Microbes Environ. Vol. 33, No. 2, 186-194, 2018 https://www.jstage.jst.go.jp/browse/jsme2 doi:10.1264/jsme2.ME17194

Microbial Diversity in Sediments from the Bottom of the , the

Takuro Nunoura1*, Manabu Nishizawa2, Miho Hirai1, Shigeru Shimamura2, Phurt Harnvoravongchai3†, Osamu Koide1, Yuki Morono4,5, Toshiaki Fukui3, Fumio Inagaki4,5,6, Junichi Miyazaki2, Yoshihiro Takaki1,2, and Ken Takai2 1Research and Development Center for Marine Biosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2–15 Natsushima-cho, Yokosuka 237–0061, Japan; 2Department of Subsurface Geobiological Analysis and Research, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2–15 Natsushima-cho, Yokosuka 237–0061, Japan; 3School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226–8501, Japan; 4Geomicrobiology Group, Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Science Technology (JAMSTEC), Nankoku, 783–0093, Japan; 5Geobiotechnology Group, Research and Development Center for Submarine Resources, Japan Agency for Marine-Earth Science Technology (JAMSTEC), Nankoku, 783–0093, Japan; and 6Research and Development Center for Drilling Science, Japan Agency for Marine-Earth Science Technology (JAMSTEC), Yokohama 236–0001, Japan (Received December 7, 2017—Accepted February 27, 2018—Published online May 25, 2018)

The Challenger Deep is the deepest ocean on Earth. The present study investigated microbial community structures and geochemical cycles associated with the trench bottom sediments of the Challenger Deep, the Mariana Trench. The SSU rRNA gene communities found in trench bottom sediments were dominated by the bacteria Chloroflexi (SAR202 and other lineages), Bacteroidetes, Planctomycetes, “Ca. Marinimicrobia” (SAR406), and Gemmatimonadetes and by the archaeal α subgroup of MGI Thaumarchaeota and “Ca. Woesearchaeota” (Deep-sea Hydrothermal Vent Euryarchaeotic Group 6). The SSU rRNA gene sequencing analysis indicated that the dominant populations of the thaumarchaeal α group in hadal water and sediments were similar to each other at the or genus level. In addition, the co-occurrence of nitrification and denitrification was revealed by the combination of pore water geochemical analyses and quantitative PCR for nitrifiers.

Key words: Hadal, Mariana Trench, nitrogen cycle, nitrification

The hadal zone of the ocean, composed of deep trenches compounds and (micro-) organisms entrained by inter trench below a water depth of 6,000 m, is similar to the abyssal and currents. Microbiological studies on hadal benthic habitats bathyal zones in temperature, salinity, and the concentrations started in the 1950s based on culture-dependent techniques of oxygen, nitrate, and other major geochemical components (1, 21), and bottom sediments in the Mariana Trench are one (21, 32, 58). On the other hand, the steep and narrow geomor- of the most intensively studied environments in the history of phology of trenches supplies the hadal zone with suspended hadal benthic microbiology (22, 23, 25, 27, 30, 33, 46–48, 59, sediments and organic compounds, possibly due to occasional 61–64, 66, 71). However, microbial diversity and functions landslides, and supports unique microbial in hadal related to the benthic geochemical cycle in the hadal sediment water and sediments (12, 20, 41, 68). The microbial commu- have not yet been examined in detail, although greater microbial nities in hadal water are characterized by an enrichment of activity and have been reported in hadal sediments potentially heterotrophic microbial populations, while those than in adjacent sediments (12). In the present in overlying abyssal water are predominated by potentially study, we investigated microbial community structures and chemolithotrophic populations (41). Microbial activity levels the nitrogen cycle as well as geochemical gradients in hadal and cell abundance are greater in trench sediments than in sediments of the Challenger Deep, the Mariana Trench. adjacent abyssal plain sediments, and the trench benthic microbial maintains the efficient mineralization of Materials and Methods organic compounds (12, 68). The Mariana arc-trench system is a non-accretionary con- Site description and sampling vergent plate margin between the Philippine Sea plate and Three dives of the ROV ‘ABISMO’ were conducted in the Challenger subducting Pacific plate (44). In contrast to the other trenches Deep of the Mariana Trench (11°22.25'N, 142°42.75'E, 10,300 m) located in the North-West Pacific region, the Mariana Trench during the JAMSTEC R/V Kairei KR08-05 cruise (June 2008) (70). is topographically isolated from other trenches by the impinging Temperature, depth, and salinity were measured using an SBE49 Ogasawara