Supporting Information

Lin et al. 10.1073/pnas.1614654114 SI Materials and Methods three available Nitrospirae MTB (Mcas, Mbav, and Mchi) is the Shotgun Metagenomic Sequencing and Data Analysis. To obtain ratio between the number of genes located in conserved content sufficient DNA for shotgun metagenomic sequencing, multiple and order and the total number of bidirectional best-hits genes displacement amplification was performed using the GenomiPhi between MGCs of HCH-1 and three Nitrospirae MTB. V2 DNA Amplification Kit (GE Healthcare) following the manufacturer’s instructions. Briefly, 1 μL of DNA was used as Implicit Phylogenomic Analysis of Nitrospirae MTB Genes. The global Nitrospirae the template and was mixed with 9 μL of sample buffer. The implicit phylogenetic pattern of the magnetotactic mixed DNA was heated at 95 °C for 3 min and cooled to 4 °C, genomes of HCH-1, Mcas, Mbav, and Mchi was inferred using before incubation at 30 °C for 90 min with 1 μL of enzyme HGTector 0.2.0 (53). Protein sequence similarity search was mixture and 9 μL of reaction buffer. To terminate the reaction, performed using DIAMOND 0.9.7 (66) against a database (gen- the sample was heated at 65 °C for 10 min. For each sample, nine erated by HGTector) that contains one representative per species amplifications were pooled to reduce potential bias. These were from all available nonredundant RefSeq prokaryotic proteomes purified using TIANquik Maxi Purification Kit (Tiangen). (October 2015), plus the MTB proteomes reconstructed in this Shotgun sequencing of metagenomic DNA was performed study. Quality cutoffs for valid hits were E value ≤ 1e-20, percent- using Illumina HiSeq 2000 using the pair-end 125 × 125 library age identity ≥ 30%, and query coverage ≥ 50%. For each protein- with a 600-bp inset size (Beijing Genomics Institute, Beijing, coding gene, the top 250 highest-scoring hits from different species China). The entire dataset of two samples is ∼5.55 Gb. Illumina were retained. For each hit, a “relative bit score” was calculated reads were trimmed to remove the adapter sequences and low- as the original bit score of the hit divided by the bit score of the quality bases, after which 86–88% of paired reads were retained query sequence aligned against itself. The overall distribution for each sample. Trimmed, paired-end reads were assembled pattern of all genes in a genome was visualized by plotting the using a multiple k-mer–based assemblies (64). Briefly, metagenomic sum of the bit scores of hits within phylum Nitrospirae against reads of each sample were individually assembled into contigs using that outside this phylum per gene. the Velvet, version 1.2.10, assembler (49) with a range of k-mers (41, 51, 61, 71, 81, and 91). The different assembles were sub- Divergence Time Estimation. Molecular-dating analyses were per- sequently merged, and the duplicated and suboptimal contigs formed using PhyloBayes, version 4.1c (63). The CAT-GTR model were removed through CD-HIT-EST (65) using a sequence was implemented for amino acid replacement, and analyses were run identity threshold of 0.95 and a word length of 8 to get the final under either the log-normal autocorrelated relaxed clock (-ln) or the assembly for each sample. Resulting contigs were filtered by a uncorrelated gamma multipliers (-ugam). For each condition, two minimal length cutoff of 1 kb. replicate chains with 20,000 generations were run. Dates were assessed by running the readdiv with the first 20% of generations Population Genome Binning of a Magnetotactic Nitrospirae from HCH. removed as burn-in for each analysis. Two different combinations of Contigs of sample HCH were sorted using BLASTn alignment age constraints were used for the divergence time estimation. For the against the NCBI genomes database (version May 2015) together first combination of age constraints, the minimum age of the root of with previously sequenced MTB draft genomes of Mcas (17), Oxyphotobacteria (oxygenic Cyanobacteria) was set at 2.32 Ga (the Mchi (18), and Mbav (18). BLASTn alignment hits with E values rise in atmospheric oxygen) (67), and the maximum age was set at − larger than 1 × 10 5 were filtered, and the taxonomical level of 3.0 Ga (40, 68). For the second combination, a minimum age of each contig was determined by the lowest common ancestor al- 1.9 Ga (the first widely accepted fossil oxygenic Cyanobacteria) (69) gorithm implemented in MEGAN, version 5 (50). All contigs and a maximum age of 2.32 Ga (postdating the rise of oxygen binned to known Nitrospirae MTB species of Mcas, Mbav, and according to ref. 70) were implemented as the oxyphotobacterial root. Mchi were selected. Due to the incomplete nature of available In addition, for the second combination another time constraint, the magnetotactic Nitrospirae draft genomes, the remaining contigs divergence time between Oxyphotobacteria and Melainabacteria, was were further classified using CLARK, version 1.1.2 (51), based included, which was set from 2.5 Ga (70) to 3.8 Ga (the end of late on reduced sets of k-mers by comparison with available genomes heavy bombardment). For all analyses, the age calibration for the or draft genomes of MTB strains. The measure of conservation last common ancestor of all taxa used in this study was set between of gene content and gene order of MGCs between HCH-1 and 2.32 and 3.8 Ga (71).

