 674 foh e S Li P 050 059 os drol xB C lla s 2 WP 833 ydro oxB mnoh W 746 1 thi ohy So ice a 0 1 th lase Limn ab 043 sulf lase Citre t rium PD 667 iosul S o itan P 950 hio ro xB stella acte ae 108 foh oxB hab s W 1 t hyd So tula ae b ce W 29 ydro Limn itan sp R 020038 737 sulfo lase Sagit race ctera P 0245379 1 th la o s s im WP 797 io dro B cte oba iosu se So hab p 63 47 07 2 1 th fohy Sox oba od ODS 47 1 thioslfoh xB itan ED P 0 319 sul e hod xB Rh 911 ydrol Cu s sp 37 2 W 950 thio drolas R e So 72 1 ase S rvib 10 00 94 1 ohy SoxB las 5A 1 ODU3 thio ulfohy o acte 3DP WP 45 sulf lase hydro p SJ 67 sulfo dr xB C r sp R2 888 1 thio ydro ulfo us s 41 1 hyd olas urvib PAE P 00 lfoh thios cocc 3 thio rolas e S act U W 274 iosu onti MM WP sulfo e S oxB er M 863 1 th B P CCB WP 0286 hyd oxB Comam delica P 005 829 TISPECIES e Sox sp 056 017 rolas Com tu W 851 UL as angia nae 6624 11 1 e So amo onas s 023 9 1 M drol xB Y r are 08 1 thiosu xB nas badia WP 457 ulfohy So acte e thiosu lfo Com sp 314 thios olase oreib iluca WP lfohy hydro amo SCN P 04 1 hydr B Lit noct 224 0484 drola lase nas 65 W 748 sulfo Sox acter C21 42046 se SoxB sp S 56 863 thio lase lfitob p IMC WP 1 t SoxB Ot CN 6 P 058 44 1 hydro Su ium s 055898 hiosul Pseudor towia 7 35 W 1004 sulfo SoxB acter 948 fohydrol hodof thio 066 1 thio rolase niceib us WP 1 th as oxyd WP 104 ohyd B Pu ontiac 01390 iosulf e Sox erax ans 1102 iosulf e Sox cter p 0703 ohydr B Ca sp Leaf P 02 1 th rolas lfitoba 01 W 1 thios olase enim 265 W 2974 lfohyd Su GAI1 P 061 ulfoh SoxB onas 2505 hiosu SoxB ter sp 49635 ydrola Ram sp S WP 0 9 1 t rolase eobac ta 9 1 thi se So libact L110 9438 lfohyd B Ros era WP osulfo xB R er sp 0479 thiosu e Sox rina temp 2 02701 hydrol amlib Leaf WP 26 1 drolas ktoma rium SB 5363 ase S acter 400 42162 ulfohy Plan bacte WP 1 thios oxB R tataou P 06 thios SoxB aceae C2150 063663 ulfohy amliba inens W 785 1 rolase obacter HTC 152 1 drolase cter ta is 37227 lfohyd Rhod erium thiosul SoxB taouin WP 0 1 thiosu e SoxB ae bact WP 020 fohydro Coma ensis 010 drolas erace 227501 lase S mona 044050 sulfohy Rhodobact la 1 thio oxB Co s comp WP 1 thio SoxB ctilucico WP sulfohy mamo osti 352483 olase acter no 057593 drolas nas thi P 068 osulfohydr lfitob 574 1 th e SoxB ooxyda W 1 thi e SoxB Su hC04 WP 06 iosulfoh Acidov ns ydrolas cter sp E 2473844 ydrolase orax sp WP 040787214 iosulfoh ulfitoba 1 thiosu SoxB V MR S7 778 1 th e SoxB S rraneus WP 0 WP 054 lfohydrola ariovor 25055 hydrolas r medite 54138706 152549 se SoxB ax parad WP 0 iosulfo lfitobacte 1 MULTISP 1 thiosul Variovo oxus 4326 1 th SoxB Su a ECIES t fohydrolas rax boro P 06729 ydrolase donghicol hiosulfohy e SoxB nicumula W thiosulfoh fitobacter drolase So beta prote ns 5887 1 SoxB Sul xB unclass obacterium WP 03790 hydrolase is ified Betap AAP51 1 thiosulfo obacter geojens WP 022979479 roteobacte 5060385 SoxB Sulfit 1 thios ria miscell WP 02 drolase is ulfohydrolas aneous 1 thiosulfohy bacter litoral WP 0540 e SoxB Ideonel 064223864 SoxB Roseo 22573 1 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d17102 gene9 32 06 h h eucoth hiothr olase SoxB 5 43 P P h B lase Sox ioth L scaffold3 P affold6774 gene14 io u y o D 1 osu B rola T W scaffold49248 gene1 4 F P So T h T W l drol ase SoxB mar rol drolase Sox x o L scaffold40476 gene3 013553334 1 thiosulfohydrolase SoxB Nitratifractor salsuginis e So mma O W 41 W d W d2 1 t sul fohyd s A 6 983 hi drolas ase Sox Thioalkalivibrio sp T WP thio ammap 0 W 764 1 th ol 1 t o W P 1 a p A1 U 432 WP yd P a 2 hy Sox y WP W P se S 208 4 ol S hy xB 0131 m 1 t h ase ydr WP 576 foh l a H P 81 W l WP P 044418689 1 thiosulfohydrolase SoxB h thio 4 tothior oxB WP WP W 012012358 1 thiosulfohydrolase SoxB W