Plateau and Caroline Ridge (19). Thus, these CTD sensor (Sea-Bird Electronics, Bellevue, WA, USA). Water topographical barriers may interrupt the transfer of organic sampling from the sea surface to near the bottom of the trench (water depth of 10,257 m) and sample processing and storage were described previously (41). A sediment core (#AB11) was obtained * Corresponding author. E-mail: [email protected]; from the trench bottom by a gravity corer on the ROV ‘ABISMO’ in Tel: +81–46–867–907; Fax: +81–46–867–9715. the same dive. Sediment cores were divided by every 5 cm above † Present address: Department of Biology, Faculty of Science, Mahidol 20 cm below the seafloor (cmbsf) and every 10 cm below 20 cmbsf, University, Bangkok, 10400, Thailand. and subsamples for molecular analyses were stored at –80°C. Pore Microbial Diversity in Hadal Sediments 187 water from each section was obtained by centrifugation at 4,000×g for PCR amplification. In the tag sequencing of the prokaryotic SSU for 10 min and filtration using a syringe cartridge filter (0.2 mm). rRNA gene, the primer set 530F/907R with 10 bp of extended tag Pore water samples for nutrient analyses were stored at –20°C. sequences in the 5′ termini was used for SSU rRNA gene amplification (39). These SSU rRNA gene fragments were amplified from envi- Geochemical analyses ronmental DNA assemblages using LA Taq polymerase with GC Pore water pH and alkalinity were measured onboard using a 794 buffer (Takara Bio, Kusatsu, Japan). Basic Titrino titration system (Metrohm, Herisau, Switzerland), and In the archaeal amoA clone analysis, the primer set arch-amoAF/ potentiometric titration was performed with 0.1 M HCl per 2 mL of arch-amoAR (10) was used with EX Taq polymerase (Takara Bio) 2– pore water. The pore water samples used for SO4 and nutrient to amplify gene fragments from the environmental DNA assem- concentration measurements were filtrated using a cartridge filter blages as described previously (40). In order to amplify hzsA, the 2– with a pore size of 0.2 μm. SO4 concentrations were analyzed using primer set hzsA 526F/hzsA 1857R (17) was applied using LA Taq the personal ion analyzer PIA-1000 (Shimadzu, Kyoto, Japan). The DNA polymerase with Mg2+ buffer as described previously (40). In remaining pore water samples were stored at –20°C. After the cruise, the amplification of mcrA, the primer set ME3MF/ME2r’ (38) was – – + NO3 , NO2 , PO4, and NH4 were analyzed spectrophotometrically used with SYBR Premix Ex Taq II (Takara Bio) in the presence of using an automated QuAAtro 2-HR analyzer (BL TEC, Osaka, Japan). MgCl2 (final 2.5 mM) as described previously (38). The contents and stable isotopic ratio of total organic carbon and In clone analyses, amplified DNA fragments were cloned into the nitrogen were estimated with a Flash EA1112 elemental analyzer pCR2.1 vector (Invitrogen), and clone libraries were constructed. (Thermo Fisher Scientific, Waltham, MA, USA) at SI Science The inserts were directly sequenced with the M13M4 primer using (Sugito-machi, Saitama, Japan). an ABI3730xl genetic analyzer with Big Dye v. 3.1 (Applied Biosystems, The stable nitrogen and oxygen isotopic compositions of pore Foster City, CA, USA). SSU rRNA gene sequences with >97% water nitrate were assessed using the denitrifier method (6, 55). identity were assigned the same clone type (phylotype) based on After the removal of nitrite by the addition of sulfamic acid to pore FastGroup II (72) and a similarity analysis in GENETYX-MAC v. water samples (14), nitrate was quantitatively converted to nitrous 15 (GENETYX, Tokyo, Japan). In the clone analysis, representative oxide using the strain Pseudomonas chlororaphis (JCM20509= SSU rRNA gene sequences were aligned and phylogenetically clas- ATCC13985), a denitrifying bacterium lacking the ability to reduce sified into certain taxonomic units using ARB (50). Archaeal amoA N2O. After the online extraction and purification of the resultant gene sequences with >96% identity were assigned the same phylo- nitrous oxide (36), nitrogen and oxygen isotopic ratios were measured type, and the phylogenetic tree was constructed by Clustal X based using a continuous-flow isotope ratio mass spectrometer (CF-IRMS) on unambiguous residues (29). (Delta V Plus, Thermo Fisher Scientific) at JAMSTEC. Corrections The SSU rRNA gene amplicons for tag sequencing were analyzed for the drift, size, and fractionation of oxygen and nitrogen isotopes by a 454 FLX Titanium sequencer (Roche, Basel, Switzerland). All during the procedures were made using international nitrate standards: of the raw tag sequences were analyzed with the shhh.flows pipeline USGS32, USGS34, USGS35, and IAEA-NO3. in MOTHUR 1.31.1 (51, 53, 54), and the primer sequences in either The isotope ratios of 13C/12C, 15N/14N, and 18O/16O are reported as or both ends of the tags were eliminated. Tag sequences shorter than δ values, in which δ=(Rsam/Rstd–1)×1,000, with R being the isotope 300 bp were removed from downstream analyses. Potentially chimeric ratios in the sample and standard, respectively. The carbon isotope sequences were surveyed using UCHIME (9). Phylogenetic assign- ratio of total organic carbon was referenced against Vienna Pee Dee ments and statistical analyses for the tag sequences were then Belemnite (V-PDB). The nitrogen isotope ratios of total nitrogen conducted. Sequencing tags were aligned using the partial order 18 and nitrate were referenced against atmospheric N2, and the δ O algorithm (POA) (SINA, http://www.arb-silva.de/aligner/) with values of nitrate were assessed relative to Vienna Standard Mean SILVA SSU Ref NR as a reference for multiple alignments (69). All Ocean Water (V-SMOW). of the aligned sequences were then clustered into operational The precision rates achieved through repeated analyses of an taxonomic units (OTUs) based on 97% sequence identity using in-house nitrate standard were typically better than 0.2‰ for δ15N MOTHUR 1.31.1 with the default parameters according to the and δ18O. average-clustering algorithm. The output was then parsed to produce occurrence tables of each OTU in each sample. The taxonomic Cell abundance position of each OTU was automatically assigned based on a BLAST The abundance of microbial cells in the sediment samples was analysis in the QIIME software package (5) using SILVA Ref NR as measured using an image-based, discriminative cell-counting tech- a reference dataset of SSU rRNA gene sequences. The sequences nique described previously (34). In brief, a paraformaldehyde-fixed that are closely related to the potential contaminants belonging to slurry sample was mildly sonicated at 5 W for 1 min with an UH-50 genera that inhabit soil and the human body and that have been ultrasonic homogenizer (SMT, Tokyo), and then treated with 1% detected from negative control experiments of environmental micro- fluoride (HF) for 20 min. An aliquot was filtered onto biology in our lab, such as Bradyrhizobium, Brevundimonas, polycarbonate filters with a pore size of 0.2 μm (Isopore; Merck Burkholderiaceae, Delftia, Erythrobacter, Lactococcus, Legionella, Millipore, Billerica, MA, USA). A microscopic count of SYBR Methylobacterium, Mycobacterium, Neisseria, Novosphingobium, Green I-stained cells was performed by an automated SYBR DiCE Propionibacterium, Sphingobium, Sphingomonas, Sphingopyxis, system (34), and >140 of acquired images were processed by Staphylococcus, Stenotrophomonas, and Streptococcus, were excluded MetaMorph software (Molecular Devices, Downingtown, PA, USA). (43). Sequences with relatively high E-values (>1.0E–30) or low identity (<90%) to the best match sequence were designated as other DNA extraction archaea or bacteria, and sequences that did not show significant Approximately 5 mL of each of the frozen sediment samples from similarity to any reference sequences were also excluded from the certain sections of the sediment core was used for DNA extraction. analysis. Alpha diversity indices (rarefaction curves, Chao1, ACE, Environmental DNA was extracted using a PowerMax Soil Shannon, Shannon evenness, and Simpson) in each library and DNA Isolation Kit (MoBio Lab, Carlsbad, CA, USA) with minor taxa/divisions were also calculated using MOTHUR 3.6. Jaccard modifications. and Bray-Curtis dissimilarity indices were estimated for each library using the vegan package in the R environment (http:// Sequencing analyses vegan.r-forge.r-project.org). Phylogenetic trees were constructed The amplification conditions and primer sequences for each PCR from the curated alignment of representative sequences using the amplification are summarized in Table S1. In clone analyses of Clustal W program (http://www.clustal.org). A weighted UniFrac archaeal and bacterial SSU rRNA genes, the primer sets Arch21F/ distance matrix of sediment samples was constructed from the Arch958R (8) and Bac27F/Bac927R (8, 28) were used, respectively, phylogenetic tree and a sample mapping files that showed the 188 Nunoura et al. frequencies of the sequence tags within OTUs. Sequencing tags for and archaeal amoA gene sequences (LC049988–LC050107 and each phylum/class were collected from the entire dataset in accor- LC050108–LC050123, respectively), and in the Short Read Archive dance with the taxonomic position of each OTU. Sub-datasets were database: environmental SSU rRNA tags (DRA006342). analyzed in the same manner as the entire dataset.

