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1 Supplementary Information for

1

Max-Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359 Bremen, Germany

2

Institute for Geology, Center for Earth System Research and Sustainability, University of Hamburg, 20146 Hamburg, Germany

3

GEOMAR Helmholtz Centre for Ocean Research, RD3 Marine Microbiology and Christian- Albrechts University of Kiel, Düsternbrooker Weg 20, D-24105 Kiel, Germany

4

Department of Medicinal Chemistry, Pharmacognosy, BioMedical Centre, Husargatan 3, Uppsala University, 751 23 Uppsala, Sweden

5

Florida State University, POB 3064326, Tallahassee, FL 32306, USA

6

Wegener Institute Helmholtz Centre for Polar and Marine Research, HGF-MPG Group for Deep Sea Ecology and Technology, Am Handelshafen 12, 27570 Bremerhaven, Germany

7

MARUM, Center for Marine Environmental Sciences, University of Bremen, 28359 Bremen, Germany

8

Israel Limnology and Oceanography Research, Tel Shikmona, 3108000, Haifa, Israel

9

Quadram Institute Bioscience, Norwich Research Park, Norwich, United Kingdom

*Corresponding authors

Maxim Rubin-Blum Nicole Dubilier

Phone: 0049 (0)421 2028 905 Phone: 0049 (0)421 2028 9032 E-mail: mrubin@mpi-bremen.de E-mail: ndubilier@mpi-bremen.de

Contents:

1. Supplementary Notes

2. Supplementary Methods

3. Supplementary Tables

4. Supplementary Figures

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2

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3

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6  

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9

µ µ

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Supplementary Figures

5 10 20 50 100 200 500 1000 2000

0.11101001000

Branching sponge library 1 coverage

Branching sponge library 2 coverage

5 10 20 50 100 200 500 1000 2000 5000

11010010005000

Branching sponge library 2 coverage

Branching sponge library 1 coverage

5 10 20 50 100 200 500 1000 2000

0.0110100

Encrusting sponge Mictlan coverage

Encrusting sponge Chapopote coverage

2 5 10 20 50 100 200 500 1000 2000

0.0010.1101000Encrusting sponge Mictlan coverage

Encrusting sponge Chapopote coverage

2030 4050 60

% GC mitochondria

mitochondria

mitochondria

mitochondria MOX

MOX

MOX

MOX Cycloclasticus

Cycloclasticus

Cycloclasticus

Bdellovibrio

Cycloclasticus

SOX

SOX SOX/JTB23 overlap

SOX/JTB23 overlap

Branchiomonas

Branchiomonas Ekhidna

Ekhidna OCS116 clade

OCS116 clade

SAR324 clade

SAR324 clade

host

host host

host KI89A clade/Turneriella/

Cytophagales sp. overlap Bdellovibrio

KI89A clade/Turneriella/

Cytophagales sp. overlap Nitrosopumilus

Nitrosopumilus

Pseudoxanthomonas sp.

Nitrosopumilus E01-9C-26 clade

E01-9C-26 clade JTB23 clade

JTB23 clade

BD7-8 clade

BD7-8 clade KI89A clade

KI89A clade

Supplementary Figure S1 Differential sequencing coverage of > 2000 bp scaffolds from the sponge metagenomic libraries. Each circle represents a metagenomic scaffold. The circles are colored according to their GC content, the diameter of these circles is proportional to scaffold length. ‘x’ symbols represent scaffolds that contain tRNAs, crossed circles represent scaffolds that contain 16S rRNA genes. Ellipses show examples of bins chosen for reassembly of sponge MOX symbionts. The sizes of the complete bins shown here (all contigs included in the ellipse) are 2.44 Mbp for the branching sponge and 2.29 Mbp for the encrusting sponge.

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1 m

empty mussel shells

sponges

inactive zone inactive zone

colony of vestimentiferans soft oil-soaked sediments,

gas and oil efusion, mussels

mussels sponges

sponges sponges

sponges

Supplementary Figure S2 A photomosaic of the Chapopote ‘bubble’ site where the sponges were collected (photos taken from 2.5 m above the seafloor). Areas dominated by sponges (green), mussels (red) and tubeworms (yellow) are highlighted.

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Methylomicrobium/Methylobacter/Methylosarcina/Methylovulum/Methylosoma spp.

Methylomarinum vadi (NR 112675.1) Methylomonas spp.

