Supplementary Materials 2

1 SUPPLEMENTARY MATERIALS 2

3 Interactions between temperature and energy supply drive microbial 4 communities in hydrothermal sediment 5 6 Running title: Temperature and energy supply drive microbial communities

8 Lorenzo Lagostina1, Søs Frandsen2, Barbara J. MacGregor3,4, Clemens Glombitza1,2,

9 Longhui Deng1, Annika Fiskal1, Jiaqi Li1, Mechthild Doll5, Sonja Geilert6, Mark Schmidt6,

10 Florian Scholz6, Stefano Michele Bernasconi7, Bo Barker Jørgensen2, Christian Hensen6,

11 Andreas Teske3, and Mark Alexander Lever1,2*

13 1Institute of Biogeochemistry and Pollutant Dynamics, Eidgenössische Technische 14 Hochschule Zürich, 8092 Zürich, Switzerland 15 2Center for Geomicrobiology, Department of BioScience, Aarhus University, DK-8000 16 Aarhus, Denmark 17 3Department of Marine Sciences, University of North Carolina at Chapel Hill, Chapel Hill, 18 NC, 27599, USA 19 4Department of Earth and Environmental Sciences, University of Minnesota, Minneapolis, 20 MN, 55455, USA 21 5Faculty of Geosciences (FB 05), University of Bremen, 28359 Bremen, Germany 22 6GEOMAR Helmholtz Centre for Ocean Research Kiel, Wischhofstraße 1-3, 24148 Kiel, 23 Germany 24 7Department of Earth Sciences Eidgenössische Technische Hochschule Zürich, 8092, 25 Zürich, Switzerland 26 27 *To whom correspondence should be addressed: Mark Alexander Lever, Eidgenössische 28 Technische Hochschule Zürich, Institute of Biogeochemistry and Pollutant Dynamics, 29 Universitätsstrasse 16, CHN G50.3, 8092 Zürich, Switzerland; phone: +41 44 632 85 27; 30 email: [email protected]. 31

32 Supplementary Methods

33 Supplementary background on study area

34 The Guaymas basin is a young rift basin in the Gulf of California that is characterized by a 35 highly productive water column, that supports high sedimentation rates of phytoplankton- 36 derived organic matter to the seafloor, and vigorous seafloor and subseafloor hydrothermal 37 activity driven by active seafloor spreading27. Tubular magmatic intrusions occur across the 38 entire basin through the 200 to 400m thick organic-rich sediment column15,27. Magmatic 39 intrusions into the organic-rich sediment lead the ‘thermogenic’ breakdown of sedimentary 40 organic matter, which releases of methane, short chain organic acids (SCOAs), aliphatic 41 and aromatic hydrocarbons, including petroleum compounds, and labile protein and 42 carbohydrate-derived organic matter1,2,16,26,30,38,42. In addition, the high temperatures caused 43 by magmatic intrusions result in sulfide production via the thermochemical reduction of 44 sulfate at temperatures >110°C19. In certain regions, vertically advecting fluids transport 45 these compounds to shallow sediments with temperatures in the growth range of microbial 46 life, where they sustain vast populations of chemoorgano- and chemolithoautotrophic 47 microorganisms, including dense mats of sulfur-oxidizing bacteria (Beggiatoaceae)31,35,41. 48 These microorganisms in turn form the basis of a rich food web that supports a high biomass 49 of macrofauna20,39. Vertical fluid advection also leads to formation of hard structures at the 50 seafloor, including hydrothermal vent chimneys and carbonate crusts12,47.

51 The abundant supplies of diverse energy substrates combined with high diversity of 52 sedimentary habitats47, and the extreme spatial and temporal variability in temperature31,32 53 results in a dynamic sedimentary environment that supports functionally, physiologically, 54 and phylogenetically very diverse microbial communities14,24,44. Decades of microbiological 55 cultivation have led to the isolation of many thermophilic and hyperthermophilic 56 microorganisms from hydrothermal vents and hydrothermal seep sediments5,6,8,22,40,45 and 57 the discovery of new catabolic pathways13,23, and have expanded the known growth 58 temperature ranges of microorganisms18,19,22. Many of the thermo- and hyperthermophiles 59 isolated occur in both hydrothermal seep sediments and nearby hydrothermal vent 60 chimneys9,24, frequently even in samples that also harbor microorganisms with much lower 61 temperature requirements11,24. Guaymas Basin sediment has also remained a treasure 62 trove of novel microbial diversity. Gene sequences of the alpha subunit of methyl coenzyme 63 M reductase (mcrA) indicate the presence of at least three novel candidate orders of

64 methane-cycling Archaea4,24. Recent genomic investigations have resulted in the proposal 65 of 5 new candidate phyla within the Bacteria and Archaea14. In situ fluid geochemistry and 66 temperature, and microbial resilience to rapidly fluctuating thermal regimes, have been 67 proposed as key drivers of microbial community structure12,32,34,46. In addition, it has been 68 proposed that high rates of thermal disturbance result in refugia for temperature-resilient 69 microorganisms that are competitively excluded in more stable environments24. The 70 existence of such refugia, combined with the fact that Guaymas Basin sediment unites 71 characteristics of hydrothermal vents, oil reservoirs, and hydrocarbon seeps may further 72 contribute to the astounding microbial diversity24.

73 Despite the impressive number of past microbiological investigations, many questions 74 concerning the controls on microbial community structure in Guaymas Basin sediment 75 remain open. For instance, only few studies have investigated in situ microbial abundances, 76 or their relationships with temperature10,34. Recent metagenomic data from 11 samples 77 indicate that Archaea dominate hydrothermal sites14, providing support to the hypothesis 78 that Archaea are better equipped for life at high temperatures than Bacteria because of 79 higher temperature resistance of archaeal ether-based lipid membranes compared to 80 bacterial ester-based lipid membranes48.

81 Most microbiological research on Guaymas Basin has so far focused on a hydrothermal field 82 with hydrothermal vents in the Southern Trough (here referred to as Seep Area (SA)). 83 Subseafloor fluid circulation through the surrounding sediment leads to the development of 84 a hot, heterogeneous methane, sulfide, and petroleum seep area, characterized by the 85 presence of Beggiatoaceae mats, hydrothermal mounds, and bare sediments47. Additional

86 research has been performed on cold methane and CO2 seeps at the northeastern transform 87 margin (Sonora Margin)9,37,49. In 2015, a second hydrothermal vent field (here referred to as 88 Non-Seep Area (NSA)) was discovered on the flank of the Northern Trough during 89 Expedition SO241 of the research vessel SONNE2. Near this vent field, extensive authigenic 90 carbonate crusts are present in the absence of clear fluid flow, indicating a previously active 91 hydrothermal and cold seep environment, in which deep fluid and thermogenic gas flow 92 terminated 7-28,000 kyrs ago16. Even though present-day vertical fluid advection was not 93 detected at locations sampled during SO241, sites surrounding the vent field maintain 94 significant temperature gradients, reaching temperatures of 60-70°C within 400-500 95 centimeters below the seafloor (mbsf)2,16. Due to the absence of new energy inputs from 96 photosynthetically produced OM or hydrothermal fluid advection, these geothermally heated 3

97 non-seep sites offer the opportunity to investigate the role of temperature in driving microbial 98 community structure under diffusion-controlled subseafloor conditions, where 99 microorganisms are severely energy-limited.

