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Supplementary Information for 1 an Ecological Basis for Dual Genetic 1 Supplementary Information for 2 An ecological basis for dual genetic code expansion in marine Deltaproteobacteria 3 Kivenson, V., Paul, BG., Valentine, DL. 4 5 1) Figures S1-S6 6 2) Tables S1-S10 7 SI Figures 8 9 10 Figure S1) Location of the barrel dumpsite and the study site off of the coast of California. 11 12 13 14 Figure S2) Apparent infauna burrows visible in the sediment near a barrel, and absent between 15 the barrel and the microbial mat ring. 16 1 17 18 Figure S3) Sampling schematic describing the site and sequencing methods used for microbial 19 analyses. 2 20 21 Figure S4) Inverse Simpsons index for diversity (bg1, bg2: background 1 and 2; nonring 16 and 22 31: outside of the microbial ring at bbl 16 and bbl 31; RING16: microbial mat at bbl 16; 23 RING31: microbial mat at bbl 31). 3 24 25 Fig S5) Phylogeny of PylB reconstructed using FastTree. This tree shows a horizontal view of 26 the tree from Fig. 3A. Branch support values are shown at all nodes across the tree. Major clades 27 are highlighted according to taxonomic classification, in either red (Euryarchaeota), blue 28 (Firmicutes), or green (Deltaproteobacteria). 4 29 30 Fig S6) Phylogeny of PylB reconstructed using RaxML. Branch support values are shown at all 31 nodes across the tree. Major clades are highlighted according to taxonomic classification, in 32 either red (Euryarchaeota), blue (Firmicutes), or green (Deltaproteobacteria). 5 33 SI Tables 34 35 Table S1. Percent abundance of top twenty taxa by location with ASV number as indicated. Nonring Nonring Ring Ring ASV bg1 bg2 bbl-16 bbl-31 bbl-16 bbl- 31 1 0 0 0 0 8 16 2 3 0 3 2 0 0 3 1 3 0 1 0 2 4 0 0 0 0 1 4 5 0 0 1 0 4 1 6 0 0 0 0 0 4 7 0 1 0 0 2 2 8 1 0 2 2 0 0 9 0 0 0 0 2 2 10 1 1 1 1 0 1 11 1 1 1 1 1 0 12 0 0 0 0 1 1 13 0 0 0 0 1 2 14 1 1 1 1 0 0 15 1 1 0 1 0 0 16 0 1 0 1 0 1 17 0 0 0 0 2 1 18 0 0 0 0 0 2 19 0 1 0 0 0 0 20 1 0 1 0 0 0 36 6 37 Table S1 continued. Matching taxonomy for each ASV of the top twenty taxa. The phylum, 38 class, family, and genus are shown. NA indicates unclassified at the given taxonomic level. AS V Phylum Class Family Genus Desulfobacteracea 1 Proteobacteria Deltaproteobacteria e Desulfobacula Gammaproteobacteri 2 Proteobacteria a NA NA 3 Lokiarchaeota NA NA NA 4 Latescibacteria NA NA NA 5 Planctomycetes Phycisphaerae NA NA 6 Bacteroidetes