H. (S.) methanophila Mictlan MOX (this study) H. (S.) methanophila Chapopote MOX (this study)

H. (S.) methanophila Chapopote MOX (HE814582.1) H. (S.) methanophila Chapopote MOX (HE814575.1) H. (S.) methanophila Chapopote MOX (HE814569.1)

MMG2 bacterium, Haakon Mosby Mud Volcano (AJ704662.1)

MMG2 genome E33, North Sea sediment enrichment (Vekeman et al. 2016)

MMG2 bacterium, hydrocarbon seep, New Zealand (JF268393.1) MMG2 bacterium, hydrocarbon seep, New Zealand (JF268387.1) MMG2 bacterium, hydrate ridge, Cascadia Margin (AJ535221.1)

MMG2 bacterium, poecilosclerid sponge, Gulf of Mexico (AB453739.1) MMG2 bacterium, hydrocarbon seep, New Zealand (JF268343.1)

MMG1 - Methyloprofundus sedimenti and the symbiotic MOX of Bathymodiolus spp.

Cycloclasticus spp.

Methylosphaera hansonii (NR_026033.1)

MMG3 Methylophaga spp.

0.02

I. methanophila Chapopote MOX (this study)

MMG2 symbiont of squat lobster Shinkaia crosnieri (AB476240.1)

> 90%

> 50%

Bootstrap support

MMG2 symbiont of folliculinid ciliate, Guaymas Basin (KU896787.1)*

MMG2 symbiont of Siboglinum cf. poseidoni (FR729025-7)*

MMG2 symbiont of Cladorhiza methanophila (SRA:SRR4269900.185564.1)*

MMG2 symbiont of Cladorhiza methanophila (SRA:SRR4269900.36609.1)*

MMG2 symbionts

MMG2 free-living/

not reported as symbiont

‘Methylomonadaceae’ I (Methylococcaceae)

‘Methylomonadaceae’ II (Methylococcaceae)

MMG1

Supplementary Figure S3 Phylogeny of the 16S rRNA genes from the symbiotic MOX of sponges collected at the Campeche and Mictlan seeps. The dataset included metagenomic 16S rRNA gene

sequences from this study and sequences from the NCBI database (94 sequences total). Bootstrap values below 50% are not shown. The tree is drawn to scale, with branch lengths representing the number of substitutions per site. The analysis included 1,262 positions. The genome of the North Sea sediment enrichment genome D14 lacked the fully assembled 16S rRNA gene sequence. Star marks the 16S rRNA gene sequences which were excluded from the tree calculation due to its short length, which would have hindered the phylogeny resolution, and whose position in the tree was estimated based on a phylogeny of separately aligned truncated sequences. MMG is Marine Methylotrophic Group.

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Gulf of Mexico, oil spill clone P192-3 (HQ738557.1) Gulf of Mexico, oil spill clone P230-1 (HQ738566.1)

Jaco Scarp , water column above methane seep, clone Jaco 0.2 27 (FJ858349.1)

Costa Rica oxygen minimum zone, hydrothermal seep clone CR-OMZwc A9 (JX569111.1) Gulf of Mexico, oil spill clone wcpmoA OC242 30 (KJ543506.1)

Gulf of Mexico, oil spill clone wcpmoA OC102 3 17 (KJ543512.1) Gulf of Mexico, oil spill clone wcpmoA OC103 003 (KJ543509.1 ) Gulf of Mexico, oil spill clone P222-1 (HQ738561.1)

Rainbow vent field on the Mid-Atlantic Ridge clone PMO-I (AY354047.1 ) I. methanophila Chapopote 2 (BSC2) MOX

I. methanophila Chapopote 1 (BSC1) MOX H. (S.) methanophila Mictlan (ESM) MOX H. (S.) methanophila Mictlan (ESM) MOX

Republic of the Congo, organic-rich lobe sediments 492CT5 pmoA B8 (KY351428.1) Guaymas Basin, Folliculinid ciliate symbiont clone GY DR399 (KX021876.1)

USA, Del Mar Seep, Folliculinid ciliate symbiont clone SD DR470 7274 (KX021877.1) Hydrate Ridge methane seep clone HR AT18-10 FA (KX021875.1)

New Zealand, methane seep, clone NZ 45 pmoA 68 (KC751370.1) Kuroshima Knoll, southern Ryukyu Arc, methane seep clone:Kuro-pmoA-17 (AB176938.1)