100 Supplementary Methods

101 DNA extraction 102 DNA was extracted according to Lever et al. (2015) (reference 25). Briefly, all samples were 103 extracted using lysis protocol II with the following specifications: 0.2 g sediment were placed 104 into screw-cap microcentrifuge tubes filled to ~15% with 0.1mm zirconium-silica beads and 105 mixed with 100 µL of 10mM sodium hexametaphosphate solution. Samples were 106 homogenized for 30s at 30 shakings per second on a Tissue Lyzer LT (Qiagen). Afterward, 107 chemical lysis for 1 hour at 50°C and 600 rpm was performed on a ThermoMixer 108 (Eppendorf). Samples were then washed two times with ice-cold chloroform-isoamlyalcohol 109 (24:1), and precipitated at room temperature in the dark with ethanol-sodium chloride 110 solution supplemented with linear polyacrylamide (LPA) as a co-precipitant (20 μg LPA mL−1 111 extract). Precipitated and dried DNA pellets were purified using the CleanAll DNA/RNA 112 Clean-Up and Concentration Micro Kit (Norgen Biotek, Madison, WI) according to the 113 manufacturer’s instructions. For further details on Lysis Protocol II, including all instrument 114 settings, see Lever et al. (2015).

115 16S rRNA gene quantification 116 Bacterial and archaeal 16S rRNA gene copy numbers were quantified by SYBR-Green- 117 based quantitative PCR (qPCR) on a LightCycler 480 Instrument II (Roche Life Science, 118 Penzberg, Germany). The Bac908F_mod (5´- AAC TCA AAK GAA TTG ACG GG-3’)25 / 119 Bac1075R (5´- CAC GAG CTG ACG ACA RCC-3´)36 primer combination was used for 120 Bacteria. The Arch915F_mod (5´-AAT TGG CGG GGG AGC AC-3´)7 / Arch1059R (5´-GCC 121 ATG CAC CWC CTC T-3´)51 was used for Archaea. qPCR reactions (final volume: 10 μL) 122 consisted of 5 µL 2 × SYBR Green I Master (Roche Life Science, Penzberg, Germany), 1 123 μg μL−1 bovine serum albumin, 10 μM of each primer, molecular-grade water, and 2 μL of 124 original DNA extract. Amplicons of 16S rRNA genes of Holophaga foetida and 125 Thermoplasma acidophilum were used as bacterial and archaeal qPCR standards. Pure 126 cultures of H. foetida and T. acidophilum were purchased from the German Collection of 127 Microorganisms and Cell Cultures and their DNA extracted using a MOBIO PowerSoil DNA 4

128 Isolation Kit (QIAGEN, Hilden, Germany. Thermal cycler protocols consisted of (1) enzyme 129 activation and initial denaturation at 95 °C for 5 min; (2) 40 cycles (Bacteria) and 50 cycles 130 (Archaea) of (a) denaturation at 95°C for 10 s, (b) annealing at 60°C (Bacteria) and 55°C 131 (Archaea) for 30 s, (c) elongation at 72°C for 15 s, and (d) fluorescence measurement at 132 72°C (Bacteria) and 81°C (Archaea) for 15 s; and (3) a stepwise melting curve from 95°C to 133 55°C in 1 min to check for primer specificity. All measurements were run in duplicate. 134 Samples with on average >3 times higher values than extraction blanks were included in 135 the manuscript.

136 16S rRNA amplicon sequencing and phylogenetic classifications 137 According to 16S rRNA gene abundances, samples were pooled in different groups for a 138 first booster PCR, performed with the aim of increasing and normalizing the gene copy 139 number. The archaeal primer pair S-D-Arch-0519-a-A-19 (5’-C AGC MGC CGC GGT AAH 140 ACC-3’; reference 43, renamed in reference 21) / Arch915RRmod (5’-GT GCT CCC CCG 141 CCA ATT-3’)7 and the bacterial primer pair S-D-Bact-0341-b-S-17 (5’-CCT ACG GGN GGC 142 WGC AG-3’) / S-D-Bact-0785-a-A-21 (5’-GAC TAC HVG GGT ATC TAA TCC-3’; both 143 reference 17, renamed in reference 21) were used for this booster PCR. Booster PCRs were 144 used to elevate gene copy numbers to similar concentrations across samples and to 145 minimize PCR cycle numbers with tailed primers, which introduce additional biases beyond 146 those of non-tailed primers3. According to the original 16S gene copy numbers determined 147 by qPCR, booster PCR cycle numbers varied from 10 to 32. PCR products of booster PCR 148 were checked on an agarose gel, and those samples with visible (preferably weakly visible) 149 correctly-sized bands were used for downstream work. Booster PCRs were repeated on 150 samples with no visible bands, increasing the cycle number by 4 additional cycles. 1µL of 151 PCR product from all successful booster PCRs was used as template for a second PCR (8 152 cycles) with frameshifted tailed primers, that were used to improve sequencing accuracy29. 153 Tailed amplicons were then cleaned using AMPure XP beads, amplicon lengths checked by 154 gel electrophoresis to confirm successful addition of adaptor (tail) sequences, and then 155 underwent Index-PCR using the Nextera DNA library Prep Kit (Illumina, San Diego, USA). 156 Indexed amplicons were cleaned using AMPure XP beads, quantified with a Tecan plate 157 reader and Tapestation (Agilent, Santa Clara, USA), and finally pooled equimolarly. Paired- 158 end sequencing (2x300 bp) was performed on a MiSeq Personal Sequencer (Illumina, San 159 Diego, USA). Raw-read ends were trimmed and pairs merged into amplicons. Subsequently 160 primers were trimmed and amplicons were quality filtered (PRINSEQ). Operational 5

161 taxonomic units (OTU) at 97% clustering were assigned using UNOISE. Taxonomic 162 assignments were performed using the SILVA database (SSURef v128) for Bacteria and 163 a manually curated in-house archaeal 16S rRNA gene database in ARB28. Sequencing data 164 were analyzed using Phyloseq package33. Statistical analyses were perfomed in R using 165 Vegan, heatmaps using package Corrplot50.

166 Supplementary Figures

Seep Area Non-Seep Area

Sulfide (µM) Sulfide (µM) 0 4000 8000 12000 16000 0 10 20 30 40 50 60 0 0 4

8 100 12 16 200 20 24 300

Depth (cm)Depth 28 MUC02 (St. 15) (Ctrl) GC13 (St. 9) (Ctrl) 32 Cold site (Ctrl) GC09 (St. 51) 36 Yellow Mat 400 GC10 (St. 58) Cathedral Hill 40 Orange mat 167 44 500

168 Supplementary Figure 1. Hydrogen sulfide concentration profiles in the Seep Area (SA) 169 and Non-Seep Area (NSA) replotted from Reference 47 (SA) and Reference 16 (NSA; also 170 see Supplementary Data 1). Note: no data exist for Everest Mound and MUC12. 171

172

173

174

175

Seep Area Non-Seep Area

δ13C-DIC δ13C-DIC -32 -28 -24 -20 -16 -12 -8 -4 0 4 -32 -28 -24 -20 -16 -12 -8 -4 0 4 0 0 4 8 80 12 160 16 20 240 24 MUC02 (St. 15) (Ctrl)

