Microbial Diversity of Deep-Sea Ferromanganese Crust

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1 Microbial Diversity of Deep-Sea Ferromanganese Crust Field in the Rio Grande Rise, Southwestern 2 Atlantic Ocean 3 4 Natascha Menezes Bergo1, Amanda Gonçalves Bendia1, Juliana Correa Neiva Ferreira1, Bramley Murton2, 5 Frederico Pereira Brandini1, Vivian Helena Pellizari1 6 7 1Department of Biological Oceanography, Oceanograophic Institute, University of Sao Paulo, Brazil 8 2Natural Environment Research Council, England 9 10 Corresponding author 11 Correspondence to Dra Vivian Helena Pellizari, [email protected] 12 13 Abstract 14 Seamounts are often covered with Fe and Mn oxides, known as ferromanganese (Fe-Mn) crusts. Future mining of 15 these crusts is predicted to have significant effects on biodiversity in mined areas. Although microorganisms have 16 been reported on Fe–Mn crusts, little is known about the role of crusts in shaping microbial communities. Here, 17 we investigated microbial community based on 16S rRNA gene sequences retrieved from Fe-Mn crusts, coral 18 skeleton, calcarenite and biofilm at crusts of the Rio Grande Rise (RGR). RGR is a prominent topographic feature 19 in the deep southwestern Atlantic Ocean with Fe-Mn crusts. Our results revealed that crust field of the RGR harbors 20 a usual deep-sea microbiome. We observed differences of microbial structure according to the sampling location 21 and depth, suggesting an influence of water circulation and availability of particulate organic matter. Bacterial and 22 archaeal groups related to oxidation of nitrogen compounds, such as Nitrospirae, Nitrospinae phyla, 23 Nitrosopumilus within Thaumarchaeota group were present on those substrates. Additionally, we detected 24 abundant assemblages belonging to methane oxidation, i. e. Ca. Methylomirabilales (NC10) and SAR324 25 (Deltaproteobacteria). The chemolithoautotrophs associated with ammonia-oxidizing archaea and nitrite-oxidizing 26 bacteria potentially play an important role as primary producers in the Fe-Mn substrates from RGR. These results 27 provide the first insights into the microbial diversity and potential ecological processes in Fe-Mn substrates from 28 the Atlantic Ocean. This may also support draft regulations for deep-sea mining in the region. 29 30 Keywords: Deep-sea Ferromanganese Crusts, Microbial Community, Biogeochemical Cycling, Rio Grande Rise, 31 Geomicrobiology. 32 33 Funding 34 This study was funded by the São Paulo Research Foundation (FAPESP), Grant number: 14/50820-7, Project 35 Marine ferromanganese deposits: a major resource of E-Tech elements, which is an international collaboration 36 between Natural Environment Research Council (NERC, UK) and FAPESP (BRA). NMB thanks the Ph.D. 37 scholarship financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - 38 Finance Code 001. 39 40 Conflict of Interest 41 The authors declare no conflict of interest. 42 43 Availability of data and material 44 The sequencing reads generated for this study can be found in the National Centre for Biotechnology Information 45 (NCBI) database under the BioProject PRJNA638744. 46 47 Acknowledgments 48 We thank the captain and the crew of the Royal Research Ship Discovery cruise DY094 for their data and sampling 49 support, as well as LECOM's research team and Rosa C. Gamba for their scientific support. 50 51 52 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.13.150011; this version posted June 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

53 INTRODUCTION 54 55 The process of biogenesis in Fe-Mn crusts formation is not well described at all. Microorganisms are suggested to 56 be potentially involved in the elemental transition between seawater and Fe-Mn substrates [15, 43], they might 57 play a role in Mn oxidation and precipitation. Microbial diversity and abundance have been studied in the Fe-Mn 58 crusts, and their associated sediment and water of Pacific Ocean seamounts [16, 24, 30,31]. These have detected 59 the potential for microbial chemosynthetic primary production supported by ammonia oxidation [12, 15, 31]. 60 Wang and Muller (2009) proposed that free-living and biofilm-forming bacteria provide the matrix for manganese 61 deposition, and coccolithophores are the dominant organisms that act as bio-seeds for an initial manganese 62 deposition in the Fe-Mn crusts. Kato et al, (2019) proposed a model of the microbial influence in the 63 biogeochemical cycling of C, N, S, Fe and Mn in Fe-Mn crusts, which indicates their contribution to crust 64 development. In contrast, other authors suggested that these endolithic and epilithic microbial communities just 65 live in/on Fe-Mn crust as a favorable physical substrate [12, 30]. 66 Fe-Mn crust occurs on rocky substrates at seamounts globally and has a slow accretion rate of typically 1–5 mm 67 per million years [10]. Seamounts and rises are most common in the Pacific Ocean where there are estimated to 68 be over 50,000 [19, 44]. Fe-Mn crusts are economically important because they mainly contain cobalt and other 69 rare and trace metals of high demand including copper, nickel, platinum, and tellurium [10]. While crusts are 70 potentially of economic value, seamounts also offer a habitat appropriate for sessile fauna, most corals and sponges 71 [9, 35]. However, the ecological roles of chemosynthetic and heterotrophic microbes in metal-rich benthic 72 ecosystems remain unknown for the Atlantic Ocean. 73 Despite the increasing efforts that have been made using high-throughput DNA sequencing of microbes living on 74 Fe-Mn crusts from the Pacific Ocean, similar studies from the Atlantic Ocean are still scarce. Considering the wide 75 distribution of Fe-Mn crusts on seamounts globally and their potential for future mineral resource supplies [10], 76 the study of their microbiome, function and resilience to environmental impacts caused by its exploitation is 77 essential [33]. 78 To better understand how microbial community structure is shaped by metallic substrates in seamounts, we 79 sampled Fe-Mn crust biofilm, crusts, encrusted coral skeletons, and calcarenite substrate from the Rio Grande Rise 80 (RGR), Southwestern Atlantic Ocean. We used the sequencing of the 16S rRNA gene to determine the microbial 81 diversity and community structure, and to predict microbial functions and ecological processes. We hypothesized 82 that there is a core microbiome among the Fe-Mn substrates, despite the influence of different environmental 83 conditions such as water masses, temperature, salinity and depth. Our results revealed a diverse microbial 84 community among the Fe-Mn substrates, including groups assigned within Thaumarchaeota, 85 Gammaproteobacteria and Alphaproteobacteria. Although differences of microbial diversity were noted 86 according to the sampling location and depth, we confirmed the presence of a core microbiome among the Fe-Mn 87 substrates mainly composed by ammonia-oxidizing Archaea. 88 89 MATERIAL AND METHODS 90 Field Sampling 91 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.13.150011; this version posted June 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