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040 7 foh osul caff ct s ulfoh lfo u o r ulfohydrolase 0 0 0 35609 1 thiosulfohydrolase SoxB 0 e 0 i 8 yd se S 054 1 c 1 Congr P P d ase s ffol u xB thi ohy 0 6 l r se SoxB l ul 1 6 drolase SoxB d 2 4 hios os f oxB 120 gene5 hydr ydrolase SoxB Ch se SoxB P M scaffold6336 gene13 G as thi y 7 7 W a r a 9 7 W 0 rolase S la n 8 1 M t olase SoxB Chloro Candidatu

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1 1 thi a ct d b o egibac m 0 W 4 m 1 5 0 P 76 90 1 t hydrolase SoxB 41 ul CI M sca par ol lase SoxB Prosthe Thiomicrospir eri u o 3 ohyd s 1 3 SoxB Chlorobaculum tepidum u 3 4 s 7 ot sulf i W 3 lfohydro i x M scaffol r 6 Thiomicros a ase olase t 7 0 p 2 5 ohydr o u 1 eobac b 1 thiosul PE f i l B a bacteriu l e 3

M scaffold221 gene54 n a Thiomicrospira 4 3 5 t M scaffold3895 gene10 6 a lfohy i thiosulfohy hios su t ial rote 12542 4844 S sulfohydrolase SoxB Thiocapsa marina t o cte

u M scaffold31929 gene1 partial Thi c p 1 t 80 m 1

1 1 io o 1 er l 1 327437 1 i SoxB C TI a a s o m KPK 79780 1 thios osul

ISPECIES thios 06597 6 hiosulf 1 th M scaffold38520 gene1 partial l Thiocystis violascens t rium L t r t t th t o b h o th o 2 h h h T h Thi P 028 o ter 24 hi 0 Allochromatium vinosum b it 0408 h t thiosulfo i mi 1 L i n i

i i P 5 i 0 a o a oralis o c o o bacteria bacterium SG8 15 o h W 262 i 8 Thiohalocapsa sp ML1 TISPECIES thiosulfohydrolase SoxB P 1 Thiorhodococcus drewsii u iu

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h o h h h 3 2 2056 50 a b ra a 8 W 026148239 y sp y y 1 y

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S W S S P se SoxB Gammaproteobacteria ba S W y x Arcobacter nitrofigilis i 063463 Arcobacter anaerophilus o c o

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la r W P oxB Gammaproteo Arcobacter sp L us x Arcobacter lanthieri x o a x x x CIB S d Arcobacter thereius f Arcobacter cibarius B l B B B

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thiosulfohydrolase SoxB Marichromatium gracile A N s N