Depth (cm)Depth MUC12 (St. 40) (Ctrl) 28 320 32 GC13 (St. 9) (Ctrl) Cold site (Ctrl) GC09 (St. 51) 36 Yellow Mat 400 GC10 (St. 58) Cathedral Hill 40 Orange mat 176 44 480

177

178 Supplementary Figure 2. Depth profiles δ13C-dissolved inorganic carbon (δ13C-DIC) in the 179 Seep Area (SA) and Non-Seep Area (NSA). All data shown in Supplementary Data 1. 180

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184

185

186

187

13 δd13CC-TOC-TOC d13C-TOC -24 -22 -20 -18 -16 -14 -12 -24 -22 -20 -18 -16 -14 -12 0 0 SA 10 100

20 200

30 300 Depth (cm) Cold site (Ctrl) MUC02

Depth (cm)Depth Yellow Mat MUC12 40 Orange mat 400 GC13 Cathedral Hill GC09 Everest Mound GC10 d13C-TOC 50 d13C-TOC 500 -24 -22 -20 -18 -16 -14 -12 -24 -22 -20 -18 -16 -14 -12 0 0 NSA 10 100

20 200

30 300 Depth (cm) Cold site (Ctrl) MUC02 Yellow Mat MUC12 40 Orange mat 400 GC13 Cathedral Hill (cm)Depth GC09 Everest Mound GC10 50 188 500

189

190 Supplementary Figure 3. Depth profiles of δ13C-Total Organic Carbon (δ13C-TOC) in the 191 SA and NSA. All data shown in Supplementary Data 1. 192

193

194

195 196 197

Station 58

0% 50% 100%

148

198 Supplementary Figure S4A, Non-Seep Area (NSA) 248 199 Station 15 348 A 0%0% 20%20% 40%40% 60%60% 80%80% 398 100%100% Bacterial taxa 0.5 448 Alphaproteobacteria 55 DeltaproteobacteriaProteobacteria, Alpha (Ctrl) 15 Bdellovibrionales 15 Desulfarculales Desulfobacterales 30 Depth (cm) Depth 30 Desulfurellales NB1-j MUC02 MUC02 38 Station 9 OTHEROther 0% 20% 40% 60% 80% 100% Sulfurimonas EpsilonproteobacteriaSulfurovum 26 26 Proteobacteria, Alpha Bdellovibrionales BD7-8_marine_group Desulfarculales Desulfobacterales Methylococcales 76 76 Desulfurellales NB1-j GammaproteobacteriaThiotrichales Other Sulfurimonas NA (Ctrl) 176 Sulfurovum BD7-8_marine_group Xanthomonadales Methylococcales Thiotrichales Other 326 NA Xanthomonadales Acetothermia Depth (cm) Depth 326 Other Acetothermia UnclassifiedHolophagae GC13 GC13 476 Holophagae Station 40Other Acidobacteria Other Acidobacteria 476 Aerophobetes Aminicenantes OtherAerophobetes 0% Atribacteria20% 40% 60%Bacteroidia 80% 100% Aminicenantes 0.5 Sphingobacteriia NA 0.5Proteobacteria, Alpha Bdellovibrionales Desulfarculales Atribacteria DesulfobacteralesOther DesulfurellalesCaldiserica NB1-j AcidobacteriaBacteroidia 2.5 Anaerolineae Dehalococcoidia 3.5Other Sulfurimonas Sulfurovum Sphingobacteriia BD7-8_marine_groupMSB-5B2 MethylococcalesOther Thiotrichales 6.5 NA (Ctrl) Elusimicrobia Clostridiales 7NA Xanthomonadales Other AcetothermiaNA Holophagae Other FirmicutesOther Acidobacteria Other 10.5 Caldiserica 11AerophobetesOmnitrophica AminicenantesParcubacteriaAtribacteria BacteroidiaPlanctomycetes SphingobacteriiaThermodesulfobacteriaNA Sum Anaerolineae

Depth (cm) Depth 16.5 17OtherThermotogae Caldiserica VerrucomicrobiaAnaerolineae Sum Dehalococcoidia Dehalococcoidia MSB-5B2 Other Sum MSB-5B2 MUC12 MUC12 29.5 Other Phyla Bacteroidetes 30Elusimicrobia ClostridialesStation 51 NA Other Other0% Firmicutes20% Omnitrophica40% 60% Parcubacteria80% 100% Elusimicrobia Planctomycetes Thermodesulfobacteria Thermotogae 37 Proteobacteria, Alpha Bdellovibrionales OtherClostridiales VerrucomicrobiaDesulfarculales Other Phyla Desulfobacterales NA 37 Desulfurellales NB1-j Other Sulfurimonas Other Firmicutes 137 Sulfurovum BD7-8_marine_group Omnitrophica 137 Methylococcales Thiotrichales ChloroflexiParcubacteria NA Xanthomonadales Anaerolineae 237 Other Acetothermia DehalococcoidiaPlanctomycetes 237 Holophagae Other Acidobacteria Thermodesulfobacteria GC09 Aerophobetes Aminicenantes MSB-5B2 Depth (cm) Depth Thermotogae Atribacteria Bacteroidia Other 337 Sphingobacteriia NA Verrucomicrobia 337 Other Station 58Caldiserica Other Phyla 0% Anaerolineae20% 40% Dehalococcoidia60% 80% 100% 48 MSB-5B2 Other Firmicutes Proteobacteria,Elusimicrobia Alpha BdellovibrionalesClostridiales Desulfarculales 48 NA Other Firmicutes 148DesulfobacteralesOmnitrophica DesulfurellalesParcubacteriaNB1-j Unclassified 148OtherPlanctomycetes Sulfurimonas ThermodesulfobacteriaSulfurovum Thermotogae Verrucomicrobia Other 248BD7-8_marine_group Methylococcales Thiotrichales 248 Other Phyla 348NA Xanthomonadales Other 348Acetothermia Holophagae Other Acidobacteria

GC10 398Aerophobetes Aminicenantes Atribacteria Depth (cm) Depth 398 448Bacteroidia Sphingobacteriia NA 448Other Caldiserica Anaerolineae Dehalococcoidia MSB-5B2 Other 200 ElusimicrobiaProteobacteria, Alpha ClostridialesBdellovibrionales NADesulfarculales OtherDesulfobacterales Firmicutes OmnitrophicaDesulfurellales ParcubacteriaNB1-j 201 PlanctomycetesOther ThermodesulfobacteriaSulfurimonas ThermotogaeSulfurovum VerrucomicrobiaBD7-8_marine_group OtherMethylococcales Phyla 10 Thiotrichales NA Xanthomonadales Other Acetothermia Holophagae Other Acidobacteria Aerophobetes Aminicenantes Atribacteria Bacteroidia Sphingobacteriia NA Other Caldiserica Anaerolineae Dehalococcoidia MSB-5B2 Other Elusimicrobia Clostridiales NA Other Firmicutes Omnitrophica Parcubacteria Planctomycetes Thermodesulfobacteria Thermotogae Verrucomicrobia Other Phyla Station 58

0% 50% 100%

48 148

248 202 Supplementary Figure S4B, Seep Area (SA) 203 Cold Site 348 B 0%0% 20%20% 40%40% 60%60% 80%80% 398100%100% Bacterial taxa 0.50.5 448 Alphaproteobacteria

(Ctrl) 4.54.5 DeltaproteobacteriaProteobacteria, Alpha 14. Bdellovibrionales 14.55 Desulfarculales Site 24. Desulfobacterales Depth (cm) Depth 24.55 Desulfurellales Yellow Mat NB1-j