Quantitative PCR (qPCR) analyses Results and Discussion The primers, probes, components of the standard mixture, and amplification conditions used for each qPCR are summarized in Table S1. The abundance of each gene was quantified as an average Pore water chemistry and organic geochemistry + – – 2– of duplicate or triplicate analyses. The 7500 Real Time PCR System The nutrient (NH4 , NO3 , NO2 , and PO4) and SO4 (Applied Biosystems) was used for all qPCR analyses. concentrations, pH, and alkalinity of pore water were analyzed The quantification of prokaryotic and archaeal SSU rRNA genes for the sediment core #AB11, which was taken from the was conducted using the primer and probe sets Uni340F/Uni516F/ Uni806R and Arch349F/Arch516F/Arch806R, respectively (60). Challenger Deep (water depth of 10,300 m) (Fig. 1 and Table – The abundances of Nitrospina, Seafloor Nitrospina-Like group S2). NO3 concentrations were depleted to less than approxi- – (SFLNG) and Nitrospira SSU rRNA genes were also examined mately 5 μM below 85 cmbsf. The NO2 profile presented two according to methods described previously (13, 40). In order to peaks: a shallow peak at 15 cmbsf and a deeper peak between quantify archaeal amoA, group-specific primer sets for groups A, 45 and 105 cmbsf. The PO4 profile presented an anomalous Ba, Bb, and D (2, 41) were used for qPCR. Betaproteobacterial inflection at 1 mbsf. The SO 2– profiles indicated that micro- amoA was also quantified as described previously (57). Amplified 4 products from qPCR using the SYBR Premix reagent were confirmed bial sulfate reduction was negligible in the sediment core. by agarose gel electrophoresis. In order to prepare qPCR mixtures, Total organic carbon (TOC) and total nitrogen (TN) ranged qPCR Quick GoldStar Mastermix Plus (Eurogentec, Seraing, Belgium) between 0.074 and 0.27 wt% and between 0.009 and 0.041 wt%, was applied for the SSU rRNA genes of archaea, all prokaryotes, respectively. The stable isotopic ratios of the organic com- and Nitrospira, and SYBR Premix Ex Taq II (Takara Bio) was used pounds δ13C and δ15N ranged between –19.0 and –22.2‰ and for archaeal and betaproteobacterial amoA genes and Nitrospina and between 11.3 and 13.5‰, respectively (Fig. 1). The TOC Seafloor Nitrospina-Like group (SFNLG) SSU rRNA genes. concentrations obtained in the present study were slightly Accession numbers lower than those reported previously for Challenger Deep 15 The nucleotide sequences obtained in this study have been deposited sediments (12). δ N values at this site were close to the in the DDBJ/EMBL/GenBank database: environmental SSU rRNA highest δ15N value of sedimentary organic compounds in the

– – + 2– Fig. 1. Pore water geochemistry: alkalinity and pH (A), and concentrations of NO3 (B), NO2 (C), NH4 (D), PO4 (E), and SO4 (F). Sediment organic geochemistry: total organic compounds (TOC) and total nitrogen (TN) (G), as well as δ13C TOC and δ15N TN (H) of sediment core #AB11 taken from the Challenger Deep, the Mariana Trench. Microbial Diversity in Hadal Sediments 189 modern ocean (65), and may have originated from primary production with highly 15N-enriched nitrate transported on the North Equatorial current.