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i TISPECIES thiosulfohydrolase SoxB Halothiobacillus p

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s t 9285 t

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d ns

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029132149 1 thiosulfohydrolase SoxB Sedimenticola selenatireducens 069014875 1 thiosulfohydrolase SoxB endosymbiont of Loripes lucinalis KPJ94871 1 thi 4 9

057955716 1 thiosulfohydrolase SoxB endosymbiont of Ridgeia piscesae 0 3

069120539 1 thiosulfohydrolase SoxB endosymbiont of Codakia orbicularis 0

WP WP P

Figure S3 Maximum-likelihood phylogeny of soxB genes retrieved from L- and M-vent chimney. The red nodes represent the soxB genes recovered from the active L-vent chimney, while the blue ones are those from the recently inactive M-vent chimney. Four soxB genes from M-vent chimney form a distinct clade (marked with red star) that different with other SoxB.

a Recovered

900

800

700

600

t h 500 p e

D 400

300

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0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 Top 50 RpS3 in the L-vent b

700

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t h 400 p D e 300

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0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49

Top 50 RpS3 in the M-vent

Figure S4 The distribution and quantity of top 50 RpS3 genes retrieved in the MAGs (metagenomic assembled genomes) for two chimney samples. (a) in the active L-vent chimeny. (b) in the recently inactive M-vent chimney. The x-axis represents each RpS3 gene identified in two samples, the y-axis is the sequencing depth for each RpS3 gene. The red bars indicate the RpS3 genes identified in the MAGs in corresponding samples, while the blue ones are those are not included in the MAGs.

Others AAK64263 1 ndhL plasmid Paenarthrobacter nicotinovorans Others CAA66830 1 quinoline 2 oxidoreductase Pseudomonas putida 1 ABK03419 1 carbon monoxide dehydrogenase large subunit apoprotein Arthrobacter sp FB24 scaffold19566 gene4 scaffold35329 gene1 1 AAP75610 1 carbon monoxide dehydrogenase form I large subunit partial Mycobacterium smegmatis 1 AAP92701 1 carbon monoxide dehydrogenase form I large subunit partial Mycobacterium tuberculosis H37Ra 1 AAP92702 1 carbon monoxide dehydrogenase form I large subunit partial Mycobacterium microti 1 AAP92703 1 carbon monoxide dehydrogenase form I large subunit partial Mycobacterium marinum 1 AAP92704 1 carbon monoxide dehydrogenase form I large subunit partial Mycobacterium gordonae 1 AAR26462 1 carbon monoxide dehydrogenase form I large subunit partial Hydrogenibacillus schlegelii

1 ABI56911 1 carbon monoxide dehydrogenase large subunit apoprotein Alkalilimnicola ehrlichii MLHE 1 Fo r 1 AIP29610 1 carbon monoxide dehydrogenase large subunit Paraburkholderia xenovorans LB400