Cold 39. 39.55 OTHEROther 0% 20% 40% 60% 80% 100% Sulfurimonas EpsilonproteobacteriaSulfurovum 0.50.5Proteobacteria, Alpha Bdellovibrionales Desulfarculales BD7-8_marine_group Desulfobacterales Desulfurellales NB1-j Methylococcales 4.54.5Other Sulfurimonas Sulfurovum GammaproteobacteriaThiotrichales BD7-8_marine_group Methylococcales Thiotrichales NA 14.514.5 Xanthomonadales NA Xanthomonadales Other Other Acetothermia Holophagae Other Acidobacteria Acetothermia Depth (cm) Depth 24.524.5 Aerophobetes Aminicenantes Atribacteria UnclassifiedHolophagae Yellow Mat Bacteroidia SphingobacteriiaCathedral Hill NA Other Acidobacteria 34.534.5Other Caldiserica Anaerolineae Other 0% 20% 40% 60% 80% 100% Aerophobetes Dehalococcoidia MSB-5B2 Other Aminicenantes Elusimicrobia Clostridiales NA Atribacteria 0.50.5 Acidobacteria Other Firmicutes Omnitrophica Parcubacteria Bacteroidia Sphingobacteriia Planctomycetes Thermodesulfobacteria Thermotogae Acidobacteria 4.54.5Verrucomicrobia Other Phyla NA Other Caldiserica 14.514.5 Sum Anaerolineae

Depth (cm) Depth Sum Dehalococcoidia BacteroidetesSum MSB-5B2 19.519.5 Cathedral Hill Orange Mat Other 0% 20% 40% 60% 80% 100% Elusimicrobia 0.5 OtherClostridiales 0.5 Proteobacteria, Alpha NA Proteobacteria,Bdellovibrionales Alpha Bdellovibrionales Desulfarculales Other Firmicutes 4.54.5 Desulfarculales DesulfobacteralesDesulfobacteralesDesulfurellales NB1-j Omnitrophica Other DesulfurellalesSulfurimonas Sulfurovum ChloroflexiParcubacteria 14.514.5BD7-8_marine_groupNB1-j Methylococcales Thiotrichales OTHER AnaerolineaePlanctomycetes NA Xanthomonadales Other Dehalococcoidia 19.519.5 Sulfurimonas Thermodesulfobacteria AcetothermiaSulfurovum Holophagae Other Acidobacteria MSB-5B2 AerophobetesBD7-8_marine_groupAminicenantes Atribacteria Thermotogae Depth (cm) Depth 24.524.5Bacteroidia MethylococcalesSphingobacteriia NA OtherVerrucomicrobia Other Thiotrichales Caldiserica Anaerolineae Orange Mat Orange Other Phyla 29.529.5 NA Everest Mound DehalococcoidiaXanthomonadalesMSB-5B2 Other Elusimicrobia0% Other20% Clostridiales40% 60% NA 80% 100%Firmicutes Other FirmicutesAcetothermia Omnitrophica Parcubacteria 0.50.5PlanctomycetesHolophagae Thermodesulfobacteria Thermotogae VerrucomicrobiaOther AcidobacteriaOther Phyla Unclassified 2.52.5 Aerophobetes Aminicenantes Other 4.54.5 Atribacteria Proteobacteria,Bacteroidia Alpha Bdellovibrionales 5.55.5 DesulfarculalesSphingobacteriia Desulfobacterales DesulfurellalesNA NB1-j Other 6.56.5 OTHER Sulfurimonas SulfurovumCaldiserica BD7-8_marine_group Depth (cm) Depth Sum Anaerolineae 7.57.5 Methylococcales Thiotrichales NA Sum Dehalococcoidia Xanthomonadales Other Sum MSB-5B2 Acetothermia 9.59.5 HolophagaeOther Other Acidobacteria Everest Mound AerophobetesElusimicrobia Aminicenantes 205204 AtribacteriaProteobacteria,Clostridiales Alpha BacteroidiaBdellovibrionales SphingobacteriiaDesulfarculalesNA NADesulfobacterales 206 SupplementaryOtherDesulfurellales FigureOther Firmicutes 4. Detailed depthCaldisericaNB1-j profiles of bacterial community structure in the OtherOmnitrophica Sulfurimonas 207 (a) NSA and (b)Sulfurovum SA,Parcubacteria focusing on the dominantBD7-8_marine_group classes, orders, and genera for phyla with Planctomycetes 208 sub-phylum classifications.MethylococcalesThermodesulfobacteria Thiotrichales NA Xanthomonadales Other Acetothermia11 Holophagae Other Acidobacteria Aerophobetes Aminicenantes Atribacteria Bacteroidia Sphingobacteriia NA Other Caldiserica Anaerolineae Dehalococcoidia MSB-5B2 Other Elusimicrobia Clostridiales NA Other Firmicutes Omnitrophica Parcubacteria Planctomycetes Thermodesulfobacteria Thermotogae Verrucomicrobia Other Phyla Station 58 0% 20% 40% 60% 80% 100%

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398 209 Supplementary Figure S5A, Non-Seep Area (NSA) Station 15 448 0%0% 20%20% 40%40% 60%60% 80%80% 100%100% A 0.5 498 Archaeal taxa 55

(Ctrl) Aenigmarchaeota 15 Aigarchaeota C3

Depth (cm) Depth 30 BathyarchaeotaMCG-1 MCG-2

MUC02 MUC02 Station 9 38 MCG-3 MCG-4 0% 20% 40% 60% 80% 100% Aenigmarchaeota Aigarchaeota MCG-16 C3 MCG-1 26 MCG-2 MCG-3 Uncl. Bathyarchaeota MCG-4 MCG-8 MCG-9 MCG-16 Other Bathyarchaeota MCG-17 MCG-21 Crenarchaeota, Thermoprotei 76 MCG-22 MCG-23 MCG-27 MCG-28 Crenarchaeota, HWCG I MCG-29 MCG-30 (Ctrl) Unclassified Other Crenarchaeota, HWCG IV 176 Desulfurococcales Thermoproteales Deeply-branching T.protei Other Thermoprotei Crenarchaeota, Other HWCG I HWCG IV Hadesarchaea Other Crenarchaeota Hadesarchaea Depth (cm) Depth 326 326 Me thanomicrobia Thermoplasmata Methanomicrobia Other Euryarchaeota Alpha GC13 GC13 Beta Pacearchaeota Thermoplasmata Ma rin e Group I Station 40 Unclassified 476 Other Thorarchaeota Other Euryarchaeota Uncl. Asgard Phylum I Other Asgardarchaeota 0%Wo esearchaeota20% 40% 60%Other Archaea80% 100% Lokiarchaeota Alpha Aenigmarchaeota Aigarchaeota Lokiarchaeota Beta 0.5 C3 MCG-1 MCG-2 MCG-3 Pacearchaeota MCG-4 MCG-8 MGI Thaumarchaeota 2.5 MCG-9 MCG-16 Crenarchaeota MCG-17 MCG-21 Uncl. Thaumarchaeota MCG-22 MCG-23