Bacteroidia Marinilabiaceae NA 7 Lokiarchaeota NA NA NA 8 Planctomycetes Planctomycetacia Brocadiaceae Candidatus_Scalindua 9 Acetothermia NA NA NA 10 Lokiarchaeota NA NA NA 11 Lokiarchaeota NA NA NA 12 Chloroflexi Anaerolineae Anaerolineaceae NA 13 Spirochaetae Spirochaetes Spirochaetaceae Spirochaeta_2 Desulfobacteracea Sva0081_sediment_gro 14 Proteobacteria Deltaproteobacteria e up Bacteroidetes_BD2- 15 Bacteroidetes 2 NA NA 16 Chloroflexi Anaerolineae Anaerolineaceae NA 17 Chloroflexi Anaerolineae Anaerolineaceae NA 18 Lokiarchaeota NA NA NA 19 Chloroflexi Anaerolineae Anaerolineaceae NA 20 Chloroflexi Anaerolineae Anaerolineaceae NA 39 7 40 41 Table S1 continued. The corresponding 16S rRNA sequence for each ASV. ASV # Corresponding 16S rRNA nucleotide sequence CACGGGGGGCGCAAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGT AGGCGGTCTTGTCGGTCAGATGTGAAAGCCCAGGGCTCAACCCTGGACG TGCATTTGAAACAGCAAGACTTGAGTACGGGAGAGGAAAGCGGAATTCC TGGTGTAGAGGTGAAATTCGTAGATATCAGGAGGAACACCGATGGCGAA GGCAGCTTTCTGGACCGATACTGACGCTGAGGCGCGAAGGCGTGGGTAG 1 CGAACAGG TACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCTCGT AGGCGGTTTGTTAAGTCGGATGTGAAAGCCCCGGGCTCAACCTGGGAAC TGCATTCGATACTGGCAAACTAGAGTATAGAAGAGGCAAGTGGAATTCC GGGTGTAGCGGTGAAATGCGTAGATATCCGGAGGAACATCAGTGGCGAA GGCGACTTGCTGGTCTAATACTGACGCTGAGGAGCGAAAGCGTGGGGAG 2 CAAACGGG AACCAGCTCTTCAAGTGGTCGGGATAATTATTGGGCTTAAAGTGTCCGTA GCCGGTTTAGTAAGTTCCTGGTAAAATCGGGTAGCTTAACTATCTATATG CTAGGAATACTACTATACTAGAGGGCGGGAGAGGTCTGAGGTACTACAG GGGTAGGGGTGAAATCTTATAATCCTTGTTGGACCACCAGTGGCGAAGG CGTCAGACTGGAACGCGCCTGACGGTGAGGGACGAAAGCCAGGGGAGC 3 GAACCGG TACGGAGGGTGCAAGCGTTGTTCGGATTTACTGGGTATAAAGGGTGCGC AGGCGGCCTGATAAGTCAGGGGTGAAATATGACGGCTCAACCGTCAAAC TGCCCCTGAAACTGCCAGGCTTGAGTCCGAGAGAGGTAGGTGGAATTCC AGGTGTAGCGGTGAAATGCGTAAATATCTGGAGGAACACCGGTGGCGAA GGCGGCCTACTGGCTCGGAACTGACGCTCAGGCACGAAAGCTAGGGGAG 4 CGAACGGG TACGAAGGTGGCAAGCGTTGTTCGGAATCACTGGGCTTAAAGCGCACGC AGGCGGAAAAGAAAGTGTGGAGTGAAATCCCTCGGCTTAACCGGGGAA CTGCTCTGCAAACTACTTTTCTTGAGGCAAGTAGGGGTACATGGAACTCT TGGTGGAGCGGTGGAATGCGTAGATATCAAGAGGAACGCCGATGGTGA AGACAGTGTACTGGGCTTGTCCTGACGCTGAGGTGCGAAAGCGTGGGGA 5 GCGAACGGG TACGGAGGGTGCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGT AGGCGGAATATTAAGTCAGTGGTGAAATCCTGTGGCTCAACCATAGAAT TGCCATTGATACTGATATTCTTGAATGCAGTTGAGGCAGGCGGAATGTGT AATGTAGCGGTGAAATGCTTAGATATTACACAGAACACCGATTGCGAAG GCAGCTTGCTAAACTGTGATTGACGCTGATGCACGAAAGCGTGGGGAGC 6 GAACAGG AACCAGCTCTTCAAGTGGTCGGGAATATTATTGGGCTTAAAGTGTCCGTA