92 The study area, RGR, is an extensive topographic feature of ∼150,000km2 in the Southwestern Atlantic Ocean [7] 93 (Fig. 1). RGR is approximately 1,000 - 4,000m deep and is located ∼1,000km to the east of the Brazil and 94 Argentine basins [7, 28]. 95 Fe-Mn crust biofilm, crust, encrusted coral skeletons, and calcarenite samples were collected during the Marine 96 E-Tech RGR2 expedition DY094 onboard the Royal Research Ship (RRS) Discovery (NOC-NERC, UK) in 97 October 2018 from the RGR (Supplementary Figure 1 and Table 1). Once onboard the vessel, the recovered 98 material was aseptically sampled from each dredge. The surface of the crust and encrusted corals samples was 99 washed with seawater filtered through a 0.2μm-pore polycarbonate membrane to remove loosely attached particles, 100 and immediately stored in sterile Whirl-Pak bags at −80°C. 101 102 DNA Extraction, 16S rRNA gene Amplification and Sequencing 103 104 DNA was extracted from the recovered samples using 5g of samples with the FastDNA™ SPIN Kit for Soil 105 (MPBiomedical), according to the manufacturer's protocol [21, 38]. DNA integrity was determined after 106 electrophoresis in 1% (v/v) agarose gel prepared with TAE 1X (Tris 0.04M, glacial acetic acid 1M, EDTA 50mM, 107 pH 8), and staining with Sybr Green (Thermo Fisher Scientific, São Paulo, Brazil). DNA concentration was 108 determined using the Qubit dsDNA HS assay kit (Thermo Fisher Scientific, São Paulo, Brazil), according to the 109 manufacturer's instructions. 110 Before sending samples for preparation of Illumina libraries and sequencing, the V3 and V4 region of the 16S 111 rRNA gene was amplified with the primer set 515F (5’–GTGCCAGCMGCCGCGGTAA-3’) and 926R (5’– 112 CCGYCAATTYMTTTRAGTTT-3’), [34] to check for the amplification of 16S using the extracted DNA. 113 Illumina DNA libraries and sequencing were performed at Biotika (www.biotika.com.br, Brazil) on a MiSeq 114 platform in a paired-end read run (2 x 250bp) following the manufacturer’s guidelines. Sequencing outputs were 115 the raw sequence data. 116 117 Sequencing Data Processing and Statistical Analyses 118 119 The demultiplexed sequences were analyzed with the software package Quantitative Insights Into Microbial 120 Ecology (QIIME 2) version 2019.4 [3]. Sequences were denoised using DADA2 [6] with the following parameters: 121 trim left-f = 19, trim left-r = 18, trunc-len-f = 287, trunc-len-r = 215. Amplicon sequence variants (ASVs) with 122 sequences less than 10 occurrences were removed. The taxonomy was assigned to the representative sequences of 123 ASVs using a Naive Bayes classifier pre-trained on SILVA release 132 clustered at 99% identity. FastTree and 124 MAFFT [17] were used to create a rooted phylogenetic tree which was used in calculating phylogenetic diversity 125 metrics. 126 Diversity and phylogenetic analyses were performed with the PhyloSeq [25], ggplot2 [45] and vegan [32] packages 127 in R software (Team, 2018). ASVs without at least two sequences occurring in two or more samples and 128 chloroplasts were removed. Alpha‐diversity metrics (e.g., observed sequence variants, Chao1 and Shannon 129 diversity) were calculated based on ASV relative abundances for each sample. To determine if there were 130 significant differences between alpha diversities, Analyses of Variance (Kruskal-Wallis) test was performed in R. 131 ASVs were normalized using the R package “DESeq2” by varianceStabilizingTransformation [22]. Beta-diversity 132 among sample groups was examined an ordinated weighted Unifrac normalized distance, tested with Permutational

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133 Homogeneity of Group Dispersions, and visualized using principal coordinate analysis (PCoA, package Phyloseq). 134 Relative abundance of taxonomic indicators was identified by the IndicSpecies [5]. The analysis was conducted 135 on ASV counts excluding ASVS < 20 reads. Predicted microbial functional groups were identified by the 136 Functional Annotation of Prokaryotic Taxa (FAPROTAX) database [23]. Statistical tests were considered 137 significant at p < 0.05. The sequencing reads generated for this study can be found in the National Centre for 138 Biotechnology Information (NCBI) database under the BioProject PRJNA638744. 139 140 RESULTS 141 Alpha and Beta Diversity Estimates at Rio Grande Rise 142 143 A total of 409,346 sequences was retrieved from eleven samples and clustered into 446 amplicon sequence variants 144 (ASVs) (0.03 cut-off). The number of sequences ranged from 10,347 to 94,103 and the ASVs ranged from 10 to 145 73 per sample (Supplementary Table 1). In general, mean richness (366 ±159, standard error, s.e.) and diversity 146 (5.38 ± 0.45, s.e.) were higher in calcarenite samples when compared to those from Fe-Mn crust biofilm, Fe-Mn 147 crust and Fe-Mn encrusted coral skeleton from the RGR (Fig. 2 and Supplementary Table 1). Richness (163±140, 148 s.e.) and diversity (3.16±1.27, s.e.) were lowest at Fe-Mn crust community compared to the other substrates with 149 greater variability between samples (see standard error, Fig. 2 and Supplementary Table 1). Although diversity 150 was higher on Fe-Mn coral samples (3.93±0.79, s.e.) compared to the crust biofilm (3.3±0.24, s.e.), the crust 151 biofilm (230.8±55.56, s.e.) community showed a higher richness than the coral (105.5±59.50, s.e.) (Fig. 2 and 152 Supplementary Table 1). Fe-Mn substrates additionally showed the variation in richness and diversity between 153 samples, as evaluated with standard error. Alpha diversity indexes were not statistically different between Fe-Mn 154 substrates (Supplementary Table 1). 155 Beta diversity among the samples and substrates was tested using the weighted Unifrac distances and ordered by 156 PCoA. In the PCoA plot, samples were clustered by substrates, expected DY94_56_01 and DY94_46_03 (Fig. 3). 157 The PCoA analysis captured 60.5% of the total variation of the prokaryotic community composition in the 158 investigated samples. Samples were also clustered by depths (Supplementary Fig. 2). The analysis of groups 159 dispersions showed a significant heterogeneity of dispersion among stations (ANOVA, P < 0.05) and depths 160 (ANOVA, P < 0.05). 161 162 Microbial Community Composition at Rio Grande Rise 163 164 Bacterial and Archaeal composition varied in abundance or occurrence among substrates and samples. For 165 example, the microbial groups Proteobacteria (classes Gammaproteobacteria and Alphaproteobacteria), 166 Thaumarchaeota (Nitrosopumilales) and Planctomycetes (classes Phycisphaerae and Planctomycetacia) were 167 abundant in all substrates, except the DY094_63_01_MB, DY094_47_01_MB, DY094_52_02_MB, 168 DY094_39_01_A and DY094_46_03_MB samples (Fig. 4). Proteobacteria was the most abundant phyla, 169 comprising about 39% of the total taxonomic composition of each sample (Fig. 5). Within Proteobacteria, 170 Gammaproteobacteria was the second most abundant class (22%), followed by Alphaproteobacteria (12%). 171 Nitrososphaeria was the most abundant class (23%), especially among the crust biofilm samples (Fig. 4 and Fig. 172 5). ASVs affiliated to Verrucomicrobia, Marinimicrobia (SAR406 clade), Calditrichaeota, Cyanobacteria and 173 Firmicutes represented less than 1% of all sequences (Fig. 4A).