(Ctrl) MCG-27 MCG-28 Other Thaumarchaeota 6.5 MCG-29 MCG-30 Unclassified Other Thorarchaeota Desulfurococcales Thermoproteales 10.510.5 Deeply-branching T.protei Other Thermoprotei Uncl. Asgard Phylum I HWCG I HWCG IV Other Asgardarchaeota

Depth (cm) Depth Other Crenarchaeota Hadesarchaea 16.5 Me thanomicrobia Thermoplasmata Woesearchaeota 16.5 Other Euryarchaeota Alpha

MUC12 MUC12 Beta Station 51 Pacearchaeota Other Archaea 29.5 Ma rin e Group I Unclassified 29.5 Other Thorarchaeota 0%Uncl. Asgard20% Phylum I 40% 60%Other Asgardarchaeota80% 100%Euryarchaeota WoAenigmarchaeota esearchaeota OtherAigarchaeota Archaea 37 C3 MCG-1 37 MCG-2 MCG-3 MCG-4 MCG-8 137 MCG-9 MCG-16 137 MCG-17 MCG-21 MCG-22 MCG-23 237237 MCG-27 MCG-28 MCG-29 MCG-30 Unclassified Other Lokiarchaeota 337337 Desulfurococcales Thermoproteales Deeply-branching T.protei Other Thermoprotei HWCG I HWCG IV GC09 387 Depth (cm) Depth 387 Other Crenarchaeota Hadesarchaea Me thanomicrobia Thermoplasmata Other Euryarchaeota Alpha 437437 Beta Pacearchaeota Ma rin e Group I Station 58 Unclassified Other Thorarchaeota 487487 Uncl. Asgard Phylum I Other Asgardarchaeota 0%Wo esearchaeota20% 40% 60%Other Archaea80% 100%Thaumarchaeota Aenigmarchaeota Aigarchaeota 48 C3 MCG-1 MCG-2 MCG-3 MCG-4 MCG-8 148 MCG-9 MCG-16 MCG-17 MCG-21 MCG-22 MCG-23 248 MCG-27 MCG-28 MCG-29 MCG-30 348 Unclassified Other 348 Desulfurococcales Thermoproteales Deeply-branching T.protei Other Thermoprotei GC10 398 HWCG I HWCG IV Depth (cm) Depth 398 Other Crenarchaeota Hadesarchaea Me thanomicrobia Thermoplasmata 448 Other Euryarchaeota Alpha Beta Pacearchaeota Ma rin e Group I Unclassified 498 Other Thorarchaeota Uncl. Asgard Phylum I Other Asgardarchaeota Wo esearchaeota Other Archaea 210 Aenigmarchaeota Aigarchaeota C3 MCG-1 MCG-2 MCG-3 211 MCG-4 MCG-8 MCG-9 MCG-16 MCG-17 MCG-21 MCG-22 MCG-23 212 MCG-27 MCG-28 MCG-29 MCG-30 Unclassified Other Desulfurococcales Thermoproteales12 Deeply-branching T.protei Other Thermoprotei HWCG I HWCG IV Other Crenarchaeota Hadesarchaea Me thanomicrobia Thermoplasmata Other Euryarchaeota Alpha Beta Pacearchaeota Ma rin e Group I Unclassified Other Thorarchaeota Uncl. Asgard Phylum I Other Asgardarchaeota Wo esearchaeota Other Archaea Station 58 0% 20% 40% 60% 80% 100%

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398 213 Supplementary Figure S5B, Non-Seep Area (NSA) Cold Site 448 B 0%0% 20%20% 40%40% 60%60% 80%80% 100%100%

0.50.5 498 4.54.5 Archaeal taxa (Ctrl) 14.5 Aenigmarchaeota 14.5 Aigarchaeota

Site C3 24.524.5 Depth (cm) Depth BathyarchaeotaMCG-1 39.5 Yellow Mat MCG-2 39.5 MCG-3 Cold 0% 20% 40% 60% 80% 100% MCG-4 Aenigmarchaeota Aigarchaeota C3 MCG-1 MCG-16 0.50.5MCG-2 MCG-3 MCG-4 MCG-8 Uncl. Bathyarchaeota MCG-9 MCG-16 Other Bathyarchaeota MCG-17 MCG-21 4.54.5MCG-22 MCG-23 Crenarchaeota, Thermoprotei MCG-27 MCG-28 MCG-29 MCG-30 Crenarchaeota, HWCG I Unclassified Other Crenarchaeota, HWCG IV 14.514.5Desulfurococcales Thermoproteales Deeply-branching Thermoprotei Other Thermoprotei HWCG I HWCG IV Crenarchaeota, Other Other Crenarchaeota Hadesarchaea Hadesarchaea 24.5Me thanomicrobia Thermoplasmata Depth (cm) Depth 24.5Other Euryarchaeota Lokiarchaeota Alpha Methanomicrobia Lokiarchaeota Beta Pacearchaeota Yellow Mat MGI Thaumarchaeota Uncl. Thaumarchaeota Thermoplasmata 34.5Other Thaumarchaeota Cathedral HillThorarchaeota 34.5Uncl. Asgard Phylum I Other Asgardarchaeota Other Euryarchaeota Wo esearchaeota Other Archaea 0% 20% 40% 60% 80% 100% Lokiarchaeota Alpha Aenigmarchaeota Aigarchaeota Lokiarchaeota Beta 0.50.5C3 MCG-1 MCG-2 MCG-3 Pacearchaeota MCG-4 MCG-8 MGI Thaumarchaeota 4.5MCG-9 MCG-16 Crenarchaeota 4.5 MCG-17 MCG-21 Uncl. Thaumarchaeota MCG-22 MCG-23 MCG-27 MCG-28 Other Thaumarchaeota 14.5MCG-29 MCG-30 Thorarchaeota 14.5 Unclassified Other Desulfurococcales Thermoproteales Uncl. Asgard Phylum I 19.5Deeply-branching Thermoprotei Other Thermoprotei Other Asgardarchaeota