1 AAD00363 1 CutL Hydrogenophaga pseudoflava m I /OMP clade 1 CAA54902 1 cut A Pseudomonas thermocarboxydovorans scaffold15107 gene4 1 AAW88346 1 carbon monoxide dehydrogenase form I large subunit partial Ruegeria sp WHOI JT 01 1 AAW88347 1 carbon monoxide dehydrogenase form I large subunit partial Ruegeria sp WHOI JT 08 WP 011048110 1 carbon monoxide dehydrogenase large subunit Ruegeria pomeroyi 1 ACH99840 1 carbon monoxide dehydrogenase large subunit partial Shinella sp FG1M5 1 AAP75604 1 carbon monoxide dehydrogenase form I large subunit partial Bradyrhizobium sp CPP 1 AAP75614 1 carbon monoxide dehydrogenase form I large subunit partial Bradyrhizobium japonicum USDA 6 1 AAR26463 1 carbon monoxide dehydrogenase form I large subunit partial Pseudomonas carboxydohydrogena 1 AEI08106 1 CO dehydrogenase large chain CoxL plasmid Oligotropha carboxidovorans OM5 1 AAP75613 1 carbon monoxide dehydrogenase form I large subunit partial Stenotrophomonas sp LUP 1 AAP75608 1 carbon monoxide dehydrogenase form I large subunit partial Stappia carboxidovorans 1 ABK91862 1 carbon monoxide dehydrogenase form I large subunit partial Stappia conradae 1 ABK91861 1 carbon monoxide dehydrogenase form I large subunit partial Stappia meyerae 1 AAP75609 1 carbon monoxide dehydrogenase form I large subunit partial Stappia sp M8 1 AAP75606 1 carbon monoxide dehydrogenase form I large subunit partial Stappia sp KB902 1 ABK91860 1 carbon monoxide dehydrogenase form I large subunit partial Photobacterium sp COX 1 ABK91876 1 carbon monoxide dehydrogenase form I large subunit partial Stappia sp BrC2 1 AAP75611 1 carbon monoxide dehydrogenase form I large subunit partial Labrenzia aggregata 1 ABK91870 1 carbon monoxide dehydrogenase form I large subunit partial Stappia sp BrT7 1 AAP75612 1 carbon monoxide dehydrogenase form I large subunit partial Stappia stellulata Others AAL63828 1 carbon monoxide dehydrogenase large subunit conjectural Pyrobaculum aerophilum str IM2 scaffold63006 gene5 partial scaffold1518 gene30 scaffold38459 gene5 scaffold22548 gene1 scaffold10155 gene14 scaffold33621 gene2 scaffold66 gene66 2 NP 773911 1 carbon monoxide dehydrogenase Bradyrhizobium diazoefficiens USDA 110 scaffold48904 gene2 partial scaffold32538 gene3 scaffold5181 gene3 AAV94806 1 carbon monoxide dehydrogenase large subunit Ruegeria pomeroyi DSS 3 scaffold36976 gene4 partial contig-127 58467 gene2 2 AAP75599 1 carbon monoxide dehydrogenase form II large subunit partial Burkholderia sp LUP Fo r contig-127 16285 gene5

contig-127 1388 gene35 m II /BMS clade scaffold32454 gene8 scaffold26589 gene3 scaffold14882 gene6 scaffold300 gene40 2 AAP75603 1 carbon monoxide dehydrogenase form II large subunit partial Xanthobacter sp COX 2 AAP75592 1 carbon monoxide dehydrogenase form II large subunit partial Stappia sp KB902 2 AAP75597 1 carbon monoxide dehydrogenase form II large subunit partial Labrenzia aggregata 2 AAP75596 1 carbon monoxide dehydrogenase form II large subunit partial Stappia sp M8 2 AAP75595 1 carbon monoxide dehydrogenase form II large subunit partial Stappia carboxidovorans 2 AAP75593 1 carbon monoxide dehydrogenase form II large subunit partial Bradyrhizobium sp CPP 2 AAP75602 1 carbon monoxide dehydrogenase form II large subunit partial Bradyrhizobium japonicum USDA 6 2 NP 387097 1 carbon monoxide dehydrogenase large subunit transmembrane protein Sinorhizobium meliloti 1021 2 AAP75598 1 carbon monoxide dehydrogenase form II large subunit partial Mesorhizobium sp NMB1 Tree scale: 0.1 2 BAB48572 1 carbon monoxide dehydrogenase large subunit Mesorhizobium japonicum MAFF 303099 2 ABK91866 1 carbon monoxide dehydrogenase form II large subunit partial Carbophilus carboxidus 2 AAP75600 1 carbon monoxide dehydrogenase form II large subunit partial Aminobacter sp COX

Figure S5 Maximum-likelihood phylogeny of coxL genes retrieved from L- and M-vent chimney. The red branches represent the coxL genes recovered from active the L-vent chimney, while the blue ones are those from recently inactive M-vent chimney. Support for internal nodes was constructed from 1000 bootstrap replicates, black dots represent those nodes with bootstrap value > 75%, which is direct proportional to their diameter.