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174 Thaumarchaeota (55%) was prevalent in crust biofilm samples (Fig. 5), followed by the phyla Proteobacteria 175 (17%), Chloroflexi (12%) and Acidobacteria (6%) (Fig. 4A). ASVs affiliated to Bacteroidetes, Marinimicrobia 176 (SAR406 clade), Actinobacteria, Calditrichaeota, Cyanobacteria and Rokubacteria represented less than 1% of all 177 sequences in crust biofilm samples (Fig. 4A). At the class level in crust biofilm, Nitrososphaeria (60-51%, 178 Nitrosopumilales), Dehalococcoidia (20-7%, order SAR202 clade), Gammaproteobacteria (10-5%, orders 179 Cellvibrionales, Nitrosococcales, UBA10353 marine group and Oceanospirillales), Alphaproteobacteria (10-3%, 180 orders Rhodospirillales, Rhodovibrionales and Sar11) and Deltaproteobacteria (6-1%, orders SAR324 clade and 181 NB1-j) were dominant, except in the DY094_39_01 sample. The bacterial class Dehalococcoidia was not 182 identified in DY094_39_01; the prevalent classes were Gammaproteobacteria (22%), Alphaproteobacteria (20%), 183 Nitrospinia (13%) and Deltaproteobacteria (4%). 184 The prevalent ASVs in the Fe-Mn crusts were Proteobacteria vAcidobacteria (14-7%) and Thaumarchaeota (17- 185 7%) and Planctomycetes (5-1%), except in the DY094_63_01 sample. The bacterial phyla Planctomycetes 186 dominated (>70%) in DY094_63_01 crust, followed by Proteobacteria (11%) and Rokubacteria (10%) (Fig. 4 and 187 Fig. 5). Representatives of the phyla Chloroflexi, Bacteroidetes, Nitrospirae, Dadabacteria, Actinobacteria and 188 Nitrospinae were detected in crusts only in the DY094_56_01 sample. At the class level, Gammaproteobacteria 189 (57-24% orders Acidiferrobacterales, Alteromonadales, MBMPE27, pItb-vmat-80 and Steroidobacterales), 190 Alphaproteobacteria (17-16%, orders Kordiimonadaceae, Rhizobiales and Rhodovibrionales), Subgroup 9 (14- 191 2%) and Nitrososphaeria (17-7%, Nitrosopumilales) were abundant in DY094_47_01 and DY094_56_01 crusts. 192 Otherwise, representatives of Phycisphaerae (80%, order Phycisphaerales), Gammaproteobacteria (10%, Sar86 193 clade) and NC10 (9%, order Methylomirabilales) were more abundant in the DY094_63_01 crust (Fig. 4A). ASV 194 related to a Colwelliaceae family were detected in the DY094_47_01 and DY094_63_01 crust samples. 195 In Fe-Mn encrusted coral skeletons, microbial communities were dominated by ASVs affiliated to Proteobacteria 196 (54-46%), Planctomycetes (17-17%), Thaumarchaeota (7-5%), Acidobacteria (9-4%) and Nitrospinae (3-1%) (Fig. 197 4A). The prevalent class in coral samples were related to Gammaproteobacteria (27% orders 198 Betaproteobacteriales, MBMPE27, BD7-8 and Steroidobacterales), Alphaproteobacteria (20%, orders 199 Kordiimonadaceae, Rhizobiales, Rhodovibrionales and Sneathiellaceae), Planctomycetacia (7%, order 200 Pirellulales), Nitrososphaeria (5%, Nitrosopumilales) and Deltaproteobacteria (5%, orders Myxococcales and 201 NB1-j), (Fig. 4A). The classes Entotheonellia (14%, order Entotheonellales) and NC10 (2%, order 202 Methylomirabilales) were only detected in the DY094_52_02_MB sample (Fig.5). Representatives of the classes 203 PAUC43f marine benthic group (5%), Dehalococcoidia (4%), Acidimicrobiia (4%), Dadabacteriia (3%), 204 Bacteroidia (1%) and Actinobacteria (0.1%) were detected only in coral from DY094_47_02_MB (Fig. 4B). ASVs 205 related to a Geodermatophilaceae family were detected only in the DY094_47_02 sample. 206 Calcarenite samples were dominated by ASVs affiliated to Proteobacteria (30%), Thaumarchaeota (20%) 207 Planctomycetes (10%), Acidobacteria (7%), Rokubacteria (5%), Nitrospirae (4%) and Gemmatimonadetes (4%). 208 ASVs associated with Actinobacteria, Calditrichaeota, Nitrospinae, Verrucomicrobia represented less than 1% of 209 all sequences in calcarinete samples (Fig. 4A). At the class level, the prevalent groups were Nitrososphaeria (21%, 210 Nitrosopumilales), Gammaproteobacteria (12%, orders Betaproteobacteriales, Gammaproteobacteria Incertae 211 Sedis, MBMPE27, BD7-8 and Steroidobacterales), Alphaproteobacteria (10%, order Rhodovibrionales), 212 Deltaproteobacteria (8%, orders Myxococcales and NB1-j), Phycisphaerae (6%), Planctomycetacia (6%, order 213 Pirellulales) and NC10 (6%, order Methylomirabilales) (Fig. 4B and Fig. 5). Representatives of Cyanobacteria

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214 (class Oxyphotobacteria) and Marinimicrobia (clade SAR406) were detected in the calcarenite sample only from 215 DY094_62_01_MB (Fig. 4B). 216 Microbial community composition among different substrates was further investigated by means of the Indicator 217 Species analysis. We compared the relative abundance and relative frequency of each ASV to identify those 218 specifically associated with only one substrate (unique) and those whose niche breadth encompasses several 219 substrates (shared). The results were visualized as a table showing the distribution of unique and shared ASV 220 among different substrates (Supplementary Table 2). Calcarinite harbored the highest set of unique ASVs (n = 24), 221 mainly belong to the oligotypes Nitrosopumilales (n = 6, Thaumarchaeota), class of uncultured 222 Alphaproteobacteria (n = 5), class Gammaproteobacteria (n = 3, families EPR3968-O8a-Bc78v and 223 Nitrosococcaceae), class Phycisphaerae (n = 2, family Phycisphaeraceae), class Nitrospira (n = 2, family 224 Nitrospiraceae) and class Gemmatimonadetes (n = 2, family Gemmatimonadaceae and class PAUC43f marine 225 benthic group) (Supplementary Table 2). 226 However, crusts, crust biofilm and encrusted coral skeletons harbored fewer unique ASVs each (n = 1, n = 1 and 227 n = 2, respectively). Those ASVs that are unique to crust and crust biofilms were ASV1055 and ASV0706, 228 belonging to the class Gammaproteobacteria and Nitrosopumilales (Thaumarchaeota), respectively. The unique 229 ASVs of encrusted coral skeletons are ASV0714 and ASV1608, from the family Pirellulaceae (phylum 230 Planctomycetes) and the order Rhizobiales (class Alpaproteobacteria). Crust and Calcarenite share only one ASV 231 that was related to the order Methylomirabilales (class NC10, ASV0807) (Supplementary Table 2). 232 233 Predicted Function Variation among the Microbial Communities 234 235 We assigned 561 out of 1875 microbial ASVs (29.92%) to at least one microbial functional group using the 236 FAPROTAX database (Supplementary Table 3). Aerobic ammonia oxidation, aerobic nitrite oxidation and 237 nitrification were functions predicted in all substrates with differences in relative abundance between samples (Fig. 238 6). Main functions associated with crust biofilm microbial communities were dark sulfide oxidation, dark oxidation 239 of sulfur compounds, nitrate respiration, nitrate reduction, nitrogen respiration and aerobic chemoheterotrophy. 240 The predicted functions in crusts and calcarenite samples were fermentation and aerobic chemoheterotrophy. 241 Manganese oxidation was predicted only in the DY094_47_02 coral sample (Fig. 6). 242 243 DISCUSSION 244 245 Our results showed that bacterial and archaeal community structure in Fe-Mn crusts are less diverse in comparison 246 with other substrates: crust biofilm, encrusted coral skeleton and calcarenite. We also observed differences in 247 microbial community structure between the sampling stations and depth, as previously reported by Kato et al. 248 (2018) for the Fe–Mn crusts from the Pacific Ocean. Three factors might be shaping the community structure and 249 composition variance on the Fe–Mn substrates from the RGR. The first is related to the water depth and location, 250 because the concentrations of elements in the crusts change depending on the water depth [1, 41]. The second 251 might be related to the geochemical differences in the composition of the studied substrates, which can promote 252 variations in the lifestyles of solid-surface attached microbes [16]. The last factor is probably associated with the 253 local circulation, as previously described for the Fe-Mn deposits from the Pacific Ocean and for the RGR [1, 16, 254 24, 31]. Further analyses are needed to clarify whether and how the physical oceanography and geochemical