Lin et al. www.pnas.org/cgi/content/short/1614654114 1of5 HCH MY MY2-3C (HM454280) 74 0 ‘Candidatus Magnetobacterium casensis’ (JMFO00000000) <1% 1%-10% ‘Candidatus Magnetobacterium bavaricum’ (X71838) 10%-50% 50%-80% 90 OTU_16 OTU_0 74 93 MHB-1 (AJ863136) MY2-1F (HM454279) MY3-11A (HM454282) 96 MY3-5B (HM454281) Nitrospiraceae 95 96 77 MY4-5C (HM454283) 91 LO-1 (GU979422) MWB-1 (JN630580) ‘Candidatus Magnetoovum chiemensis’ CS-04 (JX402654) OTU_12

99 ‘Candidatus Thermomagnetovibrio paiutensis’ HSMV-1 (GU289667) OTU_5

81 BW-2 (HQ595728) SS-5 (HQ595729) Gammaproteobacteria 92 100 Acinetobacter indicus strain A648 (NR 117784) OTU_10 99 Acinetobacter seohaensis strain SW100 (NR 115299) 100 Magnetospira sp. QH-2 (EU675666) Magnetospira thiophila MMS-1 (EU861390) Rhodospirillaceae

100 Magnetospirillum gryphiswaldense MSR-1 (Y10109) Magnetospirillum magneticum AMB-1 (D17514) 99 98 Magnetospirillum magnetotacticum MS-1 (M58171) 56 Magnetovibrio blakemorei MV-1 (L06455) OTU_7 78 97 OTU_3 77 OTU_6

99 Magneto-ovoid bacterium MO-1 (EF643520)

87 Magnetococcus marinus MC-1 (L06456) 75 OTU_13 OTU_8 98 68 54 OTU_15 OTU_1 Magnetococcaceae 74 OTU_11 100 OTU_14 OTU_2

99 81 OTU_9 79 OTU_4 0.02 ‘Candidatus Magnetococcus yuandaducum’ (FJ667777)

Fig. S1. Phylogenetic tree of operational taxonomic units (OTUs at 97% threshold similarity) for 16S rRNA gene clone libraries of MTB communities from the city moat of Xi’an in Shaanxi province (HCH) and Lake Miyun near Beijing (MY). The evolutionary history was inferred by using the maximum-likelihood method based on the Kimura two-parameter model with 100 bootstraps. On the right-hand side, a heatmap shows the relative abundance and distribution of each OTU from this study.

Lin et al. www.pnas.org/cgi/content/short/1614654114 2of5

AM M1 B-1_WP_00862263

MamA S MamB

- 1

_ 6

MSR-1_AAL0999 W P

5 _ 0667

0 O 4

0 8

QH-2_CCQ72998 6 MV-1_CAV30810

2 1

2 6 1

SO-1_EME6831 3 9 QH-2_ 9

S-1_BAH77607 1 MS-1_KIM00482 R W 0 ML-1_AFZ7701 ML-1_AFX8897 P_0460206810 0 RS-1_WP_01586273 AMB-1_WP_011383402 0 1_magnetite_CC 99 BW-1_magnetite_AET24905 100 HK-1_magnetite_KPA1903 MV-1_CAV30807 W- 4 B 4 HK-1_magnetite_KPA19045 0 77 MSR-1_WP_041633591 10 8 100 83 10 0.3 7 0 0.2 98 5 SS-5_AFX8898 100 0 10 9 7 0 90 9 2 _CCO0671 W-1_greigite _WP_011713875 100 B MC-1 100 100 IT-1_AHG23879 B13_WP_041904061HK-1_gMMP_ADV17394 BW-1_greigite_AET24910