Outgroups

Nitrospiraceae bacterium isolate UBA8124 BMS3Abin10 BMS3Bbin08 Nitrospiraceae bacterium isolate SURF_45 Nitrospiraceae bacterium isolate SURF_23 Nitrospiraceae bacterium isolate SURF_11 Nitrospira bacterium SG8_35_1 BMS3Abin09 BMS3Bbin09 BMS3Abin06 Nitrospiraceae bacterium isolate UBA11860 Nitrospiraceae bacterium isolate UBA 6907 Nitrospiraceae bacterium isolate UBA 6902 BMS3Abin07 BMS3Bbin05 Candidatus Magnetoovum chiemensis strain CS-04 Candidatus Magnetobacterium casensis Candidatus Magnetotactic genus Candidatus Magnetobacterium bavaricum Nitrospiraceae bacterium UBA3562 Nitrospiraceae bacterium UBA 3568 M-MetaBat-199 M-MetaBat-26 BMS3Abin08 BMS3Bbin06 M-MetaBat-107 Sulfide Mineral clade Nitrospirae-3 M-MetaBat-13 BMS3Bbin07 Nitrospirae-2 Nitrospiraceae bacterium UBA 9159 Nitrospiraceae bacterium isolate SURF_35 Nitrospiraceae bacterium UBA1546 Nitrospiraceae bacterium UBA 8922 Nitrospiraceae bacterium UBA 9646 Nitrospiraceae bacterium UBA 12094 Thermodesulfovibrio sp. RBG_19FT_COMBO_42_12 Nitrospira bacterium SM23_35 Nitrospiraceae bacterium UBA2194 Nitrospiraceae bacterium palsa_1316 Nitrospirae bacterium Nbg-4 Nitrospiraceae bacterium UBA6898 Nitrospiraceae bacterium UBA6905 Nitrospira bacterium HGW-Nitrospira-1 Nitrospiraceae bacterium metabat2.164 Thermodesulfovibrio sp. N1 Nitrospiraceae bacterium UBA2600 Thermodesulfovibrio thiophilus DSM 17215 Thermodesulfovibrio yellowstonii DSM 11347 Thermodesulfovibrio islandicus DSM 12570 Thermodesulfovibrio Thermodesulfovibrio aggregans Thermodesulfovibrio aggregans JCM 13213 Thermodesulfovibrio aggregans TGE-P1 Nitrospira sp. Ru_enrich_NS Nitrospiraceae bacterium UBA665 Nitrospiraceae bacterium UBA9935 Nitrospiraceae bacterium UBA10224 Nitrospiraceae bacterium fen_1313 Nitrospiraceae bacterium fen_1311 Nitrospiraceae bacterium fen_1307 Nitrospiraceae bacterium fen_1312 Nitrospiraceae bacterium fen_1304 Nitrospiraceae bacterium fen_1306 Nitrospiraceae bacterium fen_1308 Nitrospiraceae bacterium fen_1305 Nitrospiraceae bacterium fen_1314 Nitrospiraceae bacterium fen_1303 Nitrospiraceae bacterium fen_1309 M-MetaBat-229 Nitrospiraceae bacterium UBA 9217 Nitrospiraceae bacteriu m UBA 12100 Nitrospiraceae bacterium UBA 12485 Nitrospiraceae bacterium UBA 9996 Leptospirillum ferrooxidans C2-3 Leptospirillum ferriphilum DSM 14647 Leptospirillum ferriphilum pb_238 Leptospirillum ferriphilum UBA 6673 Leptospirillum ferriphilum UBA 6674 Leptospirillum ferriphilum UBA 6657 Leptospirillum sp. Group II CF-1 Leptospirillum GroupII C75 Leptospirillum Leptospirillum rubarum Leptospirillum ferriphilum sp-CI Leptospirillum ferriphilum ML-04 Leptospirillum ferriphilum ZJ Leptospirillum ferriphilum YSK Leptospirillum ferriphilum DX M-MetaBat-18 Nitrospirae-1 Nitrospira sp. Bin_34_1 Nitrospira sp. Bin_6_1_1 Nitrospira sp. Bin_24_1 Nitrospiraceae bacterium UBA2166 Nitrospira sp. bin75 Nitrospira sp. SG8-3 Nitrospiraceae bacterium UBA 8639 Nitrospira sp. RSF151 Nitrospira sp. OLB3 Nitrospira sp. NOB Nitrospira sp. UBA 8978 Nitrospira sp. ND1 Nitrospira defluvii Nitrospira sp. RSF127 Nitrospira sp. UBA4129 Nitrospira sp. UW-LDO-02 Nitrospira sp. UBA667 Nitrospira sp. UBA5699 Nitrospira japonica Ch1 Nitrospira sp. RCB Nitrospira sp. palsa_1310 Nitrospira sp. palsa_1315 Nitrospira sp. CG24E Nitrospira sp. RDF48 Nitrospira sp. RSF8 Nitrospira sp. CG24E RSF3 Nitrospira sp. CG24C Nitrospira sp. RSF7 Nitrospira sp. SCGC AG-212-E16 Nitrospira sp. RSF6 Nitrospira sp. CG24A Nitrospira sp. RSF11 Nitrospira Nitrospira sp. ST-bin5 Nitrospira lenta Nitrospira sp. CG24D Nitrospira sp. RSF13 Nitrospira moscoviensis NSP M-1 Nitrospira sp. RCA Candidatus Nitrospira inopinata Nitrospira sp. UBA2082 Nitrospira sp. UBA6909 Nitrospira sp. ST-bin4 Nitrospira sp. SG-bin2 Nitrospira sp. SG-bin1 Nitrospira sp. HN-bin3 Nitrospira sp. HN-bin3 Candidatus Nitrospira nitrosa Nitrospira sp. UW-LDO-01 Nitrospira sp. UBA11158 Nitrospira sp. UBA5702 Nitrospira sp. UBA5698 Nitrospira sp. UBA2083 Nitrospira sp. SG-bin1 Candidatus Nitrospira nitrificans Nitrospira sp. RSF9 Nitrospira sp. RSF12 Nitrospira sp. RSF-1 Nitrospira sp. CG24B Nitrospirae bacterium Ga0074138 Tree scale: 0.1 Nitrospira sp. RSF5