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255 composition of Fe-Mn substrates in the RGR are associated with the differences observed in microbial 256 communities. 257 Microbial assemblages in samples from the RGR showed the dominance of the classes Gammaproteobacteria, 258 Alphaproteobacteria and Deltaproteobacteria, as reported for Pacific Fe-Mn crusts [21, 38, 42, 46]. However, at 259 higher taxonomic resolution, we detected differences in the microbial community composition between samples 260 from the RGR and those from the deep Pacific Ocean. Samples from the RGR showed higher abundance of the 261 orders Steroidobacterales (family Woeseiaceae), uncultured MBMPE27, Methylomirabilales and Nitrospirales, 262 when compared to Fe-Mn crusts from other regions [16, 24]. 263 Previous studies have reported that dissolved ammonia in seawater is an important nutrient in the Fe-Mn crusts 264 from the Pacific Ocean [15, 16, 30, 31]. Based on the recovered ASVs and the functional predictions performed 265 in this study, nitrification (i.e., ammonia oxidation and nitrite oxidation) and carbon fixation might be the main 266 processes occurring in the Fe-Mn substrates from the RGR. We detected a high proportion of 267 chemolithoautotrophic ammonia-oxidizing Archaea (Nitrososphaeria class) in the Fe-Mn substrates, especially in 268 our Fe-Mn crust endolithic biofilm samples, as well as chemolithoautotrophic nitrate-oxidizing Bacteria within 269 Nitrospirae and Nitrospinae. High proportions of these archaeal ammonia oxidizers (class Nitrososphaeria) and 270 bacterial nitrate, urea and cyanate oxidizers (Nitrospirae and Nitrospinae) have also been reported in abyssal Fe– 271 Mn deposits in the South Pacific Gyre [15], in the Clarion and Clipperton Zone [30, 31, 38] and in the Peru basin 272 [26]. 273 Shiraishi et al. (2016) suggested that Nitrososphaeria might be involved in manganese oxidation in Fe-Mn nodules, 274 as they have multi-copper oxidase, a gene that is utilized by most known manganese oxidizers. Also, previous 275 study proposed that Mn-reducing bacteria, such as species within Shewanella and Colwellia, are the major 276 contributing microorganisms in the dissolution of Mn in Fe–Mn crusts [2]. Although we have not detected ASVs 277 related to Shewanella, we identified ASVs in our results associated with acetate-oxidizing manganese reducers 278 (Colwelliaceae) and manganese oxidizers (Geodermatophilaceae). These manganese oxidizers and reducers were 279 in low abundance and detected only in Fe-Mn crusts and encrusted coral skeletons from stations 47 and 63. Further, 280 we detected a low abundance of ASVs in the Fe-Mn crust biofilm associated with iron reducers from the 281 Magnetospiraceae family. Magnetospiraceae and Colwelliaceae families have been reported previously across the 282 Pacific Nodule Provinces and the Peru Basin [26, 38]. Other possible mechanisms for Mn release on the Fe-Mn 283 substrate surface are acidification through the production of nitrite by ammonia oxidizers and organic acid 284 production by fermenting microorganisms [16]. Indeed, we detected ASVs related to ammonia oxidizers and 285 fermenting microorganisms, including members of the orders Bacillales, Pseudomonadales and Azospirillales. 286 Biofilms with filamentous microorganisms associated with micro-stromatolitic growth bands have been reported 287 in the Fe-Mn nodules and crusts surfaces from Pacific Ocean [4, 13, 40, 42]. Recently, Thaumarchaeota affiliated 288 metagenome-assembled genome (MAG) was recovered from Fe-Mn crust [15] and proteins associated with 289 biofilm formation and surface adhesion capabilities were detected [15]. Kato et al (2019) suggested that these 290 biofilms probably contribute to the Fe-Mn crust formation by adsorbing Fe and Mn oxide particles from seawater 291 [15]. Moreover, endolithic microorganisms within coral skeletons have been previously described [47], but those 292 associated with Fe-Mn substrates are poorly studied. We found within the microbial community of our Fe-Mn 293 encrusted coral skeleton an ASV belonging to the order Phycisphaerales (phylum Planctomycetes), which was 294 previously associated with tropical stony corals found mainly in cold waters [18]. Gammaproteobacteria are