New Zealand, methane seep, clone NZ 45 pmoA 68 (KC751370.1) Escarpment cold seep site, clone BR1pmoA30 (EU982958.1)

Siboglinum cf. poseidoni symbiont, clone W10 M11 (FR729005.1)

Republic of the Congo, organic-rich lobe sediments clone 492CT5 pmoA A8 (KY351416.1) Republic of the Congo, organic-rich lobe sediments 491CT12 pmoA C6 (KY351183.1) Escarpment cold seep site, clone BR1pmoA11 (EU983006.1)

Methylococcaceae bacterium SF-BR (AB453965.1 )

Republic of the Congo, organic-rich lobe sediments clone 486CT9 pmoA G10 (KY351033.1) Republic of the Congo, organic-rich lobe sediments clone 486CT9 pmoA E5 (KY351009.1) North Sea MMG2 isolate D14 (LT555367.1 )

North Sea MMG2 isolate E33 (LT555366.1)

North American continental margin methane sep clone Bubsed 2H (EU444857.1) Shinkaia crosnieri epibiont, clone ihePR661 145 (AB794867.1)

MMG1 - Methyloprofundus sedimenti and Bathymodiolus spp. MOX Methylomarinum vadi strain IT-4(AB302947.2 )

Methylomarinum vadi strain: T2-1 (AB453964.1) Methylomarinum sp. SSMP1 (KU740209.1)

Methylobacter sp. BB5.1 (AF016982.1)

Methylobacter tundripaludum (AJ414658.1) Methylomicrobium sp. 5B (AF307139.1 )

Methylomicrobium japanense (AB253367.1) Methylomicrobium alcaliphilum (FO082060.1) Methylovulum psychrotolerans (CP022129.1)

Methylovulum miyakonense (AB501285.1) Methylosoma sp. TFB (KX282487.1) 0.050

Methylomonas spp.

Branch bootstrap support:

> 90%

> 80%

> 50%

MMG2 symbionts

MMG1 MMG2 free-living/ not reported as symbiont

‘Methylomonadaceae’ I

‘Methylomonadaceae’ II (Methylococcaceae)

(Methylococcaceae)

Supplementary Figure S4a Phylogeny of pmoA genes from the symbiotic MOX of sponges collected at the Campeche and Mictlan seeps. The dataset included pmoA sequences from this study and from the NCBI database (54 nucleotide sequences total). The tree is drawn to scale, with branch lengths

representing the number of substitutions per site. The analysis included 344 positions.

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Methylmonas denitrificans Methylomo

nas ko yamae

Methylomonas met hanica

Methylmo nas lenta

Methylomarinum vadi North S

ea MM G2 g

enome E33

North Sea MM

G2 genome D14

Bathymodiolus sp. SMAR MOX Methyloprofundus sedimenti

Bathymodiolus azoricus MOX Bathymodiolus platifrons MOX I. methanophila MOX H. (S.)

methanophila Chapop ote MOX H. (S.) met

hanophila Mictlan MO X Methylobacter l

uteus Methy

lobacter w hitten

buryi Methylom

icrob ium

alcali philum Meth

ylom icrob

ium bur

yatense

etM

lovhy

ulum

iy m

onak

ense

luvulohyetM

m p

sychrotolerans

Methylosarcina fi bra ta

Methylosarcina lac

us

Methylomicrobium agile

Nitrosococcus halophilus Nitrosococcus oceani Nitrosococcus watsonii Methyloglobulus morosus Methylocystis rosea Methylocystis bryophila

Beijerinckiaceae sp.

Methylomicrobium agile Methylobacter tun

dripaludum Methylobacter l

uteus Methylov

ulum miyakonense Methylmo

nas d enitrifica

ns Methylomo

nas met hanica Candida

tus N itros

pira ni trific

ans Can

dida tus N

itros pira ni

trosa

0.20

Methylobacter tundripaludum

AmoCAB Am

oCAB

PxmABC PmoCAB Branch bootstrap support:

> 95%

> 80%

> 50%

‘Methylomonadaceae’ I

‘Methylomonadaceae’ II

MMG1 MMG2 free-living

MMG2 symbionts

‘Methylomonadaceae’ II and I

Supplementary Figure S4b Phylogeny of concatenated PmoCAB and PxmABC proteins (39 amino acid sequences total). Sequences were first aligned for each subunit, following concatenation of individual alignments in the ABC order. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis included 886 positions.