GCCGGTTTGGTAAGTTCCTGGTTAAATCTGGCAGCTTAACTGTCAGTCAG CTAGGAATACTACTTTACTAGAGGGTGGGAAAGGTTTGAGGTACTCCAG GGGTAGCGGTGAAATGCGATAATCCTTGGGGGACCACCAGTGGCGAAGG CGTCAGACTGGAACACGCCTGACGGTGAGGGACGAAAGCCAGGGGAGC 7 GAACGGG 8 42 Table S1 continued. The corresponding 16S rRNA sequence for each ASV. ASV # Corresponding 16S rRNA nucleotide sequence TACAGAGGTGGCAAGCGTTGTTCGGAATTATTGGGCGTAAAGAGCACGT AGGTGGGTTTGTAAGTCAGATGTGAAAGCCTTCTGTTCAACGGAAGAAT TGCATCTGAAACTGCGAGTCTTGAGTGTAGGAGGGGAGAATGGAACTTC TGGTGGAGCGGTGAAATGCGTAGATATCAGAAGGAACGCCGGCGGCGA AAGCGATTCTCTGGCCTATTACTGACACTCAGTGTGCGAAAGCTAGGGG 8 AGCAAACGGG GACGAGGGATGCAAGCGTTATCCGGAATTACTGGGCGTAAAGGACGTCT AGGCGGTTGGATAAGTCATTTGTGAAATCCCAGGGCTTAACCCTGGAAG GTCTTGTGATACTGTCCGGCTTGGGTGTAGGAGAGGAGAGCGGAACTCA CAGAGTAGCGGTGGAATGCGTAGATACTGTGAGGTACCCCGATGGCGA AGGCAGCTCTCTGGCCTATTACCGACGCTGAAGCGTGAAAGCGTGGGGA 9 GCAAAGGGG AACCAGCTCTTCAAGTGGTCGGGATTATTATTGGGCTTAAAGTGTTCGTA GCCTGTTTAGTAAGTTCTTGGTTAAATCGGATAGCTTAACTATCTGTCTG CTAAGAATACTACTATACTAGGGGGCGGGAGAGGTCTGAGGTACTCCAG GGGTAGCGGTGAAATGCTATAATCCTTGGGGGACCACCAGTGGCGAAG GCGTCAGACTGGAACGCGCCCGACGGTGAGGGACGAAAGCCAGGGGAG 10 CGAACCGG AACCAGCTCTTCAAGTGGTCGGGATTATTATTGGGCTTAAAGTGTTCGTA GCCTGTTTAGTAAGTTCCTGGTTAAATCGGATAGCTTAACTATCTGTCTG CTAGGAATACTACTATACTAGGGGGCGGGAGAGGTCTGAGGTACTCCAG GGGTAGCGGTGAAATGCTATAATCCTTGGGGGACCACCAGTGGCGAAG GCGTCAGACTGGAACGCGCCCGACGGTGAGGGACGAAAGCCAGGGGAG 11 CGAACCGG TACGTAGGAGGCGAGCGTTATCCGGATTTATTGGGCGTAAAGCGCGTGC AGGTGGTTTGGTAAGTTGGGTATGAAATCTTCTGGCTTAACTAGGAGAG GTTGCTCAAAACTGCCAGACTAGAGGACGATAGAGGAAGGTGGAATTC CCGGTGTAGTAGTGAAATGCGTAGATATCGGGAGGAACACCAGTGGCG AAGGCGGCCTTCTGGGTCGTTCCTGACACTAAGACGCGAAAGCATGGGT 12 AGCAAACGGG CACGTATGGGGCGAGCGTTGTTCGGAATCATTGGGCGTAAAGGGCGCGC AGGCGGTTATATAAGCCTGGTGTGAAATACTGCAGCTCAACTGCAGAAC CGCACTGGGAACTGTATGACTGGAGTTCAAGAGGGGAAGCTGGAATTCC TGGTGTAGGGGTGAAATCTGTAGATATCAGGAAGAACATCAGTGGCGA AGGCGAGCTTCTGGCTATGAACTGACGCTGAGGCGCGAAAGCGTGGGG 13 AGCAAACAGG 43 9 44 Table S1 continued. The corresponding 16S rRNA sequence for each ASV. ASV # Corresponding 16S rRNA nucleotide sequence CACGGGGGGTGCAAGCGTTATTCGGAATCACTGGGCGTAAAGAGCGCGT AGGCGGTCTCTTAAGTCAGATGTGAAAGCCCGGGGCTCAACCCCGGAAG TGCATTTGAAACGAAGGGACTTGAGTATGGGAGAGGGAAGTGGAATTCC TGGTGTAGCGGTGAAATGCGTAGATATCAGGAGGAACACCGGTGGCGAT GGCGACTTCCTGGACCAATACTGACGCTGAGGCGCGAAGGCGTGGGGAG 14 CAAACAGG TACGGAGGATGCAAGCGTTATCCGGATTTATTGGGTTTAAAGGGTACGTA GGCGGAAAATTAAGTCAGTAGTGAAATCCTGCAGCTTAACTGTAGAACT GTTATTGATACTGGTTTTCTTGAATATAGTTGAGGTAGGCGGAATGTGTA