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295 common members of coral microbiomes [18]. Our main contributing gammaproteobacterial family were related 296 to the Woeseiaceae (order Steroidobacterales), in contrast to other studies of deep-sea corals [18, 47]. Members of 297 the Woeseiaceae family were abundant in all Fe-Mn substrates in this study. The Woeseiaceae family is 298 cosmopolitan in deep-sea sediments [29] and was also reported across an Fe-Mn nodule field in the Peru basin 299 [26]. The fact isolated Woeseiaceae members show a chemoorganoheterotrophic lifestyle [8], suggested a role in 300 organic carbon remineralization in the deep-sea [11]. 301 In our Fe-Mn substrates, we also detected higher abundance of ASVs belonging to methane oxidation, i. e. 302 Methylomirabilales and SAR324. Members of the order Methylomirabilales, within phylum NC10, were abundant 303 in Fe-Mn crusts and calcarenite, and are capable of nitrite-dependent anaerobic methane oxidation (Versantvoort 304 et al., 2018). SAR324 of the Deltaproteobacteria was abundant in our crust biofilm and crusts and is known to 305 have wide metabolic flexibility, including carbon fixation through Rubisco and an ability to oxidize alkanes, 306 methane and/or sulfur to generate energy [20, 36]. 307 In addition to carbon, the results indicate that nitrogen metabolism is important in Fe-Mn substrates from the RGR. 308 This is probably because the RGR is in the oligotrophic South Atlantic Gyre, with a low concentration of organic 309 carbon and high concentrations of nitrate, phosphate and Fe-Mn substrates [27]. Organic carbon compounds in the 310 deep-sea might be recalcitrant for microbial life [14], whereas Mn oxides are known to abiotically decompose 311 recalcitrant organic carbon (i.e., humic substances) to simple carbon compounds, such as pyruvate, acetaldehyde, 312 and formaldehyde [39]. These simple carbon compounds can be used as carbon sources by deep-sea 313 microorganisms, as members of family Woeseiaceae and classes Methylomirabilales and SAR324 [39]. 314 Finally, this study is the first to report microbial diversity in Fe-Mn substrates from the deep Atlantic Ocean. Our 315 results reveal that: (1) the microbial community compositions among Fe–Mn substrates are different according to 316 the water depths and sampling location, (2) populations associated with nitrogen and carbon metabolisms are likely 317 important contributors for the ecological process occurring in Fe-Mn substrates, (3) the microbial community 318 composition detected on the RGR are not similar to those described in Fe-Mn crusts from the Pacific Ocean, except 319 for a higher proportion of ammonia-oxidizers, which suggest the commonality predominance of this group in Fe- 320 Mn substrates from both the Pacific and Atlantic oceans, and (4) a similar microbial community composition was 321 detected in an oligotrophic Fe-Mn nodules field from Peru basin, i.e. Woeseiaceae, Methylomirabilales, 322 Rhodovibrionales, BD2-11 terrestrial group and SAR324, Subgroup9. Thus, further monitoring of the benthic- 323 pelagic coupling microbiome, such as metagenomics and metatranscriptome, and measurement of the 324 oceanographic conditions in the RGR are needed to better elucidate the ecological processes involved with the 325 formation of Fe-Mn substrates. 326 327 REFERENCES 328

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FIGURES

RGR

Fig. 1 Study area and collection stations during the oceanographic expedition RGR2 to the RGR, Atlantic Ocean. High resolution bathymetry performed during the cruise

Chao1Chao1 ShannonShannon 6

500

400

300 Description

Measures A_Crustbiofilm B_Crust C_Coral

Diversity 3 D_Calcarenite Alpha Diversity Measure Alpha Diversity Alpha 200

100

0 A_Crustbiofilm B_Crust C_Coral D_Calcarenite A_Crustbiofilm B_Crust C_Coral D_Calcarenite Crust Coral Calcarinite Crust biofilm Crust Coral Calcarinite DescriptionCrust biofilm Fig. 2 Alpha diversity medians (number of ASVs, Chao1 and Shannon indexes) of microbial communities in the Fe-Mn crust biofilm-like, crusts, encrusted coral skeletons and calcarenites on the RGR

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SamplesSamples TaxaTaxa

DY094_52_02 DescriptionLegend TaxaTaxa 0.10 CrustA_CrustbiofilmBiofilm CrustB_Crust CoralC_Coral CalcareniteD_Calcarenite

DY094_47_02 PhylumPhylum 0.05 DY094_62_02 Samples Acidobacteria DY094_62_01 Actinobacteria Bacteroidetes

DY094_56_01 Calditrichaeota DY094_46_03 Chloroflexi 0.00 DY094_39_01 Cyanobacteria

Axis.2 [16.9%] Axis.2 Dadabacteria Entotheonellaeota Firmicutes

DY094_61_01 Gemmatimonadetes Marinimicrobia (SAR406 clade) −0.05 DY094_45_01 Nitrospinae Nitrospirae Planctomycetes Proteobacteria Rokubacteria Thaumarchaeota −0.10 DY094_63_01 Verrucomicrobia

DY094_47_01

−0.1 0.0 0.1 −0.1 0.0 0.1 Axis.1 [43.6%] Fig. 3 Principal coordinate analysis (PCoA) of the microbial community of Fe-Mn crust biofilm, crusts, encrusted coral skeletons and calcarenites from the RGR. The results were based on the amplicon sequencing data of the 16SrRNA genes using weighted Unifrac distance. The taxa correlating with the community differences (at phylum level) are also shown in the plot on the right (Taxa)

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(a) A_Crustbiofilm B_Crust C_Coral D_Calcarenite 100

CrustA_Crustbiofilmbiofilm CrustB_Crust C_CoralCoral CalcariniteD_Calcarenite 100100

7575

5050 % Abundance % Abundance Abundance %

2525

0 aDY094_39_01_A00 bDY094_45_01_MB cDY094_46_03_MB dDY094_61_01_MB eDY094_47_01_MB gDY094_56_01_MB hDY094_63_01_MB iDY094_47_02_MB jDY094_52_02_MB lDY094_62_01_MB mDY094_62_01_MB_1 aDY094_39_01_A bDY094_45_01_MB cDY094_46_03_MB dDY094_61_01_MB eDY094_47_01_MB gDY094_56_01_MB hDY094_63_01_MB iDY094_47_02_MB jDY094_52_02_MB lDY094_62_01_MB mDY094_62_01_MB_1 DY094_39_01 DY094_45_01 DY094_46_03 DY094_61_01 DY094_47_01 DY094_56_01 DY094_63_01 DY094_47_02 DY094_52_02 DY094_62_01 DY094_62_02

A_Crustbiofilm < 1% Low abund. AncK6 B_CrustChloroflexi Entotheonellaeota MarinimicrobiaC_Coral (SAR406 clade) Planctomycetes ThaumarchaeotaD_Calcarenite 100 < 1% Low abund. AncK6 Chloroflexi Entotheonellaeota Marinimicrobia (SAR406 clade) Planctomycetes Thaumarchaeota Phylum Acidobacteria Bacteroidetes Cyanobacteria Firmicutes Nitrospinae Proteobacteria Verrucomicrobia Phylum Acidobacteria Bacteroidetes Cyanobacteria Firmicutes Nitrospinae Proteobacteria Verrucomicrobia Actinobacteria Calditrichaeota Dadabacteria Gemmatimonadetes Nitrospirae Rokubacteria Actinobacteria Calditrichaeota Dadabacteria Gemmatimonadetes Nitrospirae Rokubacteria (b)

CrustA_Crustbiofilmbiofilm B_CrustCrust C_CoralCoral D_CalcareniteCalcarinite 100100

7575

50 % Abundance

5050 Abundance % Abundance %

2525

0 aDY094_39_01_AbDY094_45_01_MBcDY094_46_03_MBdDY094_61_01_MBeDY094_47_01_MBgDY094_56_01_MBhDY094_63_01_MB iDY094_47_02_MB jDY094_52_02_MB lDY094_62_01_MB mDY094_62_01_MB_1 00 aDY094_39_01_ADY094_39_01bDY094_45_01_MBDY094_45_01cDY094_46_03_MBDY094_46_03dDY094_61_01_MBDY094_61_01 eDY094_47_01_MBDY094_47_01 gDY094_56_01_MBDY094_56_01 hDY094_63_01_MBDY094_63_01 iDY094_47_02_MBDY094_47_02 jDY094_52_02_MBDY094_52_02 lDY094_62_01_MBDY094_62_01 mDY094_62_01_MB_1DY094_62_02