Depth (cm) Depth 19.5 HWCG I HWCG IV Other Crenarchaeota Hadesarchaea Woesearchaeota Me thanomicrobia Thermoplasmata 24.5Other Euryarchaeota Orange MatLokiarchaeota Alpha Other Archaea Cathedral Hill 24.5 Lokiarchaeota Beta Pacearchaeota MGI Thaumarchaeota Uncl. Thaumarchaeota 0%Other Thaumarchaeota20% 40% 60%Thorarchaeota80% 100%Euryarchaeota Uncl. Asgard Phylum I Other Asgardarchaeota 0.5AenigmarchaeotaWo esearchaeota AigarchaeotaOther Archaea 0.5C3 MCG-1 MCG-2 MCG-3 MCG-4 MCG-8 4.5MCG-9 MCG-16 4.5MCG-17 MCG-21 MCG-22 MCG-23 MCG-27 MCG-28 14.514.5MCG-29 MCG-30 Unclassified Other Lokiarchaeota 19.5Desulfurococcales Thermoproteales 19.5Deeply-branching Thermoprotei Other Thermoprotei HWCG I HWCG IV Other Crenarchaeota Hadesarchaea Depth (cm) Depth 24.5Me thanomicrobia Thermoplasmata 24.5Other Euryarchaeota Lokiarchaeota Alpha Orange Mat Orange Lokiarchaeota Beta Pacearchaeota 29.5MGI Thaumarchaeota Everest MoundUncl. Thaumarchaeota 29.5Other Thaumarchaeota Thorarchaeota Uncl. Asgard Phylum I Other Asgardarchaeota 0%Wo esearchaeota20% 40% Other60% Archaea80% 100%Thaumarchaeota 0.5Aenigmarchaeota Aigarchaeota 0.5C3 MCG-1 MCG-2 MCG-3 2.5MCG-4 MCG-8 2.5MCG-9 MCG-16 4.5MCG-17 MCG-21 4.5MCG-22 MCG-23 MCG-27 MCG-28 5.5MCG-29 MCG-30 5.5Unclassified Other 6.5Desulfurococcales Thermoproteales 6.5Deeply-branching Thermoprotei Other Thermoprotei HWCG I HWCG IV Depth (cm) Depth 7.5Other Crenarchaeota Hadesarchaea 7.5Me thanomicrobia Thermoplasmata Other Euryarchaeota Lokiarchaeota Alpha 9.5Lokiarchaeota Beta Pacearchaeota Everest Mound 9.5MGI Thaumarchaeota Uncl. Thaumarchaeota Other Thaumarchaeota Thorarchaeota 214 Uncl.Aenigmarchaeota Asgard Phylum I OtherAigarchaeota Asgardarchaeota WoC3 esearchaeota OtherMCG-1 Archaea MCG-2 MCG-3 MCG-4 MCG-8 215 SupplementaryMCG-9 Figure 5. Detailed depthMCG-16 profiles of archaeal community structure in the MCG-17 MCG-21 216 (a) NSA andMCG-22 (b) SA, focusing on the dominantMCG-23 classes, orders, and genera for phyla with MCG-27 MCG-28 217 sub-phylumMCG-29 classifications. MCG-30 Unclassified Other Desulfurococcales Thermoproteales Deeply-branching Thermoprotei Other13 Thermoprotei HWCG I HWCG IV Other Crenarchaeota Hadesarchaea Me thanomicrobia Thermoplasmata Other Euryarchaeota Lokiarchaeota Alpha Lokiarchaeota Beta Pacearchaeota MGI Thaumarchaeota Uncl. Thaumarchaeota Other Thaumarchaeota Thorarchaeota Uncl. Asgard Phylum I Other Asgardarchaeota Wo esearchaeota Other Archaea 218 Supplementary Figure S6A, Cren- and Aigarchaeota

LV−Arc28, AM943618, hypersaline lagoon sediment Terrestrial Hot Spring Crenarchaeota (THSC) A 100 SP−AS3−5, JF428802, shrimp pond sediment 100 Guaymas_16S_ZOTU8079 95 Guaymas_16S_ZOTU594 95 Guaymas_16S_ZOTU4307 99 Guaymas_16S_ZOTU6954 64 R−UA−92, JQ258685, tropical marine sedimen t Guaymas_16S_ZOTU976 100 MD3059K−14 (THSCG), GQ927605, tropical ocean sediment 100 Mothra_A2−18,, GQ267151, mothra sediment 100 Guaymas_16S_ZOTU6198 100 Guaymas_16S_ZOTU8505 100 Guaymas_16S_ZOTU9567 77 Guaymas_16S_ZOTU2899 79 Guaymas_16S_ZOTU2463 95 Guaymas_16S_ZOTU2665 100 Guaymas_16S_ZOTU416 100 Guaymas_16S_ZOTU934 100 Guaymas_16S_ZOTU668 100 Guaymas_16S_ZOTU863 ‘HWCG V’ 95 Papm3A51, AB213102, hydrothermal fluid 100 VulcPIw.73, DQ300340, geothermal well 95 FS448_11A,, HQ635908, deep−sea hydrothermal vent Guaymas_16S_ZOTU356 79 Guaymas_16S_ZOTU378 58 AB019733, deep−sea hydrothermal vent sediment 100 20b−7, AJ299162, marine sediment 68 Guaymas_16S_ZOTU5245 87 Fhm3A36, AB293219, hydrothermal sulfide structure 87 p816_a_3.80, AB302037, hydrothermal sediment 99 p816_a_3.18, AJ299151, hydrothermal sediment 100 FZ2bA108_(HWCG_IV), AY166111, black smoker HWCG IV

100 FZ2bA12_(HWCG_IV), AY166102, black smoker Crenarchaeota 95 PNG_TB_4A2.5H2_A64, EF100627, hydrothermal sediment 95 Guaymas_16S_ZOTU1408 Guaymas_16S_ZOTU112 Exp331_INH_117A_06, AB825726, hydrothermal subseafloor sediment 56 P10a5RS63, FN666143, hot spring 77 OUTa18, DQ228584, sulfide microbial incubation 100 Pbsc2A26, AB293228, hydrothermal sulfide 95 Exp331_INH_117A_21, AB825730, hydrothermal subseafloor sediment 90 TVG11AR05, GQ848428, hydrothermal vent sediment 95 4489RNA90, JF937790, hydrothermal sediment 95 Guaymas_16S_ZOTU1406 100 Guaymas_16S_ZOTU2570 100 Guaymas_16S_ZOTU8350 100 Guaymas_16S_ZOTU1135 95 Exp331_INH_117A_02, AB825725, hydrothermal subseafloor sediment 100 Guaymas_16S_ZOTU139 ‘SSHG’ 95 Guaymas_16S_ZOTU2983,

95 Guaymas_16S_ZOTU8093 HWCG I 77 Guaymas_16S_ZOTU3542 100 Guaymas_16S_ZOTU8865 100 Guaymas_16S_ZOTU6711 100 ERBTW20082009OTU096, KF923316, fumarolic soil 80 SUBT−13_(HWCG_II), AF361212, terrestrial hot spring 100 TOTO−A6−15_(HWCG_II), AB167488, hydrothermal vent 100 p816_a_1.46, AB302013, hydrothermal sediment 85 Guaymas_16S_ZOTU1358 95 ARC_OTU_3, KP091005, hydrothermal seep sediment 99 Guaymas_16S_ZOTU2804 100 BY127, EF156492, terrestrial hot spring mat 100 SK859, DQ834111, terrestrial hot spring 95 pUWA36, AB007308, hot water environment 85 BY168 (Terr. Hot Spring Grp (T, EF156533, terrestrial hot spring microbial mat pISA9, AB019732, hydrothermal vent sediment 95 Exp331_INH_114U_11, AB825710, hydrothermal subseafloor sediment 100 pIR3AC09, AY354121, hydrothermal vent sediment 100 Exp331_INH_114U_82, AB825718, hydrothermal subseafloor sediment 60 Exp331_INH_117A_15, AB825729, hydrothermal subseafloor sediment 95 Exp331_INH_117A_08, AB825727, hydrothermal subseafloor sediment F2apm1A20, AB284822, hydrothermal fluid, 93 WT−2007−339−22−1, KT453565, sub lacustrine thermal vent 78 1301A09_162, JX194308, borehole fluid 53 EnqDayyy, KC004007, subseafloor sediment 100 A257−45, FN554068, hydrothermal vent 53 LX3−A101, KP784731, hot spring from geothermal field 96 Desulfurococcus_kamchatkensis, EU167539 90 Sulfolobus acidocaldarius, NR_043400, terrestrial hot spring Thermo- 88 Thermofilum pendens, NR_029214, solfataric hot spring 53 Vulcanisaeta distributa, NR_040876, terrestrial hot spring protei W8A−3, KM221275, hot spring 4489RNA39, JF937779 , hydrothermal sediment ARC_OTU_53, KP091028, hydrothermal seep 93 SSE_L4_D01_(Aigar,_G4), EU635909, hot spring 95 Guaymas_16S_ZOTU905 100 p816_a_3.39_(HWCG_I), AB302032, hydrothermal sediment lhc2925_(Aigar,_G5), KT028698, hot spring 100 YNP_ObP_A17, DQ243752, terrestrial hot spring lhc1936, KT028708, hot spring 50 Candidatus Caldiarchaeum_subterraneum, JN881568, hydrothermal vent biofilm 98 HL1env.02, EU239990, hot spring sediment Candidatus 98 YNP_SBC_BP4_A45, HM448111, terrestrial hot spring 96 GBS_L2_A09, DQ490009, hot spring Aigarchaeota 98 YNP_SBC_QL1_A9, HM448141, terrestrial hot spring 100 YNP_ObP_A10, DQ243744, hot spring 100 LHC3_L2_F12, EU924220, hot spring 95 SSW_L4_G06, EU635916, hot spring Guaymas_16S_ZOTU2387 219 0.10 220 221

