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Supplementary Material Supplementary Material 1 Supplementary Figures and Tables 1.1 Supplementary Figures Deltaproteobacterium PSCGC (2) Deltaproteobacteria Gammaproteobacteria bacterium isolate NP949 Gammaproteobacteria Deltaproteobacteria bacterium spp. (4) Deltaproteobacteria Streptomyces sp. CNQ-509 Actinobacteria candidate division KSB1 bacterium RBG_16_48_16 candidate division KSB1 Dethiobacter alkaliphilus AHT-1 Firmicutes-Clostridia Desulfuromonas sp. DDH-964 Deltaproteobacteria Desulfuromonas spp. (2) Deltaproteobacteria OTU-487 (Deltaproteobacteria bacterium 37-65-8) Spirochaetes bacterium (2) Spirochaetes Methanocella sp. RC-I Methanomicrobia Methanoregula boonei 6A8 Methanomicrobia candidate division OP9 bacterium SCGC AAA255-N14 Atribacteria Methanoregulace aearchaeon JGI M3C4D3-001-G22 Methanomicrobia Methanomicrobia (5) Methanofollis liminatans GKZPZ Methanomicrobia OTU-267 (uncultured microorganism) Candidatus Aminicenantes bacterium RBG_13_59_9 Aminicenantes Methanomassiliicoccus luminyensis B10 Thermoplasmata Anaerolineae bacterium CG_4_9_14_0_8 Chloroflexi Elusimicrobia bacterium GW (2) Elusimicrobia candidate division WOR-3 bacterium SM23_42 Methanomassiliicoccales archaeon RumEn M1 Thermoplasmata OTU-858 (uncultured microorganism) OTU-544 (uncultured bacterium) Theionarchaea archaeon DG-70-1 Theionarchaea Chloroflexi bacterium RIFOXYD12_FULL_57_15 Chloroflexi OTU-201 (uncultured bacterium) OTU-481 (uncultured bacterium) Methanomicrobia (6) Lentisphaerae bacterium (2) Lentisphaerae Fimicutes (5) Bdellovibrionales bacterium GWB1_55_8 Deltaproteobacteria Spirochaetes bacterium (3) Spirochaetes Deltaproteobacteria bacterium (2) OTU-2602 (Deltaproteobacteria bacterium GWA2_54_12) Phycisphaerae bacterium SG8_4 Planctomycetes Deltaproteobacteria bacterium JGI (4) candidate division WOR-1 bacterium RIF (4) Desulfobulbus mediterraneus Deltaproteobacteria Desulfobulbus japonicus Deltaproteobacteria Deltaproteobacteria bacterium MLMS-1 Desulfarculus sp. SPR Deltaproteobacteria Desulfovibrio sp. J2 Deltaproteobacteria OTU-25 (not assigned) Desulfovibrio aespoeensis Aspo-2 Deltaproteobacteria Desulfovibrio desulfuricans ND132 Deltaproteobacteria Desulfococcus spp. (2) Deltaproteobacteria Desulfovibrio sp. isolate SP109 91617 Deltaproteobacteria Desulfobulbus propionicus Deltaproteobacteria OTU-139 (Desulfobulbus propionicus) Composite genome from Trout Bog TBhypo.metabat.2922.v2 unknown Desulfobulbaceae bacterium PR2_D12 Deltaproteobacteria OTU-952 (Desulfobulbus propionicus) Desulfovibrio bizertensis Deltaproteobacteria Desulfofustis glycolicus Deltaproteobacteria Bacteria (33) Lentisphaerae bacterium RIFOXYC12_FULL_60_16 candidate division WOR-1 bacterium RIF (2) Spirochaetes bacterium RBG_16_49_21 Elusimicrobia bacterium RIF spp. (5) Marinilabilia salmonicolor MSL42 Bacteroidetes Bdellovibrionales bacterium RIFOXYC1_FULL_54_43 Deltaproteobacteria Candidatus Raymondbacteria spp. (6) Deltaproteobacteria bacterium RIF spp. (6) Nitrospirae bacterium spp. (3) Nitrospirae bacterium GWC2_56_14 Nitrospirae OTU-30603 (uncultured microorganism) OTU-47 (uncultured microorganism) OTU-126 (Nitrospirae bacterium RBG_19ft_Combo_55_12) OTU-141 (uncultured microorganism) OTU-787 (Nitrospirae bacterium RBG_19ft_Combo_55_12) OTU-110 (uncultured microorganism) OTU-113 (uncultured microorganism) OTU-211 (Nitrospirae bacterium RBG_19ft_Combo_55_12) OTU-13 (uncultured microorganism) Nitrospirae bacterium RBG_19FT_COMBO_55_12 Nitrospirae OTU-477 (uncultured bacterium) OTU-154 (uncultured bacterium) Candidatus Firestonebacteria bacterium RIFOXYA2_FULL_40_8_2713315861 Firestonebacteria Bacteria (10) Lentisphaerae bacterium RIFOXYA12 FULL_48_11 Lentisphaerae Firmicutes (12) Lentisphaerae bacterium GWF2_57_35 Lentisphaerae Spirochaetes bacterium GWB1_36_13 Spirochaetes Dehalococcoides mccartyi DCMB5 Chloroflexi OTU-42 (Deltaproteobacteria bacterium 37-65-8) Syntrophorhabdus aromaticivorans UI Deltaproteobacteria Desulfocurvus vexinensis Deltaproteobacteria Syntrophobacterales bacterium RBG_19FT_COMBO_59_10 Deltaproteobacteria OTU-536 (Syntrophus sp. GWC2_56_31) OTU-2945 (Syntrophus sp. GWC2_56_31) Deltaproteobacteria bacterium RBG_16_58_17 Deltaproteobacteria Syntrophobacterales bacterium GWC2_56_13 Deltaproteobacteria OTU-9666 (uncultured microorganism) Geobacter spp. (3) Deltaproteobacteria OTU-84 (uncultured microorganism) OTU-1349 (uncultured microorganism) Geobacter_sp._M18_Deltaproteobacteria OTU-164 (uncultured microorganism) OTU-266 (uncultured microorganism) OTU-504 (uncultured microorganism) OTU-158 (Desulfuromonadales bacterium GWD2_54_10) OTU-571 (uncultured microorganism) OTU-421 (uncultured microorganism) Syntrophus sp. G20 Deltaproteobacteria Pelobacter seleniigenes Deltaproteobacteria OTU-829 (uncultured microorganism) Composite genome from Trout Bog Hypolimnion pan-assembly TBhypo.metabat.433 unknown Desulfuromonadales bacterium GWC2_61_20 Deltaproteobacteria OTU-837 (Desulfuromonadales bacterium GWC2_61_20) Geobacter daltonii Deltaproteobacteria Geobacter uraniireducens Rf4 Deltaproteobacteria Geobacter argillaceus ATCC BAA-1139 Deltaproteobacteria Geobacter spp. (5) Deltaproteobacteria Geobacter metallireducens (2) Deltaproteobacteria OTU-198 (uncultured microorganism) OTU-288 (uncultured microorganism) OTU-1700 (Geobacter uraniireducens) Geobacter sp. OR-1 Deltaproteobacteria OTU-303 (uncultured microorganism) OTU-227 (uncultured microorganism) Geobacter pickeringii Deltaproteobacteria OTU-230 (uncultured Geobacteraceae bacterium) OTU-408 (uncultured microorganism) OTU-247 (not assigned) Deltaproteobacteria bacterium RIFOXYA12_FULL_58_15 Deltaproteobacteria OTU-237 (uncultured microorganism) Nitrospirae bacterium GWC2_56_14_2 Nitrospirae OTU-6 (Geobacteraceae bacterium enrichment culture clone B2_FE-17) OTU-15299 (uncultured microorganism) OTU-569 (uncultured microorganism) Bacteria (9) Deltaproteobacteria (4) Desulfocarbo indianensis SCBM Deltaproteobacteria Desulfovibrio putealis DSM 16056 Deltaproteobacteria Bacteria ferrireducans S3R1 Deltaproteobacteria OTU-202 (Anaerocolumna jejuensis) Pelobacteraceae bacterium GWC2_58_13 Deltaproteobacteria Geopsychrobacter electrodiphilus Deltaproteobacteria OTU-1099 (Geothermobacter sp EPR-M) Geoalkalibacter ferrihydriticus Deltaproteobacteria Bacteria (37) Candidatus Marispirochaeta associata strain JC231 Spirochaetes OTU-5 (uncultured microorganism) Syntrophus