ATGTAGCGGTGAAATGCTTAGATATTACACAGAACACCGATTGCGAAGG CAGCTTACTAAGCTATGATTGACGCTGAGGTACGAAAGCGTGGGGAGCG 15 AACAGG AACGTAGGATCCGAGCGTTATCCGAATTCACTGGGCGTAAAGCGCGTGT AGGCGGTTCGGTAAGTTGGATGTGAAAGCTCCCGGCTCAACTGGGAGAG GACGTTCAAAACTGTTGGACTAGAGGGCGGAAGAGGGAGGTGGAATTCC CGGTGTAGTGGTGAAATGCGTAGATATCGGGAGGAACACCAGTGGCGAA GGCGGCCTCCTGGGCCGCACCTGACGCTCAGACGCGAAAGCTAGGGTAG 16 CAAACGGG TACGTAGGAGGCAAGCGTTATCCGGATTCATTGGGCGTAAAGCGCGTGC AGGTGGTTTGGTAAGTTGGGTATGAAATCTTCTGGCTTAACTAGGAGAGG TTGCTCAAAACTGTCAGACTAGAGGACGATAGAGGAAGGTGGAATTCCC GGTGTAGTAGTGAAATGCGTAGATATCGGGAGGAACACCAGTGGCGAAG GCGGCCTTCTGGGTCGTTCCTGACACTAAGACGCGAAAGCATGGGTAGC 17 AAACGGG AACCAGCTCTTCAAGTGGTCGGGAATATTATTGGGCTTAAAGTGTCCGTA GCCGGTTTGAACAGTTCCTGGTTAAATCTGGTAGCTTAACTATCAGTCAG CTAGGAATACTATCTTACTAGAGGGTGGGAAAGGCTTGGGGTACTCCGG GGGTAGCGGTGAAATGCGATAATCCTCGGGGGACCACCAGTGGCGAAGG CGCCAAGCTGGAACACGCCTGACGGTGAGGGACGAAAGCCAGGGGAGC 18 GAACGGG CACGTAGGATCCGAGCGTTATCCGAATTTACTGGGCGTAAAGCGCGTGTA GGCGGCCGGGTAAGTTGGACGTGAAAGCTCCTGGCTCAACTAGGAGAGG TCGTTCAAAACTGCCTGGCTAGAGGGCGACAGAGGGAGGTGGAATTCCC GGTGTAGTGGTGAAATGCGTAGATATCGGGAGGAACACCAGTGGCGAAG GCGGCCTCCTGGGTCGCCCCTGACGCTCAGACGCGAAAGCTAGGGGAGC 19 AAACGGG GACATAGGAGGCGAGCGTTATCCGGATTTATTGGGCGTAAAGTGCGTTG AGGCGGCATTGTAAGTTGGACGTGAAAGCTCCCGGCTTAACTGGGAGAG GTCGTTCAATACTGCAAGGCTAGAGGGCAGTAGAGGGGGGTGGAATTCC CGGTGTAGTGGTGAAATGCGTAGATATCGGGAGGAACACCAGTGGCGAA GGCGGCCCCCTGGACTGTTACTGACGCTGAAGGCGAAAGCTAGGGTAGC 20 AAACGGG 45 10 46 Table S2. Assembly statistics following whole genome sequencing. Assembly shown was 47 performed with Megahit with standard parameters unless otherwise indicated (with minimum 48 contig length of 1000 bp). Sample name Raw Total Max Mean N50 Most Reconstruc reads assembly contig contig abundant ted file length length length 16S ASV genome(s)? size percent abundance Core 12 10.6 G 400,023 10,690 1,481 1,359 3% No (background) Core 13 9.2 G 9,475,213 34,633 1,364 1,288 3% No (background) Core 18 9.2 G 27,849,371 16,830 1,575 1,506 3% No (outside ring, bbl-31) Core 7 12.6 G 9,734,423 43,818 1,466 1,360 3% No (outside ring, bbl-16) Core 19 15.2 G 106,625,211 167,842 2,185 2,228 16% Yes (ring, bbl-31) Core 8 22 G 126,963,432 331,719 1,972 1,957 8% Yes (ring, bbl-16) 49 11 50 Table S3. Properties and taxonomic identification of the reconstructed genomes. NCBI Taxonomy Gene used Size Mean GC (Order unless for NCBI Bin name Mb Coverage % otherwise specificed) Taxonomy CORE_8_RING_Bin_00001
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