222 Supplementary Figure S6B, Crenarchaeotal Class Thermoprotei only

FZ2aA55, AY166115, black smoker chimney Guaymas_16S_ZOTU2512 53 Guaymas_16S_ZOTU116 B 53 53 a Guaymas_16S_ZOTU1212 Guaymas_16S_ZOTU63 53 Guaymas_16S_ZOTU531 53 53

53 Guaymas_16S_ZOTU3356 Thermoproteales Guaymas_16S_ZOTU135 Thermo- Guaymas_16S_ZOTU12 Guaymas_16S_ZOTU166 filaceae 53 53 53 53 Guaymas_16S_ZOTU1351 53 Guaymas_16S_ZOTU5522 Thermofilum n Thermofilum pendens

Thermoprotei Guaymas_16S_ZOTU22 s Guaymas_16S_ZOTU3216 y s Guaymas_16S_ZOTU1533 y i Pyrobaculum neutrophilum 21 Pyrobaculum aerophilum Pyrobaculumferrireducens Pyrobaculum igneiluti Pyrobaculum islandicum Pyrobaculum Thermoproteus neutrophilus Thermoproteus Thermoproteus Thermo- 51 Thermoproteus l Thermoproteus tenax Thermoproteus thermophilus prote- Vulcanisaeta distributa Caldivirga maquilingensis aceae Thermocladium modestius 66 Sulfolobus solfataricus Sulfolobus yangmingensis Sulfolobus acidocaldarius Sulfuracidex metallicus Sulfolobus Sulfolobales 53 53 Caldisphaera_draconis Caldisphaera lagunensis Acidilobus saccharovorans Acidilobales Acidilobus sulfurireducens Acidilobus aceticus Desulfurococcus kamchatkensis Desulfurococcus amyloticus Desulfurococcus fermentans Desulfurococcus mobilis Desulfurococcus Thermosphaera aggregans Staphylothermus achaiicus Staphylothermus marinus 53 Stetteria hydrogenophila Desulfuro- 52 Thermogladius cellulolyticus coccaceae Ignicoccus islandicus Ignicoccus pacificus Aeropyrum camini y Aeropyrum pernix Thermodiscus maritimus Desulfuro- Desulfurococcus Fervidicoccus fontis Fervidi- coccales coccaceae Guaymas_16S_ZOTU1223 Pyrodictium abyssi Pyrodictiaceae Pyrodictium brockii 52 Guaymas_16S_ZOTU652 53 Guaymas_16S_ZOTU1211 Deeply-branching 53 63 Thermoprotei 53 Caldiarchaeum subterraneum 223 224 225

226 Previous 2 pages 227 Supplementary Figure 6. Phylogenetic trees of (a) Terrestrial Hot Spring Crenarchaeota 228 (THSC) and Hot Water Crenarchaeote Group I (HWCG I) and Aigarchaeota, and (b) the 229 crenarchaeotal class Thermoprotei. Trees are based on a manually optimized archaeal 16S 230 rRNA gene alignment and were constructed by ARB Neighbor-Joining using Jukes-Cantor 231 Correction combined with a column filter that excluded insertions and hypervariable regions. 232 Subsurface Hydrothermal Group (SSHG) and Deeply-Branching Thermoprotei are newly 233 classified groups within the HWCG I and Thermoprotei, respectively. Bootstrap values 234 (1,000 repetitions) of ≥50% are shown at branch nodes. 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 16

3A025, EF203529, harbor sediment 92 YS16Ac31, AB329761, hydrothermal deposit MCG-12 51 60 67 MCG-11 62 52 MCG-9 79 MCG-3 2 59 80 MCG-2 38 23 88 9 MCG-8 97 28 MCG-19 6 100 57 MCG-4 1 93 90 90 90 MCG-27 79 1 90 60 90 19 BA1_ba1_80 subgroup 99 95 BA2_ba2_11 subgroup 100 8 9 56 73 73 MCG-5 70 91 99 93 MCG-6 73 58 90 MCG-1 90 90 100 52 93 91 100 MCG-1b 82 82 68 MCG-10 90 99 0 56 5 98 MCG-28 35 100 38 MCG-13 60 90 5 100 90 100 MCG-24 61 90 89 9 MCG-14 90 1 57 35 90 100 MCG-26 90 90 95 79 MCG-17 8 92 59 MCG-16 15 100 77 3 96 99 MCG-25 100 99 82 5 100 93 62 0 MCG-29 100 100 100 MCG-21 31 57 98 YS16Ac22, AB329759, hydrothermal deposit MCG-22 30 93 99 YS18As31, AB329810, hydrothermal deposit 100 90 MCG-23 81 8 90 90 82 90 90 100 MCG-30 90 90 90 C3 (MCG-15) 260 0.10 261 Supplementary Figure 7. Phylogenetic tree highlighting novel Bathyarchaeota subgroups 262 (MCG-24 through -30), along with their closest relatives. The tree was produced by ARB 263 Neighbor-Joining using Jukes-Cantor Correction and is based on a manually optimized 264 archaeal 16S rRNA gene alignment with a column filter to exclude insertions and 265 hypervariable regions. Note: C3 are also known as MCG-15. Bootstrap values (1,000 266 repetitions) of ≥50% are indicated at branch nodes. 17

Bacteria:archaea ratio VS environmental variables

-0.34 100 100 y = 3.7695x 100 -0.002x tio 100 a 100 b 100 c y = 2.481e y = 25.13x-1.111 ra R² = 0.3885 R² = 0.023 R² = 0.6746 1010 1010 1010

chaea 1 1 1 r 1 1 1 ia:a r 0.10.1 0.10.1 0.10.1 e t c 0.01 0.01 0.01

Ba 0.01 0.01 0.01 0 220 440 660 8080 100100 0 11000.00 2000.000 33000.00 4400.00 55000.00 6000.00 0 100100 220000 330000 44000 55000 Temperature (°C) Temperature gradient (°C m-1) Depth (cmbsf)

-1.171 -0.299 y = 14.003x 0.215 y = 0.6219x

tio y = 1.4072x 100 100 100 R² = 0.2996 100 100 R² = 0.3006 100 ra d e f R² = 0.0792 1010 1010 1010 chaea r 1 11 1 ia:a r

e 0.10.1 0.10.1 0.10.1 t c

Ba 0.010.01 0.010.01 0.010.01 00 5 110 115 220 2255 3030 3535 00 1 1 22 33 44 5 5 66 0.0 55.0 1100.0 1155.0 2200.0 25.0 30.0 Sulfate (mM) Methane (mM) DIC (mM)