spp. (3) Deltaproteobacteria Desulfovibrio (2) Deltaproteobacteria uncultured Desulfarculaceae 191 Deltaproteobacteria Kosmotoga pacifica SLHLJ1 Thermotogae Aminicenantes (candidate phylum OP8) (12) Atribacteria (candidate phyla OP9) (3) Methanococcoides methylutens Methanomicrobia Thermococci (3) Dehalococcoidia bacterium DG-22 Chloroflexi Dehalococcoidia bacterium (4) Chloroflexi Dehalococcoidia bacterium SCGC AG-205-I13 Chloroflexi Dehalococcoidia bacterium SCGC AG-205-M21 Chloroflexi OMZ North Pacific GP0053648 LPjun08P41300mDRAFT Nitrospina spp. (3) Candidatus Bathyarchaeota archaeon RBG_13_38_9_2 Bathyarchaeota Candidatus Omnitrophica bacterium CG1_02_41_171 Omnitrophica (paralog) Thermosulfurimonas dismutans Thermodesulfobacteria (paralog) 0.6 Supplementary Figure 1. Maximum likelihood tree of hgcA sequences from the updated reference package showing pplacer placements of the top 50 OTUS from New Horizon (NH) hgcA amplicon sequences (Matsen et al., 2010). Nearest neighbor shown in bold. NH hgcA sequences are shown in blue with classification based on the lowest common ancestor (LCA, 50 % cut-off) of protein BLAST hits to NCBI non-redundant protein database (Altschul et al., 1990); LCA calculated in MEGAN v6 (Huson et al., 2016). Miseq amplicons Clones 100 10 Total hits (%) Total 1 0.1 ORNL_HgcAB_uni_F F11 F35 F47 F23 F46 F34 F10 F22 F38 F44 F36 F14 F12 F32 F26 F41 F5 F29 F2 F48 F8 F20 F17 F37 F24 F25 F39 F40 F28 F13 F16 F27 F45 F43 F1 F4 F15 F33 F19 F21 F42 F9 F3 F30 F6 F18 F31 F7 Supplementary Figure 2. Percent occurrence of each of the 48-degenerate forward (ORNL- HgcAB-uni-F) oligonucleotide sequence in the environmental clone hgcAB sequences (Table S2) and Miseq hgcAB amplicons from sites NH. Clones were generated with both the Christensen 2016 primer set (ORNL-HgcAB-uni-F, ORNL-HgcAB-uni-R; n = 119) and the new less degenerate primer set (ORNL-HgcAB-uni-F, ORNL-HgcAB-uni-32R; n = 509). Forward primer binding sites were found in 531 clones of the total 628 clone library. A subset of sequences were pulled from amplicon libraries produced from six separate NH sediment samples using ORNL- HgcAB-uni-F and ORNL-HgcAB-uni-R (n = 87,500) and ORNL-HgcAB-uni-F and ORNL- HgcAB-uni-32R (n = 87,500). Forward primer binding sites were found in 166,984 of the 175,000 amplicon sequences searched. Primers are listed in Table S2. Data is shown on a log scale. Dashed line indicates equal distribution of forward primer sequences across amplicon sequences: 2.08%. Condition A 1) 96 2) 32 3) equi_26 4) equi_32 SP P NH + SP P NH + SP P NH + SP P NH + Condition B 1) 96 2) 32 3) equi_26 4) equi_32 SP P NH + SP P NH + SP P NH + SP P NH + Condition C 1) 96 2) 32 3) equi_26 4) equi_32 SP P NH + SP P NH + SP P NH + SP P NH + Condition D 1) 96 2) 32 3) equi_26 4) equi_32 SP P NH + SP P NH + SP P NH + SP P NH + Condition E 1) 96 2) 32 3) equi_26 4) equi_32 SP P NH + SP P NH + SP P NH + SP P NH + Supplementary Figure 3. Gel image showing PCR amplification of hgcAB from SPRUCE soil (SP), NH periphyton (P), and NH sediment (NH) using PCR conditions A—E (Table S4) with 96 degenerate primer set (ORNL-HgcAB-uni-F, ORNL-HgcAB-uni-R), 32 degenerate primer set (ORNL-HgcAB-uni-F,
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