Figure S6 Phylogeny of 6 Nitrospirae MAGs (metagenomic assembled genomes) recovered from M-vent chimney. The phylogenetic tree is based on 37 concatenated ribosomal proteins and collapsed at the genus level. The red nodes represent those MAGs recovered from M-vent chimney, the green ones are those from subseafloor massive sulfide deposits [72] and the organe ones are those retrieved from inactive chimneys [37]. The green-shded branches are proposed as “Sulfide mineral” clade of Nitrospirae in this study.

Outgroups Halothiobacillus neapolitanus Ignatzschineria larvae DSM 13226 Wohlfahrtiimonas chitiniclastica Cardiobacterium hominis Cardiobacterium sp. isolate bin 12 Cardiobacterium valvarum M-MetaBat-218 L-MetaBat-25 M-MetaBat-162 L-MetaBat-93 L-MaxBin-115 M-MetaBat-77 Thiomicrospira pelophila Thiomicrospira aerophila Piscirickettsiaceae Thiomicrospira cyclica Hydrogenovibrio marinus Thiomicrorhabdus chilensis Gamma-3 Gamma-4 L-MaxBin-137 Thiomicrorhabdus arctica Woeseia sp. SP227 Woeseia sp. SP2978 Woeseia oceani XK5 M-MetaBat-209 Woeseiaceae bacterium CPC1462 Woeseiaceae Woeseiaceae bacterium CPC1328 Woeseiaceae bacterium SP56 Woeseiaceae bacterium RS417 Dokdonella fugitiva Dokdonella Koreensis Dokdonella immobilis Gamma-01 Marinicella sediminis Gamma-2 Oceanospirillales Marinicella litoralis DSM25488 Marinicella litoralis KMM3900 Thiothrix lacustris Ectothiorhodosinus magna Ectothiorhodosinus mongolicus Gamma-5 Gamma-6 Gamma-7 Gamma-8 Gamma-09 Gamma-10 Ectothiorhodospiraceae Gamma-11 Gamma-12 M-MetaBat-2 M-MetaBat-93 Gamma-13 Gamma-15 Gamma-14 Thiogranum longum Beggiatoa alba M-MetaBat-112 Thiotrichaceae M-MetaBat-99 Thiotrichales bacterium SG8 50 Gamma-17 M-MetaBat-56 Nitrosococcus halophilus Nitrosococcus oceani Chromatiaceae Methylophaga lonarensis Methylophaga thiooxydans Piscirickettsiaceae Gamma-26 M-MetaBat-81 M-MetaBat-125 Candidatus Tenderia electrophaga Candidatus Tenderia electrophaga-like Gamma-16 M-MetaBat-201 Gamma-18