< 5% Low abund. Bacteroidia hydrothermal vent metagenome Oxyphotobacteria Subgroup 21 TK30 Acidimicrobiia < 5% Low BD2abund.−11 terrestrialBacteroidia group Ignavibacteriahydrothermal vent metagenome P9X2b3D02Oxyphotobacteria SubgroupSubgroup 21 22 TK30 uncultured bacterium Acidobacteriia AcidimicrobiiaBlastocatellia (SubgroupBD2−11 terrestrial 4) groupJG30−KFIgnavibacteria−CM66 PAUC43fP9X2b3D02 marine benthic groupSubgroupSubgroup 22 26 uncultured unculturedbacterium bacterium 405006−B04 Actinobacteria AcidobacteriiaCalditrichia Blastocatellia (SubgroupMarine 4) BenthicJG30−KF Group−CM66 A PhycisphaeraePAUC43f marine benthic group SubgroupSubgroup 26 5 uncultured unculturedbacterium 405006 bacterium−B04 AD261−B2 Actinobacteria Calditrichia Marine Benthic Group A Phycisphaerae Subgroup 5 uncultured bacterium AD261−B2 AKAU4049 Dadabacteriia Marinimicrobia bacterium SCGC AAA003−E22 Pla3 lineage Subgroup 6 uncultured deep−sea bacterium Class AKAU4049 Dadabacteriia Marinimicrobia bacterium SCGC AAA003−E22 Pla3 lineage Subgroup 6 uncultured deep−sea bacterium AlphaproteobacteriaClass AlphaproteobacteriaDehalococcoidiaDehalococcoidia NC10 NC10 Pla4Pla4 lineage SubgroupSubgroup 9 9 uncultured uncultureddelta proteobacterium delta proteobacterium Alteromonas macleodiiAlteromonasDeltaproteobacteria macleodii Deltaproteobacteria NitrososphaeriaNitrososphaeria PlanctomycetaciaPlanctomycetacia ThermoanaerobaculiaThermoanaerobaculiavadinHA49vadinHA49 Anaerolineae AnaerolineaeEntotheonelliaEntotheonellia NitrospiniaNitrospinia RhodothermiaRhodothermia ThermoleophiliaThermoleophilia VerrucomicrobiaeVerrucomicrobiae AT−s3−28 AT−s3−28 GammaproteobacteriaGammaproteobacteriaNitrospiraNitrospira SubgroupSubgroup 11 11 TK10 TK10 NA NA Bacilli Gemmatimonadetes OM190 Subgroup 15 TK17 Bacilli Gemmatimonadetes OM190 Subgroup 15 TK17 Fig. 4 The relative abundances of bacterial and archaeal taxonomic composition for (a) phylum and (b) class in the Fe-Mn crust biofilm, crusts, encrusted coral skeletons and calcarenites from the RGR. Only phyla and classes with more than 0.1% of abundance are represented. Phyla with relative abundances below 1% and classes with relative abundances below < 5% were grouped together into low abundance groups. Gray boxes at the top indicate sample substrates, i.e. crust biofilm, crust, encrusted coral skeleton and calcarenite

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Dadabacteria,Dadabacteriia,Dadabacteriales d0bdf71414c2cf3a6120044a8964b67c Nitrospirae,Nitrospira,Nitrospirales,Nitrospiraceae,Nitrospira,uncultured CytophagaX487938ee8db7ee225af7d436a786f35dsp.

Bacteria Nitrospirae,Nitrospira,Nitrospirales,Nitrospiraceae,Nitrospira,uncultured Cytophagad466a177444aa6de6e66e0deea85276dsp. Gemmatimonadetes,PAUC43f marine benthic group,uncultured bacteriumX7b5a4e76aa7ec0f07c2467f531d46ebd Gemmatimonadetes,BD2-11 terrestrial group X7102cae1cf4c0ebb2aa31aae83262db3 Proteobacteria,Gammaproteobacteria,UBA10353 marine group,uncultureda94a0644df888e8f6feb44023d6ec062marine microorganism 172c45f35d297ad0ee3b81127a498d00 Proteobacteria,Gammaproteobacteria a39e79aac5bccc4c2fcd7c890b503c18 7b5a4e76aa7ec0f07c2467f531d46ebd Proteobacteria,Gammaproteobacteria,MBMPE27,uncultured organismo eb1454cd3ea4e04645b7daa7bd870a62 32ecf533195692df5ae8f9541a3e6c74 93b26efe732c96ce11cd8c3fb2e06952 Proteobacteria,Gammaproteobacteria,SAR86 clade X1a756b47e4ba964da43b4534a0444d67 1a756b47e4ba964da43b4534a0444d67 Proteobacteria,Gammaproteobacteria,Steroidobacterales,Woeseiaceae,WoeseiaX1a39399e7ac7e34c44fd0cc95fee60b2 f52f08c2b73e4a5a0834a3832879ddb5 Proteobacteria,Gammaproteobacteria,Steroidobacterales,Woeseiaceae,WoeseiaX53b3094f4dcaca5bc9ab15befe087570 2ddc02abbee1b90a2ca309b1330657f2 Description Proteobacteria,Gammaproteobacteria,Steroidobacterales,Woeseiaceae,Woeseiaf678c23a94c5f321b59710249866a0d3 8f1247e1b8844b5ce2b17c28eef9c327 A_Crustbiofilm Proteobacteria,Gammaproteobacteria X62afa4a8c7e0f3ca0c472e5cbc608605 5ab8fc66f58ffe9d7dc761532711525c B_Crust Proteobacteria,Alphaproteobacteria,Rhodovibrionales,Kiloniellaceae,unculturedX8f1247e1b8844b5ce2b17c28eef9c327 c888a8be422c59d73606a87756bd28ec RelativeC_Coral 4a45810e794a2a2afcf7569cb3d09a4a Proteobacteria,Alphaproteobacteria,SAR11 clade,Clade I e997d2495b0d4675a1ed2438f0dc0b74 abundanceD_Calcarinita 942ede8b42183a4d89ef36f3f2a14099 Proteobacteria,Deltaproteobacteria,SAR324 clade(Marine group B) X0d7a1e88c49785f4f8b2bd0bf06059d0 (%) 260bdbb00143bd6ba0259b5aef72d4f0 value Proteobacteria,Deltaproteobacteria,SAR324 clade(Marine group B) fd754428af0482e40e7f86c88924009b 17987bbab15fa638153d93f2c7ae191e 0 Proteobacteria,Deltaproteobacteria,SAR324 clade(Marine group B) X72a7bb71aba22d70542ac8d718c74657 ASVs f1b553dc635274f2b8bcdfee10d892b5 25 Acidobacteria,Subgroup 9 f5f05c9d3db98b79993e5f848bc320f8 dc993a8d5a98020a0c869e37bed160bb ASVs Entotheonellaeota,Entotheonellia,Entotheonellales,Entotheonellaceae d491eaf7faf3577d844a09e033dab493 f5f05c9d3db98b79993e5f848bc320f8 50 Planctomycetes,Phycisphaerae,Phycisphaerales,Phycisphaeraceae,UraniaX9c835d2f90cf1a421ee16a306d2375cb-1B-19-marine-sediment d491eaf7faf3577d844a09e033dab493 Verrucomicrobia,Verrucomicrobiae,Arctic97B-4 marine group,uncultured marinee8baf93b0e1d51a1a26ec86c5666d372 bacterium 76fb46fb184f15a6d783ecf2f9d2b279 9c835d2f90cf1a421ee16a306d2375cb 75 Rokubacteria,NC10,Methylomirabilales,Methylomirabilaceae,wb1-A12 X6bb0d31020ab800d7122fac54a1adf20 db385e7c282235f90d8d5c0ef97d13eb Calditrichaeota,Calditrichia,Calditrichales,Calditrichaceae,JdFR-76 deb7f7382a716ba071797610e09eb326 dc01aef0374e18ba98426e78c1808521 Thaumarchaeota,Nitrososphaeria,Nitrosopumilales,Nitrosopumilaceae X3bb32e6ab5921715dd78b8f38d6f92f0 100 ab222d722fb80b6e78a4ea13c62e8b0a