IT-1_AHG23882 98 MMP_ADV17392 reig HCH-1 ite_KP 100 MC-1_WP_011713872 7 5 A17996 SS-5_AFX88984 B13_WP_041904060

Mchi_ 100

IM4131 9

KJR43885 0 99 HCH Mcas_A Mbav_KJU8484 9 -1

Mchi_

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Mcas_AIM4131

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0

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100 AMB-SO-1_EM

AMB-1_WP_0113834286 2 MS-1_WP_041039444

8 10 5 0 10 HK-1_magnetit 94 MSR-1_CAE120 0 ML-1_AFZ77020 34 95 0.2 9 7 RS-1_BAH77598 83 9 9 100 75 00 BW- 1 HY02427 0 BW-1_AE 1_ SS-5_A T ma 0.2 24914_magne gn eti 10 tite te_ 0 AE IT-1_AHG23888MC-1_WP_01171388 B13 T2 QH-2_CCQ7299 49 BW-1_AE 13

MMP_ADV1737 9 8 T24915_greigi 5 0 1 MV-1_CAV30818 3 10

0 te_KPA1428 98

10 HCH-1 te Mcas_AIM4132 0 HK-1_magneti

10

5 HCH-1 ML-1_AFZ77032

8 JU84843

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5 BW-1_AET24921_magnetite IT- 5

R 1_ te_KPA19047 S-1_BAH77600 447 A

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AV ML-1_AFX88981 3 MC-1_ Alphaproteobacteria 97 100 0.2 7 6 MV-1_C Gammaproteobacteria SS-5_AFX88991QH-2_CCQ729 77 9 3 Deltaproteobacteria 8 2 MS-1_WP_009869051 86 AMB-1_WP_0113834 SO-1_ Nitrospiraceae MSR-1_CAM78030

5 8 7 93 3 E ME68313 01 Latescibacteria 1 3 HCH-1 BW-1_AET24922_greigite 7

IM4132

_A

MMP_ADV1739 Mbav_KJU8485

Mcas

B13_WP_041904056

Fig. S2. Bootstrap consensus trees of five magnetosome proteins (MamABEKP) based on the maximum-likelihood method. Only full-length protein sequences were included in this analysis. Bootstrap values are expressed as percentages, and only values of more than 75% are shown. MSR-1, Magnetospirillum gryphiswaldense MSR-1; AMB-1, Magnetospirillum magneticum AMB-1; SO-1, Magnetospirillum sp. SO-1; QH-2, Magnetospira sp. QH-2; MC-1, Magnetococcus marinus MC-1; BW-1, Candidatus Desulfamplus magnetomortis BW-1; MMP, Ca. Magnetoglobus multicellularis; RS-1, Desulfovibrio magneticus RS-1; ML-1, alkaliphilic magnetotactic strain ML-1; MV-1, Magnetovibrio blakemorei MV-1; IT-1, Ca. Magnetofaba australis strain IT-1; SS-5, Gammaproteobacteria magnetotactic strain SS-5; HK-1, Ca. Magnetomorum sp. HK-1; B13, Latescibacteria bacterium SCGC AAA252-B13.

Lin et al. www.pnas.org/cgi/content/short/1614654114 3of5 MamAMY-2 MamB MY-2 100 100 HCH-1 HCH-1 Mchi_KJR43885 Mchi_KJR43883 76 MY-3 MY-3 75 97 96 Mcas_AIM41317 MY-23 91 99 MY-22 100 Mcas_AIM41319 76 MY-23 MY-22 85 Mbav_KJU84845 Mbav_KJU84847

Proteobacteria Proteobacteria

0.2 0.2

MamE MY-2 MamK 100 89 MY-2 HCH-1 HCH-1 MY-3 MY-3 100 Mcas_AIM41315 98 99 Mcas_AIM41328 MY-22 99 MY-22 Mbav_KJU84843 82 MY-23 MY-23 Proteobacteria Proteobacteria 0.1 0.1

MamP HCH-1 96 MY-2 MY-3

98 Mcas_AIM41323 92 MY-22

98 Mbav_KJU84851 MY-23

Proteobacteria

0.2

Fig. S3. Bootstrap consensus trees of magnetosome proteins MamABEKP from the four additional Nitrospirae MGCs of MY and those full-length proteins from all available Nitrospirae MTB based on the maximum-likelihood method. Bootstrap values are expressed as percentages, and only values of more than 75% are shown.