15 18 Interpretation of δ NNO3– and δ ONO3– 15 15 The δ N value of nitrate in pore water (δ NNO3–) ranged 18 between 4.4 and 17.8‰, and the δ ONO3– value ranged 15 18 between 2.8 and 20.7‰. δ NNO3– and δ ONO3– values increased with depth up to 75 cmbsf (Fig. 2A and Table S2). The δ15N–δ18O relationship of pore water nitrate revealed signatures to estimate the potential contributions of the in situ nitrate supply (nitrification) and consumption (dissimilatory nitrate reduction) to the dynamic nitrogen cycle in the subsur- face biosphere (40, 67). The δ15N–δ18O relationship of pore 15 water nitrate changed at a depth of 50 cmbsf. The δ NNO3–– 18 δ ONO3– relationship exhibited a slope of 3.2 (±0.5) in the shallower interval above 50 cmbsf, and 0.95 (±0.10) in the deeper interval between 50 cmbsf and 80 cmbsf. The 15 18 down-core decrease in the slope of the δ NNO3––δ ONO3– relationship was also observed for trench bottom sediments in the Ogasawara Trench (40). 15 18 The similar increases observed in δ NNO3– and δ ONO3– values with decreases in nitrate concentrations in the deeper interval may be explained by equivalent nitrogen vs oxygen isotope fractionation during denitrification (i.e., the preferential breakage of 14N-16O bonds by respiratory nitrate reductase) 18 (15) (Fig. 2B). In contrast, a preferential increase in δ ONO3– 15 over δ NNO3– with decreases in nitrate concentrations in the shallower interval required the involvement of nitrification because the δ15N and δ18O values of nitrate from nitrification 15 18 (δ NNTR and δ ONTR) are set independently of each other. In the shallower interval, ammonium from the remineralization of organic matter and nitrite did not accumulate with depth, except for 12.5 cmbsf and 17.5 cmbsf, as discussed below. 15 15 Thus, δ NNTR was expected to be closer to the δ N of ambient organic matter (11‰; Fig. 1H) because the δ15N of ammonium is generally similar to that of host organic matter 15 18 – 18 Fig. 2. (A) δ N and δ O profiles of pore water NO3 in the sediment (±1‰) (11, 49). In contrast, δ ONTR is mainly set by the δ18O core #AB11 taken from the Challenger Deep, the Mariana Trench. (B) 15 18 values of seawater (0‰) and dissolved O2 (≥23.5‰) as well The relationship between pore water nitrate δ N and δ O values. In as kinetic isotope effects during the multistep oxidation of comparisons, based on experimental results, fractionation lines constraining changes in nitrate δ15N and δ18O values during nitrate reduction were ammonium to nitrite (3, 7, 37). Using available data on plotted (slopes 18ε/15ε=1) (14, 16). 18 oxygen isotope effects during nitrification, the δ ONTR value in oceanic sediments was estimated to have a value between 18 18 –6 and 0‰ when the δ O value of pore water O2 (δ OO2) interval, phosphate concentrations and alkalinity synchronously was equal to that of bottom seawater (ca. 27‰). The calcula- increased with depth, while ammonia concentrations did not 18 18 tion of δ ONTR was shown below. Furthermore, the δ ONTR change significantly (Fig. 1). Nitrate concentrations decreased value in oceanic sediments is expected to increase to ≥5‰ with depth. These contrasting changes suggest nitrification 18 with depth due to downward increases in δ OO2 by aerobic coupled with an ammonia supply from the remineralization of 18 18 respiration ( ε=20±3‰) (24). For example, the δ ONTR organic matter via aerobic and anaerobic (=nitrate) respiration value was predicted to increase to 6~11‰ when 88% of in the shallower interval. In contrast, in the deeper interval, dissolved O2 in bottom seawater was consumed in sediments phosphate and ammonium concentrations and alkalinity did 18 (δ OO2=70‰). Therefore, the depth profile of dual isotope not change significantly with depth, whereas nitrate concen- compositions and nitrate concentrations in pore water may be trations decreased with depth. These changes do not support mostly explained by the coupling of nitrification and denitri- nitrification, but suggest denitrification. fication in the shallower interval, whereas denitrification in 18 18 Calculation of the δ ONTR value the deeper interval though the depth profile of δ OO2 was not measured at this site. The δ18O value of nitrate produced from the sequential oxi- 18 A metabolic shift from the coupling of nitrification and dation of ammonia via nitrite (δ ONTR) is expressed as follows denitrification to denitrification with depth is consistent with when the intermediate nitrite pool does not accumulate (4): 18 18 18 18 the nutrient concentration profiles in pore water. In the shallower δ ONTR=(1–XNOB) (2/3*δ ONO2–,pro+1/3*[δ OH2O– εk,H2O,NOB]) 190 Nunoura et al.