Thaumarchaeota,Nitrososphaeria,Nitrosopumilales,Nitrosopumilaceae,unculturedX3d32055d938d6303e70fc4302d98c7d2 DCM863 6bb0d31020ab800d7122fac54a1adf20 Archaea Thaumarchaeota,Nitrososphaeria,Nitrosopumilales,Nitrosopumilaceae c5de2445abc7a2b461f4f526c8941184 d74e78b76046c4967e85f6ce8a309071 Thaumarchaeota,Nitrososphaeria,Nitrosopumilales,Nitrosopumilaceae fae997cbbae2ae61fdbbcfc23ded392b 5303a9f287db1fe3c836119beb5048a3 Thaumarchaeota,Nitrososphaeria,Nitrosopumilales,Nitrosopumilaceae,unculturedX5ca9c7c01b53a5caa40c71b3c1f2a1f6 marine archaeon 22799a1108aa6df7f27e4ce45b20be1d Thaumarchaeota,Nitrososphaeria,Nitrosopumilales,Nitrosopumilaceae b7cb5c9cd9cf896f45567b4e4d6a681a 19095d2c56afed5d0cd37d4d86ffb085 582d4ed267fd8f5b0dae2ea186829946 Thaumarchaeota,Nitrososphaeria,Nitrosopumilales,Nitrosopumilaceae,CandidatusX05c5422837223ef0648f3b3ef90d582f Nitrosopumilus 313421781896b1f418bb6b2b3e48b498 Thaumarchaeota,Nitrososphaeria,Nitrosopumilales,Nitrosopumilaceae,CandidatusX5fdbe921b7dfe6ae6c6493698474ec6c Nitrosopumilus 2 3 4 5 1 6 7 8 9 10 11 12 Thaumarchaeota,Nitrososphaeria,Nitrosopumilales,Nitrosopumilaceae,CandidatusX6c6ef46ca4c8f9ae4cb31ff08b6f0b73 Nitrosopumilus Samples Thaumarchaeota,Nitrososphaeria,Nitrosopumilales,Nitrosopumilaceae,CandidatusX6e5df728f168278052dbaba6f64c2c9b Nitrosopumilus

DY094_39_01 DY094_45_01 DY094_46_03 DY094_61_01 DY094_63_01N1 DY094_56_01N2 DY094_47_01N3 DY094_47_02N5 DY094_52_02N6 DY094_62_01N8 DY094_62_02N9 N10 N11 N12 N13 Samples Crust biofilm Crust Coral Calcarinite Fig. 5 Classification of the most abundant ASVs for Archaea and Bacteria (> 0.1% relative abundance) in the Fe-Mn crust biofilm, crusts, encrusted coral skeletons and calcarenites. The size of circles is related to the relative abundance of each ASV. ASVs are organized by phylum. Gray boxes at the bottom indicate sample substrates, i.e. crust biofilm, crust, encrusted coral skeleton and calcarenite

Microbial Predicted Function

methanotrophy

methanol_oxidation

methylotrophy

aerobic_ammonia_oxidation

aerobic_nitrite_oxidation

nitrification

sulfate_respiration

respiration_of_sulfur_compounds

nitrogen_fixation

cellulolysis

dark_sulfide_oxidation % relative dark_thiosulfate_oxidation 100

dark_oxidation_of_sulfur_compounds 75

manganese_oxidation 50 Function fermentation 25

aerobic_chemoheterotrophy 0

hydrocarbon_degradation

nitrate_respiration

nitrate_reduction

nitrogen_respiration

chloroplasts

photoautotrophy

photoheterotrophy

phototrophy

ureolysis

chemoheterotrophy

CrustA_Crustbiofilmbiofilm B_CrustCrust C_CoralCoral D_CalcareniteCalcarinite aDY094_39_01_ADY094_39_01bDY094_45_01_MBDY094_45_01cDY094_46_03_MBDY094_46_03dDY094_61_01_MBDY094_61_01eDY094_47_01_MBDY094_47_01gDY094_56_01_MBDY094_56_01hDY094_63_01_MBDY094_63_01iDY094_47_02_MBDY094_47_02jDY094_52_02_MBDY094_52_02lDY094_62_01_MBDY094_62_01mDY094_62_01_MB_1DY094_62_02 Sample_ID Fig. 6 Mean of relative abundances of microbial functional groups in Fe-Mn crust biofilm, crusts, encrusted coral skeletons and calcarenites. Relative abundances are depicted in terms of color intensity from white (0) to dark green (100)

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TABLE

Table 1. Samples list, sample type, latitude, longitude and depth from RGR.

Sample ID Sample Type Water mass Station LAT LONG Depth (m) DY094_63_01 Fe-Mn Crust SACW 63 30.704 35.704 663 DY094_56_01 Fe-Mn Crust SACW 56 30.838 36.016 664 DY094_47_01 Fe-Mn Crust SACW 47 30.875 35.981 762 DY094_46_01 Fe-Mn Crust SACW 46 30.854 35.005 685 DY094_47_02 Fe-Mn encrusted coral skeletons SACW 47 30.875 35.981 762 DY094_52_02 Fe-Mn encrusted coral skeletons AIA 52 31.009 35.943 903 DY094_62_01 Calcarenite SACW 62 30.698 35.742 661 DY094_62_02 Calcarenite SACW 62 30.698 35.742 661 DY094_39_01 Fe-Mn Crust Biofilm SACW 37 30.969 35.912 881 DY094_45_01 Fe-Mn Crust Biofilm SACW 39 30.005 35.245 758 DY094_46_03 Fe-Mn Crust Biofilm SACW 46 30.854 35.005 685 DY094_61_01 Fe-Mn Crust Biofilm SACW 42 30.689 35.746 711

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SUPPLEMENTARY MATERIAL

Supplementary Figure 1 Examples of (a) Fe-Mn crust biofilm on Fe-Mn crust, (b) Fe-Mn crust, (c) calcarenite and (d) Fe-Mn encrusted coral skeletons collected during the scientific expedition on the RRS Discovery on the RGR

a b

c d

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Supplementary Table 1. Sum, mean, standard error (S. error) for number of sequences, number of amplicon sequence variants (ASVs), Chao1 and Shannon indexes for Fe-Mn crust, crust biofilm, encrusted coral skeleton and calcarenite from the RGR.