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bc

f cf cf

f e f

embryo

embryo

5 μm

n

n n

bc cf

cf cf

f

f sp

sp

sp

sp sp

sp

n f

f cf

cf

cf bcbc sp

bc bc

sp bc sp

sp

5 μm 20 μm

a b c

d

ma mi

5 μm bl

li

1 μm li

li li

5 μm

n

he ho co

MOX

Supplementary Figure S5 TEM images of H. (S.) methanophila embryo tissue a, Overview of an embryo.

mi=micromere cells, ma=macromere. b, electron-dense blastomeres (bl) with lipid droplet inclusions (li).

c, Lipid droplet inclusions in blastomeres. d, nucleolated larval cells with homogeneous (ho) and heterogeneous yolk (he), n = nucleus, co = collagen fibers. e and f, methane-oxidizing symbionts accumulated between follicle-like cells (f) and embryo, and on the periphery of follicle-like cells.

bc=bacteria in bacteriocytes; cf= collagen fibrils; n=nucleus; sp=sponge cell.

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1290 618 823*(97)

608* 7819*

2385*

(1361)***

11 522 733 70

10 31

117 118 44

Sponge MOX symbionts

(4 genomes) MMG1 MOX

(3 genomes) North Sea

enrichment MMG2 (2 genomes)

Methylomonadaceae isolates (18 genomes) 1402

119 13

15 14

298 45

344 2

109

137 8

2 111 7

I. met hano

phila C hapop

ote 1 M OX

I. methanophila Chapopote 2 MOX H. (S.) methanophila Mictlan MOX

H. (S.) met

hanophila Chapopote MOX

a

b

**

Supplementary Figure S6 Pangenome analysis of methane-oxidizing bacteria (MOX). Orthologous gene counts are shown. a, Comparison of the sponge MOX symbiont genomes assembled in this study. b, Comparison between the genomes of the sponge MOX symbionts and related free-living and symbiotic MOX. “Sponge MOX symbionts” group includes the genomes of MOX from the four sponge individuals analyzed in this study. “North Sea enrichment MMG2” group includes the two genomes of the Marine Methylotrophic Group 2 bacteria enriched from the North Sea sediments. “Methylomonadaceae isolates”

group comprises the 18 representative genomes of Methylomonadaceae (main text, Figure 4), which were downloaded from NCBI. “MMG1” group comprises genomes of Methyloprofundus sedimenti, as well as those of MOX hosted by mussels Bathymodiolus sp. SMAR and B. platifrons. * clade-specific genes; **

unique to the sponge MOX symbiont core genome; *** mussel symbionts only (not including M.

sedimenti).

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H. (S.) methanophila

BGC0000837: Arylpolyene Vf biosynthetic gene cluster Vibrio fischeri (40% of genes show similarity) BGC0000836: Arylpolyene Ec biosynthetic gene cluster Escherichia coli (25% of genes show similarity)

halogenase

VF0841

c1204

beta-ketoacyl synthase beta-ketoacyl synthase short-chain dehydrogenase

acyl carrier protein (ACP1) acyl carrier protein (ACP2) acyl transferase

histidine ammonia-lyase

VF0860

c1186

1 2 3 4 5 log(TPM+1)

Supplementary Figure S7: Aryl polyene synthesis cluster in H. (S.) methanophila MOX symbiont genome. Vibrio fischeri and Escherichia coli Vf and Ec aryl polyene synthesis domains are shown as references. Homologs are shown in the same color. The second alignment is colored according to the average expression values of respective genes (color legend is shown in figure on bottom right).

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1 2500 5000 7500 10000 12500 14616 bp

1 2500 4603 bp

consensus MPI

consensus MPI query 1 query 1

query 1

H. (S.) methanophila Cycloclasticus

I. methanophila methane-oxidizing symbiont H. (S.) methanophila methane-oxidizing symbiont

Autotransporter beta-domain DUF285: related to leucine rich repeats Cadherin-like beta sandwich domain

Fibronectin type 3 domain PKD domain

consensus MPIquery 2 query 2

a

b

ORU94421.1 (98%) (94%)

(86%)

ORU92747.1 (81%) ORU91689.1 (51%) ORU94544.1 (75%) ORU94587.1 (45%) ORU94767.1 (42%) (87%)

(84%) (84%) (76%) (85%) (46%) (85%) (62%) (46%)

(51%) (57%) (57%) (48%) (43%) (37%) (47%) (43%)