Gammaproteobacteria bacterium RBG 16 57 12 z Gammaproteobacteria bacterium SG8 11 M-MetaBat-104 Unclassified Gammaproteobacteria Gamma-20 M-MetaBat-225 Gamma-19 M-MetaBat-51 Thiohalomonas denitrificans Gammaproteobacteria bacterium SG8 47 Gamma-22 Gamma-21 Thiohalophilus thiocyanatoxydans Gamma-23 Thiohalophilus-like Gamma-25 Gamma-24 Tree scale: 0.1 M-MetaBat-151

Figure S7 Phylogeny of 20 Gammaproteobacteria MAGs (metagenomic assembled genomes) recovered from M- and L-vent chimney. The phylogenetic tree is based on 37 concatenated ribosomal proteins and collapsed at the genus level. The red nodes represent those MAGs recovered from L-vent chimney, and blue for M-vent chimney. The organe ones are those retrieved from inactive chimneys [37].

Outgroups

Crenarchaeota archaeon JdFR-12 Crenarchaeota archaeon 13 1 20CM 2 53 14 Crenarchaeota SCGC AAA261-N23 Crenarchaeota JGI-OTU-1 Crenarchaeota archaeon SCGC AAA471-L13 Thermoprotei archaeon B36 G2 Thermoprotei archaeon B15 G16 Thermoproteus sp.CP80 Pyrobaculum sp.WP30 Vulcanisaeta sp.OSP 8 Caldivirga sp.MU80 Thermocladium sp.ECH B Thermoproteaceae L-MetaBat-236 L-MaxBin-38 L-MaxBin-140 Ignicoccus islandicus L-MaxBin-089 Ignicoccus hospitalis Staphylothermus marinus F1 L-MaxBin-049 Thermogladius calderae Thermogladius calderae 1633 Desulfurococcaceae Thermosphaera aggregans DSM 11486 Desulfurococcus mucosus Desulfurococcus amylolyticus 1221n Zestosphaera tikiterensis L-MaxBin-41 Ignisphaera aggregans DSM 17230 Fervidicoccus fontis Kam940 Fervidicoccaceae Metallosphaera yellowstonensis MK1 Sulfolobus acidocaldarius DSM 639 Sulfolobus metallicus Sulfolobaceae Sulfolobus islandicus L.S.2.15 Pyrolobus fumarii L-MetaBat-44 L-MetaBat-77 Pyrodictiaceae Hyperthermus butylicus Pyrodictium delaneyi Aeropyrum pernix K1 Aeropyrum camini Acidilobus saccharovorans uncultured Acidilobus sp.OSP8 Caldisphaera lagunensis Acidilobales Tree scale: 0.1 Caldisphaera sp.ZAV-20

Figure S8 Phylogeny of 8 Crenarchaeota MAGs (metagenomic assembled genomes) recovered from L-vent chimney. The phylogenetic tree is based on 37 concatenated ribosomal proteins and collapsed at the family level. The red nodes represent those MAGs recovered from L-vent chimney.