18 18 5 6 –1 +(δ OH2O+ εeq)(XNOB) (eq. 1), where XNOB is the fraction of between 1.8×10 and 9.4×10 copies g sediment. These results nitrite oxygen atoms that have equilibrated with water catalyzed showed that microbial abundance generally decreased with 18 18 by NOB. δ ONO2–,pro is the δ O value of nitrite produced increases in depth with a few exceptions; however, several from archaeal and bacterial ammonia oxidation. The kinetic gaps were observed between direct cell counts and SSU isotope effect for oxygen atom incorporation from water is rRNA gene copy numbers. These gaps may have been the 18 denoted as εk,H2O,NOB, whereas the equilibrium fractionation result of biases in DNA extraction, PCR inhibitors in environ- of oxygen isotopes between nitrite and water is denoted as mental DNA solutions, primer and probe mismatches, and 18 εeq (15‰ at 4°C). variance in SSU rRNA copy numbers, as discussed previ- Previous culture experiments showed that ambient water ously (e.g., 18, 31, 35). The relative abundance of the archaeal and dissolved oxygen are oxygen sources for nitrite produced SSU rRNA gene in all of the prokaryotic SSU rRNA genes during archaeal and bacterial ammonia oxidation (7, 37). In estimated by qPCR was higher in surface sediments and their experiments, the δ18O value of nitrite produced from lower in core bottom sediments. 18 archaeal and bacterial ammonia oxidation (δ ONO2–,pro) was 18 18 18 SSU rRNA gene diversity analyses expressed as follows: δ ONO2–,pro={0.5(δ OO2– εk,O2,AO)+ 18 18 18 18 0.5(δ OH2O– εk,H2O,AO)}(1–XAO)+(δ OH2O+ εeq)(XAO)= SSU rRNA gene diversity was analyzed using cloning and 18 18 18 18 0.5(1+XAO)δ OH2O+0.5(1–XAO)(δ OO2– εk,O2,AO– εk,H2O,AO) tag-sequencing techniques. Samples at depths of 5, 12.5, 45, 18 +(XAO) εeq (eq. 2), where XAO is the fraction of nitrite oxygen 65, 100, and 123 cmbsf were used for the SSU rRNA gene atoms that have equilibrated with water catalyzed by AOA or clone analysis, and those at depths of 5, 12.5, 65, and 123 AOB. The kinetic isotope effects for oxygen atom incorpora- cmbsf were used for SSU rRNA gene tag sequencing. 18 tion from dioxygen and water are denoted as εk,O2,AO and A total of 267 bacterial SSU rRNA gene sequences (42 to 18 εk,H2O,AO, respectively. 47 sequences for each sample) and 322 archaeal SSU rRNA 18 18 Using experimental data on XAO, εk,O2,AO+ εk,H2O,AO, and gene sequences (44 to 90 sequences for each sample) were 18 18 εeq (37), the potential δ ONO2–,pro value in trench bottom obtained in the clone analysis (Table S3 and S4). Bacterial 18 sediments was estimated (–1‰ to 7‰ if δ OO2=27‰). The SSU rRNA gene communities were generally similar to each 18 potential δ ONTR value in trench bottom sediments was then other, except for the sample from a depth of 123 cmbsf. estimated from equation 1 using the experimental values of Sequences from Alphaproteobacteria, Chloroflexi, and 18 XNOB (0.02) and εk,H2O,NOB (15‰) (3). We assumed 27‰ as Gemmatimonadetes were detected as the dominant popula- 18 the δ OO2 value of bottom seawater in the Mariana Trench tions in all of the bacterial clone libraries, and the relative –1 (170 μmol O2 kg ) based on the empirical relationship abundance of Chloroflexi phylotypes increased in the sample 18 between δ OO2 and O2 concentrations in the deep ocean (26). from a depth of 123 cmbsf. The SAR202 group was abundant 18 The experimental value of εeq at 4°C was previously in the Chloroflexi population in the libraries from depths reported to