ORU91839.1 (59%)

Supplementary Figure S8 (a,b) Structure of and occurrence of eukaryote-like protein (ELP)-encoding sequences in the genomes of sponge symbionts (a, b) and in sponge metagenomes (c, d). a, The structure of the longest ELP in H. (S.) methanophila MOX symbiont. Eukaryote-like domains (ELDs), which comprised leucine-rich repeats and cadherin-like domains, as well as an autotransporter beta-domain, which encodes a pore-forming element of the type V secretion system are shown. BLAST hits for this sequence (query 1) within the genome of H. (S.) methanophila MOX and Cycloclasticus symbionts are shown below. b, The structure of the longest ELP in I. methanophila MOX symbiont. Eukaryote-like domains (ELDs), which comprised leucine-rich repeats cadherin-like, polycystic kidney disease (PKD) and fibronectin type 3 domains are shown. BLAST hits for this sequence (query 2) within the genome of H. (S.) methanophila MOX are shown below. In panels a and b, identity values at amino acid level

between the query and the sequences identified by BLAST are shown in parentheses. Consensus sequence and the mean pairwise identity (MPI) over all pairs in the column are included (100%, green; 30-99%, yellow; below 30%, red).

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Supplementary Figure S8 (c, d) Structure of and occurrence of eukaryote-like protein (ELP)-encoding sequences in the genomes of sponge symbionts (a, b) and in sponge metagenomes (c, d). c, d: Scaffolds that contain ELPs (green circles) in the metagenomes of H. (S.) methanophila (c) and I. methanophila (d), revealed by BLAST analysis using the longest assembled ELP genes as queries. In panel c, red circles mark scaffolds that contain the autotransporter beta domains identified by BLAST using the respective partial ELP sequence as a query.

5 10 20 50 100 200 500 1000 2000

5 10 20 50 100 200 500 1000 2000

0.0110100H. (S.) methanophila Chapopote coverage

H. (S.) methanophilaMictlan coverage

0.11101001000

I. methanophila library 2 coverage

mitochondria MOX

Cycloclasticus

host

Nitrosopumilus

E01-9C-26 JTB23

SUP05 BD7-8

KI89A

mitochondria MOX

CycloclasticusAqs2-like SUP05/JTB23 overlap

Ekhidna OCS116

SAR324 host

KI89A/Turneriella/

Cytophagales sp. overlap Bdellovibrio

Nitrosopumilus

2030 4050 60

% GC

2030 4050 60

% GC scaffold with an ELP BLAST hit

scaffold with an autotransporter beta domain BLAST hit

scaffold with an ELP BLAST hit

I. methanophilalibrary 1 coverage

c

d

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H. (S.) methanophila Mictlan MOX H. (S.) methanophila Chapopote MOX I. methanophila Chapopote MOX

MMG1 - Methyloprofundus sedimenti and Bathymodiolus spp. MOX Thermithiobacillus tepidarius (WP_028989412.1)

Thioalbus denitrificans (WP_114279615.1)

Sedimenticolaceae (free living Sedimenticola sp. + symbionts of tubeworms and bivalves) Candidatus Thioglobus autotrophicus (WP_053951905.1)

Nitrosomonadales bacterium (PWB46437.1) Methylophilaceae spp.

Rhodocyclaceae spp.

Thiohalorhabdus denitrificans (WP_054965502.1) Burkholderia pseudomallei (WP_085538356.1)

Nitrosomonadales spp.

Beijerinckiaceae sp. (WP_009337376.1 ) Thioalkalivibrio paradoxus (WP_006747611.1) Thioalkalivibrio nitratireducens (WP_015258541.1) Methylococcaceae bacterium AMb10-2013 (PZN76546.1)

Methylosarcina fibrata (WP 020564491.1) Methylomonas methanica (WP 064044676.1) Methylobacter tundripaludum (WP_104423249.1)

Rhodobacteraceae spp.

Kordimonadales spp.

Thioglobaceae (SUP05) symbionts of Bathymodiolus spp. and clams Oceanospirillales spp.

0.10

Methylomonas denitrificans (WP_036278592.1) Crenothrix polyspora (WP 087145999.1)

Crenothrix sp. D3 (TE97995.1) Branch bootstrap support:

> 90%

> 50%

‘Methylomonadaceae’

MMG1 MMG2 sponge symbionts

Supplementary Figure S9 Phylogeny of narG genes from the symbiotic MOX of sponges collected at the Campeche and Mictlan seeps (highlighted in green). The dataset included narG sequences from this study and from the NCBI database (73 amino sequences total). The tree is drawn to scale, with branch lengths representing the number of substitutions per site. The analysis included 1167 positions.