100100 100 -0.276 100

tio y = 2.0175x g y = 2.8486x-0.311 h i y = 1.363x-0.15 ra R² = 0.1535 10 R² = 0.1342 1010 10 R² = 0.0257

chaea 11

r 1 1 ia:a r 0.1 0.10.1

e 0.1 t c

0.010.01 Ba 0.01 0.01 0.0 11000.0 22000.0 330000.0 44000.0 55000.0 660000.0 77000.0 88000.0 00.0 5050.0 100100.0 150150.0 220000.0 250250.0 33000.0 0.0 2020.0 4040.0 6060.0 8080.0 100100.0 120120.0 140140.0 Acetate (µM) Formate (µM) Propionate (µM)

y = 0.7458x0.7226 y = 9.9911x1.5604 R² = 0.0366 y = 7007.5x-3.63 tio 100 j k 100 l y = 0.7458x0.7226 100 R² = 0.262

ra R² = 0.4259 R² = 0.0366 10 10 10 chaea r 1 1 1 ia:a r

e 0.1 0.1 t 0.1 c

Ba 0.01 0.01 0.01 0.0 2.0 4.0 6.0 8.0 1010.0 1212.0 1414.0 1616.0 1818.0 0.00 0.20.20 0.40.40 0.60.60 0.80.80 1.01.00 0.0 5.0 1010.0 1515.0 2020.0 2525.0 3030.0 TOC (%) TN (%) C/N

m Sites 100 y = 8E-05e-0.475x Seep area (SA) Non-seep area (NSA) Controls R² = 0.0417 Cathedral Hill GC10 GC13 (NSA) 10 Everest Mound GC09 Cold site (SA) 1 Yellow Mat MUC02 (NSA) Orange Mat MUC12 (NSA) 0.1

0.01 -25 -24 -23 -22 -21 -20 -19 -18 -17 δ13C-TOC (‰) 267 268 269 Supplementary Figure 8. Relationships between BARs and environmental variables. 270 Environmental variables include (a) temperature, (b) temperature gradient, and (c) sediment 271 depth, concentrations of (d) sulfate, (e) methane, (f) dissolved inorganic carbon (DIC), (g) 272 acetate, (h) formate, and (i) propionate, as well as (j) total organic carbon (TOC), (k) total 273 nitrogen (TN), (l) TOC/TN (C/N), and (m) δ13C-TOC. Trendlines reflect best-fit functions in 274 Microsoft Excel, which was also used to calculate coefficient of determination (R2) values.

100 B2/A3 y = 13.472e-0.051x R² = 0.730

B1/A3 y = 13.906e-0.051x R² = 0.693 10 B2/A1 y = 13.946e-0.057x R² = 0.695

B1/A1 y = 13.511e-0.057x R² = 0.736 1

0.1

Bacteria:Archaea ratio

B2/A2 y = 3.7328e-0.043x R² = 0.699

0.01 B1/A2 y = 3.6163e-0.043x R² = 0.666 0 10 20 40 60 80 100 Temperature (°C)

Sites Depth (cmbsf) Seep area (HSA) Non-seep area (HNSA) 10 100 200 300 400 Controls Primers used

B1: Bac806F_mod2/Bac908R B1 / A1 B2 / A1 B2: Bac908F_mod/Bac1075R B1 / A2 B2 / A2 A1: Arc806F/Arc958R A2: Arch915F_mod/Arch1059R B1 / A3 B2 / A3 A3: Arc806F / Arc915 R mod 275 276 277 278 Supplementary Figure 9. Bacteria-to-Archaea 16S rRNA gene copy ratios (BARs) vs. 279 temperature. Calculated for different bacterial and archaeal qPCR primer combinations on 280 a subset of samples. The best-fit trendline in all cases follows an exponential function that 281 was fitted in Microsoft Excel, which was also used to calculate the coefficient of 282 determination (R2) values. Results obtained with the primer pairs B2 and A2 on all samples 283 are shown in Figures 1 and 2 and in Supplementary Figure S9. 284 285 286

Bacteria; ZOTU - NMDS-Bray Bacteria; Class - NMDS-Bray a 2 b 2 ia

r 1 2

e 1 1 t 2 c NMDS2 NMDS2 0

Ba 0 ) 3 C e (° r

3 −1 tu

−2 ra −2 0 2 −2 −1 0 1 2 NMDS1 NMDS1 empe T

Archaea; ZOTU - NMDS-Bray Archaea; Class - NMDS-Bray 75

2 50 c 2 1 d 25 1 1 3 0 0 chaea NMDS2 NMDS2 r

A 1 3 −1 −1 2

−2

−2 −1 0 1 2 −1 0 1 NMDS1 NMDS1 Sites Seep Area (1) Non Seep Area (2) Controls (3)

Yellow Mat GC09 Cold site (SA) Cathedral Hill GC10 MUC02 (NSA) Orange Mat MUC12 (NSA) Everest Mound GC13 (NSA) 287 288 289 Supplementary Figure 10. Non-metric multi-dimensional scaling (NMDS) plots calculated 290 with Bray-Curtis algorithms of ZOTU- and class-level bacterial and archaeal community 291 structure across all sites. Cold control sites from both locations are grouped together in the 292 legend for easier viewing. 293 294 295 296 297

a Thermotogae Thermodesulfobacteria Caldiserica Acetothermia Sulfurimonas Sulfurovum Bdellovibrionales Parcubacteria Aerophobetes Atribacteria Verrucomicrobia Desulfurellales Methylococcales Thiotrichales Clostridiales Sphingobacteriia Desulfobacterales Bacteroidia Xanthomonadales Anaerolineae Holophagae Alpha-Proteobacteria BD7.8 marine group NB1-j Omnitrophica Dehalococcoidia MSB-5B2 Aminicenantes Elusimicrobia Desulfarculales Planctomycetes OC DIC C/N T mate TN % r OC % adient r Sulfate o C- T Acetate F 13 Methane T-g Propionate Temperature

b Aigarchaeota Desulfurococcales Thermoproteales Other M. sarcinales MCG-29 MCG-23 HWCG-IV MCG-21 MCG-28 MCG-30 Deeply branching Thermoprotei HWCG-I MCG-16 MCG-22 MCG-27 MCG-3 MCG-9 ANME-2 Methanomicrobia ANME-1-Gba ANME-1-AT Hadesarchaea MCG-4 MCG-1 MCG-8 C3 MCG-17 MCG-2 ANME-1a-b Uncl. M. microbia Lokiarchaeota (Alpha Subgroup) Thermoplasmata Woesearchaeota Lokiarchaeota (Beta Subgroup) MG-I Thaumarchaeota Thorarchaeota Aenigmarchaeota OC DIC C/N T mate TN % r OC % adient r Sulfate o C- T Acetate F 13 Methane T-g Propionate Temperature 0 1

298 299 Supplementary Figure 11. Heat map showing correlations between relative abundances 300 of key bacterial and archaeal groups and geochemical variables based on Spearman’s rank 301 correlation coefficients. Only significant (p<0.05) correlations are colored.

302 Supplementary References

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