Sample No. of Sequences No. of ASVs Chao1 Shannon Fe-Mn Crust Biofilm DY094_39_01 12906 42 120 3.07 DY094_45_01 10347 36 153 4.00 DY094_46_03 49568 49 303 3.24 DY094_61_01 47539 62 347 2.91 Sum 120360 189 923 13.22 Mean ± SD 38236.33 ± 5.59 47.25 ± 5.59 230.75 ± 55.56 3.30 ± 0.24 Fe-Mn Crust DY094_63_01 38870 10 20 1.38 DY094_56_01 23042 73 443 5.62 DY094_47_01 52797 10 26 2.48 Sum 114709 93 489 9.48 Mean ± SD 38236.33 ± 8595.37 31.00 ± 21.00 163.00 ± 140.00 3.16 ± 1.27 Fe-Mn Coral skeleton DY094_47_02 27988 41 165 4.72 DY094_52_02 94103 16 46 3.14 Sum 122091.00 57.00 211.00 7.85 Mean ± SD 61045.50 ± 33057.50 28.50 ± 12.50 105.50 ± 59.50 3.93 ± 0.79 Calcarenite DY094_62_01 33177 36 207 4.93 DY094_62_02 19009 71 525 5.82 Sum 52186.00 107.00 732.00 10.75 Mean ± SD 26093.00 ± 7084.00 53.50 ± 17.50 366 ± 159 5.38 ± 0.45

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Supplementary Table 2. Index Species result with significant amplicon sequence variants (ASVs) and taxonomy organized by substrate.

ASVs Taxonomy and Substrate stat P value Fe-Mn Crust ASV1055 Bacteria_Proteobacteria_Gammaproteobacteria 0.911 0.01 Fe-Mn Crust biofilm ASV0706 Archaea_Thaumarchaeota_Nitrososphaeria_Nitrosopumilales_Nitrosopumilaceae_uncultured 0.877 0.01 Fe-Mn encrusted coral skeletons ASV0714 Bacteria_Proteobacteria_Alphaproteobacteria_Rhizobiales_KF-JG30-B3 0.975 0.04 ASV1608 Bacteria_Planctomycetes_Planctomycetacia_Pirellulales_Pirellulaceae_Bythopirellula 0.919 0.04 Calcarenite ASV0769 Bacteria_Planctomycetes_Phycisphaerae_Phycisphaerales_Phycisphaeraceae Urania-1B-19 marine sediment group 1.000 0.04 ASV0719 Archaea_Thaumarchaeota_Nitrososphaeria_Nitrosopumilales_Nitrosopumilaceae_Candidatus Nitrosopumilus 1.000 0.04 ASV0303 Archaea_Thaumarchaeota_Nitrososphaeria_Nitrosopumilales_Nitrosopumilaceae_Candidatus Nitrosopumilus 1.000 0.04 ASV1012 Bacteria_Gemmatimonadetes_Gemmatimonadetes_Gemmatimonadales_Gemmatimonadaceae_uncultured 1.000 0.04 ASV0591 Bacteria_Acidobacteria_uncultured Acidobacteriales bacterium 1.000 0.04 ASV1396 Bacteria_Nitrospirae_Nitrospira_Nitrospirales_Nitrospiraceae_Nitrospira 0.999 0.04 ASV1159 Archaea_Thaumarchaeota_Nitrososphaeria_Nitrosopumilales_Nitrosopumilaceae 0.999 0.04 ASV1062 Bacteria_Proteobacteria_Alphaproteobacteria_uncultured 0.998 0.04 ASV0529 Bacteria_Proteobacteria_Gammaproteobacteria_EPR3968-O8a-Bc78v 0.997 0.04 ASV0883 Bacteria_Proteobacteria_Alphaproteobacteria_Thalassobaculales_uncultured 0.997 0.04 ASV1622 Bacteria_Proteobacteria_Gammaproteobacteria 0.993 0.04 ASV0573 Archaea_Thaumarchaeota_Nitrososphaeria_Nitrosopumilales_Nitrosopumilaceae 0.993 0.04 ASV0551 Archaea_Thaumarchaeota_Nitrososphaeria_Nitrosopumilales_Nitrosopumilaceae_Candidatus Nitrosopumilus 0.992 0.04 ASV1437 Bacteria_Planctomycetes_Phycisphaerae_Phycisphaerales_Phycisphaeraceae_Urania-1B-19 marine sediment 0.989 0.04 ASV1789 Bacteria_Proteobacteria_Alphaproteobacteria_uncultured 0.988 0.04

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ASV1709 Bacteria_Gemmatimonadetes_PAUC43f marine benthic group_uncultured bacterium 0.987 0.04 ASV0716 Bacteria_Proteobacteria_Alphaproteobacteria_uncultured 0.985 0.04 ASV1856 Bacteria_Proteobacteria_Alphaproteobacteria_uncultured 0.980 0.04 ASV0333 Archaea_Thaumarchaeota_Nitrososphaeria_Nitrosopumilales_Nitrosopumilaceae 0.971 0.04 ASV0085 Archaea_Thaumarchaeota_Nitrososphaeria_Nitrosopumilales_Nitrosopumilaceae_CandidatusNitrosopumilus_uncultured 0.969 0.04 ASV0255 Bacteria_Proteobacteria_Deltaproteobacteria_Myxococcales_bacteriap25 0.948 0.04 ASV1024 Bacteria_Planctomycetes_Phycisphaerae_Phycisphaerales_Phycisphaeraceae_Urania-1B-19 marine sediment group 0.941 0.04 ASV1811 Bacteria_Nitrospirae_Nitrospira_Nitrospirales_Nitrospiraceae_Nitrospira 0.940 0.04 ASV0093 Bacteria_Proteobacteria_Gammaproteobacteria_Nitrosococcales_Nitrosococcaceae_Inmirania_uncultured 0.843 0.04 Crust and Calcarenite ASV0807 Bacteria_Rokubacteria_NC10_Methylomirabilales_Methylomirabilaceae_wb1-A12 0.877 0.0106

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Supplementary Table 3. Microbial functional groups predicted in the Fe-Mn crust biofilm, crust, encrusted coral skeleton and calcarenite from the RGR, and the number of bacterial and archaeal amplicon sequencing variants (ASVs) identified for each functional group.

Microbial Function Predicted ASVs assigned chemoheterotrophy 119 aerobic_chemoheterotrophy 113 nitrification 124 aerobic_ammonia_oxidation 81 aerobic_nitrite_oxidation 43 predatory_or_exoparasitic 10 intracellular_parasites 10 fermentation 9 animal_parasites_or_symbionts 9 human_pathogens_all 8 nitrate_reduction 3 dark_oxidation_of_sulfur_compounds 3 nitrogen_fixation 2 dark_sulfide_oxidation 2 nitrogen_respiration 2 nitrate_respiration 2 phototrophy 2 ureolysis 2 chloroplasts 2 methylotrophy 2 sulfate_respiration 1 respiration_of_sulfur_compounds 1 hydrocarbon_degradation 1 manganese_oxidation 1 dark_thiosulfate_oxidation 1 cellulolysis 1 fish_parasites 1 methanotrophy 1 methanol_oxidation 1 cyanobacteria 1 oxygenic_photoautotrophy 1 photoautotrophy 1 photoheterotrophy 1