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retention time

intensity

C16:1w8/w7

C16:2D5,9

istd C26:3D5,9,?

C14

C16:1w6/w5

C18:1w8/w7

C20:1w8/w7 C22:1w8/w7C24:1w8/w7

C26:3D5,9,?

C26:2D5,9

C26:1w8/w7

C26:3D5,9,? (brominated)

C26:2D5,9 (brominated)

16:2D5,9 istd

C14

24:1ω9

C28:3D5,9,?

C28

C26

C24 22:1ω9

C22 22:1ω7 20:1ω8

20:1ω7 20:1ω6

C20

24:1ω7 18:1ω7

20:2∆5,9 18:1ω9

18:2∆5,9

C18

C17

C15 16:1ω8 16:1ω7/6/5

a

b

²⁸D⁵,²⁴

retention time

intensity

istd

istd c

c c 27∆5

²⁷D⁵,²⁴

²⁹D⁵,²⁴E

²⁹D⁵,²⁴Zcdiplopterol

²⁸D⁵,²⁴ 27D5

diplopterol

c c c c

c

d

C16

Supplementary Figure S10 Partial gas chromatograms (total ion current) of fatty acid (a, b) and alcohol fractions (c, d) in H. (S.) methanophila (a, c) and I. methanophila (b, d). Istd= internal standard. Symbols and detailed nomenclature; fatty acids (a, b): gray circles= saturated fatty acids, white circles=

monounsaturated fatty acids, black stars= demospongic acids, red stars= brominated demospongic acids, white triangle= iso-fatty acids, black triangle= anteiso-fatty acids, ω= position of double bond counting from the methyl-end of the chain, Δ= position of double bonds counting from the carboxylic acid end.

Nomenclature alcohols (c, d): 27Δ5=cholest-5-en-3β-ol (cholesterol), 27Δ5,24= cholesta-5,24-dien-3β-ol (desmosterol), 28Δ5,24= 24-methylcholesta-5,24-dien-3β-ol, 29Δ5,24= 24-ethylcholesta-5,24E-dien-3β-ol (fucosterol), 28Δ5,24= 24-ethylcholesta-5,24Z-dien-3β-ol (iso-fucosterol), c= contaminants.

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GADP Pyruvate

dxs 1-deoxy-D-xylulose 5-phosphate synthase

DOXP

ispC 1-deoxy-D-xylulose 5-phosphate reductoisomerase MEP

2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase ispD

CDP-ME

4-diphosphocytidyl-2-C-methyl-D-erythritol kinase ispE

CDP-MEP

2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase ispF

MEcPP

4-hydroxy-3-methyl-2-(E)-bute- nyl-4-diphosphate synthase ispG

HMB-PP

4-Hydroxy-3-methyl-2-(E)- butenyl-4-diphosphate reductase ispH

Isopentenyl pyrophosphate (IPP) ? Dimethylallyl pyrophosphate (DMAP)

ispA ispB Farnesyl/octaprenyl diphosphate synthase

Farnesyl/octaprenyl diphosphate

Farnesyl-diphosphate farnesyltransferase fdft

Squalene-hopene cyclase Squalene

shc

The non-mevalonate pathway (MEP/DOXP)

Squalene monooxygenase 2,3-oxidosqualene sterol cyclase

Sterol 14-demethylase

4-methylated sterols

1 2 3 4

hpnG Hopanoid-associated phosphorylase

Hopanoid biosynthesis associated radical SAM protein HpnH Present in Bathymodiolus MOX

hpnH

Aminotransferase in hopanoid biosynthesus cluster argD

hpnN Hopanoid biosynthesis associated RND transporter like protein HpnN MOX hopanoid

Hopanoid synthesis

Scale [Log (TPM+1)]

Supplementary Figure S11 Hopanoid synthesis in sponge MOX symbionts. The reconstruction is based on genomes of I. methanophila and H. (S.) methanophila MOX and on transcriptomes of H. (S.)

methanophila MOX. Enzymes and their subunits are abbreviated with the name of the encoding gene.

Boxes are colored according to the expression value of a respective transcript (color legend for expression values is shown on the bottom of figure).

24

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