EHO41485.1 phosphate-selective porin O and P [Caldithrix abyssi DSM 13497] M-MetaBat-201-Gammaproteobacteria M-MetaBat-151-Gammaproteobacteria M-MetaBat-126-Calditrichaeota M-MetaBat-96-Calditrichaeota M-MetaBat-128-Deltaproteobacteria M-MetaBat-126-Calditrichaeota M-MetaBat-200-Calditrichaeota M-MetaBat-154-Oligoflexia M-MetaBat-197-Deltaproteobacteria UOGF01000110.1 6 Nitrospirae-1 ALP52279.1 [Candidatus Tenderia electrophaga] Electrotrophic Gammaproteobacteria M-MetaBat-104-Gammaproteobacteria-1 M-MetaBat-225-Gammaproteobacteria M-MetaBat-51-Gammaproteobacteria M-MetaBat-104-Gammaproteobacteria-2 M-MetaBat-51-Gammaproteobacteria-2 M-MetaBat-107-Nitrospirae “Sulfide mineral” calde M-MetaBat-13-Nitrospirae Nitrospirae GH01000144.1 8 Nitrospirae-2 M-MetaBat-58-Deltaproteobacteria WP 008242986.1 [Sulfurovum sp. AR] KIM11389.1 [Sulfuricurvum sp. PC08-66] M-MetaBat-86-Campylobacteria AKN78226.1 [Mariprofundus ferrooxydans PV-1] WP 018283531.1 [zeta proteobacterium SCGC AB-137-C09] Zetaproteobacteria WP 038247608.1 [Ghiorsea bivora] Marine FeOB L-MetaBat-131-Aquificae WP 039960854.1 [endosymbiont of Riftia pachyptila] WP 040820377.1 [endosymbiont of Tevnia jerichonana] WP 006367388.1 [Chlorobium ferrooxidans] WP 011358173.1 [Pelodictyon luteolum] WP 013028406.1 [Sideroxydans lithotrophicus] WP 013028404.1 [Sideroxydans lithotrophicus] WP 013292418.1 [Gallionella capsiferriformans] Betaproteobacteria WP 018292269.1 [Gallionella sp. SCGC AAA018-N21] Freshwater FeOB WP 013028405.1 [Sideroxydans lithotrophicus] WP 012513365.1 [Hydrogenobaculum sp. Y04AAS1] BAP89290.1 [Burkholderiales bacterium GJ-E10] WP 031596628.1 [Ferrovum myxofaciens] AAM52228.1 [Thiobacillus denitrificans] WP 009206871.1 [Sulfuricella denitrificans] WP 009770369.1 [Paraburkholderia terrae] WP 031319384.1 [Pseudomonas fluorescens] WP 018992664.1 [Azoarcus toluclasticus] WP 017775840.1 [Paraburkholderia kururiensis] WP 024974009.1 [Ralstonia pickettii] WP 027478826.1 [Curvibacter gracilis] WP 006158001.1 [Cupriavidus basilensis] L-MetaBat-84-Betaproteobacteria M-MetaBat-115-Betaproteobacteria WP 026439021.1 [Acidocella facilis] WP 029311772.1 [Acidiphilium angustum] WP 006892594.1 [Methylobacter tundripaludum] WP 011379976.1 [Nitrosospira multiformis] WP 013104837.1 [Thiomonas] M-MetaBat-60-Alphaproteobacteria M-MaxBin-158-Alphaproteobacteria WP 020648557.1 [Solimonas variicoloris] WP 028312810.1 [Derxia gummosa] BMS3Bbin07 BDTQ01000098.1 3 BMS3Abin08 BDTA01000123.1 4 M-MetaBat-23-Bacteroidetes M-MetaBat-167-Bacteroidetes M-MetaBat-24-Bacteroidetes ACD03837.1 [Acidihalobacter prosperus] WP 038090550.1 [Acidihalobacter prosperus] WP 012537628.1 [Acidithiobacillus ferrooxidans] Tree scale: 1 WP 014029249.1 [Acidithiobacillus ferrivorans]

Figure S9 Maximum-likelihood phylogeny of cyc2 genes retrieved from L- and M-vent chimney. The red branches represent the cyc2 genes recovered from the active L-vent chimney, while the blue ones are those from recently inactive M-vent chimney. the green ones are those from subseafloor massive sulfide deposits [72] and the organe ones are those retrieved from inactive chimneys [37]. The other reference sequences are come from Kato et al., 2015 [74]. Support for internal nodes was constructed from 1000 bootstrap replicates, black dots represent those nodes with bootstrap value > 70%, which is direct proportional to their diameter.