Calibartion constraints and Mean divegence time 95% mean confidence intervals molecular clock models:

Time_constraint_2_ugam_chain2

Time_constraint_2_ugam_chain1

Time_constraint_2_ln_chain2

Time_constraint_2_ln_chain1

Time_constraint_1_ugam_chain2

Time_constraint_1_ugam_chain1

Time_constraint_1_ln_chain2

Time_constraint_1_ln_chain1 Great Oxidation Event (GOE) Archean Proterozoic Phanerozoic 4 3.5 3 2.5 2 1.5 1 0.5 0 (Ga)

Fig. S4. Summary of mean divergence dates for the Nitrospirae and Proteobacteria phyla estimated using Bayesian relaxed molecular-clock analyses with two different time constraints and two different molecular clock models (see SI Materials and Methods for details). Two replicated chains were run for each condition. The input phylogenomic tree used here is shown in Fig. S5.

Lin et al. www.pnas.org/cgi/content/short/1614654114 4of5 p_Aquificae_Sulfurihydrogenibium_azorense_Az-Fu1_NC_012438 90 p_Aquificae_NC_014926_Thermovibrio_ammonificans 100 p_Aquificae_NC_015185_Desulfurobacterium_thermolithotrophum

p_Cyanobacteria_c_Melainabacteria_Caenarcanum_bioreactoricola Aquificae

80 p_Cyanobacteria_c_Melainabacteria_MEL_B2 p_Cyanobacteria_c_Melainabacteria_MEL_B1 100 p_Cyanobacteria_c_Melainabacteria_MEL_A1

100p_Cyanobacteria_c_Melainabacteria_Gastranaerophilaceae_MH_37 100 100 100p_Cyanobacteria_c_Melainabacteria_MEL_C1 p_Cyanobacteria_c_Melainabacteria_Gastranaerophilaceae_Zag_1 100 p_Cyanobacteria_c_Melainabacteria_Gastranaerophilus_phascolarctosicola p_Cyanobacteria_c_Melainabacteria_Gastranaerophilaceae_Zag_111 100 Melainabacteria p_Cyanobacteria_c_Melainabacteria_Obscuribacter_phosphatis p_Cyanobacteria_Gloeobacter_violaceus_PCC_7421_NC_005125 p_Cyanobacteria_Nostoc_sp_PCC_7120_NC_003272 100 100 p_Cyanobacteria_Nodularia_spumigena_CCY9414_NZ_CP007203 76 p_Cyanobacteria_Cylindrospermum_stagnale_PCC_7417_NC_019757 100 p_Cyanobacteria_c_Oxyphotobacteria_NZ_AJWF00000000_Anabaena_sp_90 p_Cyanobacteria_Synechococcus_sp_JA-2-3B_NC_007776 100 p_Cyanobacteria_Synechococcus_sp_JA-3-3Ab_637000313 p_Nitrospirae_NC_017094_Leptospirillum_ferrooxidans_C2-3 100 p_Nitrospirae_NC_018649_Leptospirillum_ferriphilum_ML-04 100 p_Nitrospirae_NC_014355_Nitrospira_defluvii 100 p_Nitrospirae_NZ_CP011801_Nitrospira_moscoviensis 90 99 p_Nitrospirae_LN885086_Candidatus_Nitrospira_inopinata 98 p_Nitrospirae_CZQA00000000_Candidatus_Nitrospira_nitrosa 98 100p_Nitrospirae_LNDU00000000_Nitrospira_sp_Ga0074138 100 p_Nitrospirae_CZPZ00000000_Candidatus_Nitrospira_nitrificans p_Nitrospirae_LJTM00000000_Nitrospira_bacterium_SG8_35_4 100 p_Nitrospirae_LJTK00000000_Nitrospira_bacterium_SG8_35_1

100 p_Nitrospirae_LJUG00000000_Nitrospira_bacterium_SM23_35 p_Nitrospirae_NZ_AUIU00000000_Thermodesulfovibrio_thiophilus_DSM_17215 Nitrospirae Oxyphotobacteria 100 100 p_Nitrospirae_BBCX00000000_Thermodesulfovibrio_aggregans_JCM_13213 p_Nitrospirae_NC_011296_Thermodesulfovibrio_yellowstonii_DSM_11347 100 p_Nitrospirae_NZ_AXWU00000000_Thermodesulfovibrio_islandicus_DSM_12570 p_Nitrospirae_JZJI00000000_Candidatus_Magnetoovum_chiemensis 100 p_Nitrospirae_LNQR00000000_Nitrospirae_bacterium_HCH-1 100 100 p_Nitrospirae_JMFO00000000_Candidatus_Magnetobacterium_casensis 100 p_Nitrospirae_LACI00000000_Candidatus_Magnetobacterium_bavaricum c_Deltaproteobacteria_NC_016629_Desulfovibrio_africanus c_Deltaproteobacteria_NC_007519_Desulfovibrio_alaskensis 100 100 c_Deltaproteobacteria_NC_011883_Desulfovibrio_desulfuricans 94 92 c_Deltaproteobacteria_NC_002937_Desulfovibrio_vulgaris 99 c_Deltaproteobacteria_AP010904_Desulfovibrio_magneticus_RS-1 c_Deltaproteobacteria_ATBP00000000_Candidatus_Magnetoglobus_multicellularis 100 c_Deltaproteobacteria_JPDT00000000_Candidatus_Magnetomorum_sp_HK-1 100 c_Deltaproteobacteria_NZ_AXAM00000000_Desulfosarcina_sp_BuS5 99 98 c_Deltaproteobacteria_NZ_BBCC00000000_Desulfosarcina_cetonica_JCM_12296 100 c_Deltaproteobacteria_NZ_ATHJ00000000_Desulfococcus_multivorans_DSM_2059 Deltaproteobacteria c_Alphaproteobacteria_CP000471_Magnetococcus_marinus_MC-1 c_Alphaproteobacteria_FO538765_Magnetospira_sp_QH-2

100 c_Alphaproteobacteria_HG794546_Magnetospirillum_gryphiswaldense_MSR-1 100 c_Alphaproteobacteria_LN997848_Magnetospirillum_sp_XM-1

100c_Alphaproteobacteria_AP007255_Magnetospirillum_magneticum_AMB-1 100 98 c_Alphaproteobacteria_JXSL00000000_Magnetospirillum_magnetotacticum_MS-1 c_Alphaproteobacteria_AONQ00000000_Magnetospirillum_caucaseum_SO-1 0.9 c_Alphaproteobacteria_NC_000963_Rickettsia_prowazekii_str_Madrid_E 100c_Alphaproteobacteria_NC_003103_Rickettsia_conorii_str_Malish_7 100 100c_Alphaproteobacteria_NC_016915_Rickettsia_rickettsii_str_Hlp 100 c_Alphaproteobacteria_NC_007940_Rickettsia_bellii_RML369-C c_Alphaproteobacteria_NC_007797_Anaplasma_phagocytophilum_str_HZ c_Alphaproteobacteria_NC_011420_Rhodospirillum_centenum_SW c_Alphaproteobacteria_NC_007643_Rhodospirillum_rubrum_ATCC_11170 Alphaproteobacteria 100 c_Alphaproteobacteria_NC_017059_Pararhodospirillum_photometricum_DSM_122

Fig. S5. Phylogenomic maximum-likelihood tree of 64 bacterial genomes. Bootstrap values are expressed as percentages, and only values of >75% are shown. Magnetotactic bacteria are displayed in blue. The Aquificae strains were used as outgroup.

Other Supporting Information Files

Table S1 (DOCX) Table S2 (DOCX) Table S3 (DOCX)

Lin et al. www.pnas.org/cgi/content/short/1614654114 5of5