be 15‰ (4). above 100 cmbsf; however, its relative abundance decreased at a depth of 123 cmbsf. Archaeal SSU rRNA gene clone Microbial abundance libraries were dominated by only one phylotype MCD_ Cell abundance above 123 cmbsf ranged between 6.3×105 AB10_5cm_A01, which belonged to the α subgroup of the and 1.5×108 cells mL–1 sediment (Fig. 3). In the qPCR analysis, Marine Group I (MGI) Thaumarchaeota, including the genus the abundance of the entire prokaryotic SSU rRNA gene Nitrosopumilus. MCD_AB10_5cm_A01 was very similar to ranged between 3.9×105 and 1.1×107 copies g–1 sediment. the hadal water phylotype MW_10000m_04 (similarity The copy number of the archaeal SSU rRNA gene ranged >98%). Subgroup γ MGI, another abundant MGI population

Fig. 3. Profiles of direct cell counts (A) and copy numbers of whole prokaryotic and archaeal SSU rRNA genes (B), amoA genes (C), and SSU rRNA genes of nitrite oxidizers (D) in sediment core #AB11 taken from the Challenger Deep, the Mariana Trench. Groups D and A and Beta (C) indicate the archaeal amoA of groups D and A and betaproteobacterial amoA, respectively. SFNLG (D) indicates the potential nitrite-oxidizing Subseafloor Nitrospina-Like Group. Microbial Diversity in Hadal Sediments 191

Fig. 4. Composition of SSU rRNA gene tags of microbial communities from hadal water and trench bottom sediment (sediment core #AB11) in the Challenger Deep, the Mariana Trench. in hadal water (41), did not dominate in benthic archaeal 16S the thaumarchaeal SSU rRNA and amoA gene clusters was rRNA gene communities. suggested as follows: group α in the SSU rRNA gene and D As a result of tag sequencing, 53886 rRNA gene tags (each in amoA, β and A, γ and Ba, and δ and Ba (41). Thus, the from 10547 to 12402 tags) were obtained from the four DNA predominance of group D was expected in the archaeal amoA assemblages after removing non-SSU rRNA gene tags, gene communities based on the SSU rRNA gene clone analysis, shorter tags, potential contaminants, and chimeric sequences while it was not the most abundant population in the amoA (Fig. 4 and Table S5). SSU rRNA gene tag populations were clone analysis. A similar contradiction between the SSU generally similar to the SSU rRNA gene communities shown rRNA and amoA gene clone analyses was also observed in a by the clone analysis. However, several differences were previous study on the Mariana Trench, and the found between the SSU rRNA gene communities obtained by contradiction was suggested to be a result of biases in the the two techniques. A notable difference was that “Candidatus amoA gene clone analysis (41). (Ca.) Woesearchaeota” (Deep-sea Hydrothermal Vent We then investigated the abundance of each group of Euryarchaeotic Group 6 [DHVE6]) (45) was dominant in the archaeal amoA genes using group-specific primer sets. As a tag communities, but was not detected in the classical clone result, only subgroups A and D were detected in qPCR, while analysis. Moreover, the abundance of the MGI tags markedly Ba and Bb were not (Fig. 3). The maximum abundance of decreased at a depth of 123 cmbsf, whereas the MGI popula- subgroup D amoA (2.6×106 copies g–1 sediment) was found in tion dominated in all of the archaeal SSU rRNA gene clone the surface layer and decreased with increasing depth. The libraries; the relative abundance of MGI tags in all of the abundance of subgroup A amoA was less than 3.8×102 copies g–1 archaeal tags in a sample from a depth of 65 cmbsf was 62%, sediment. Considering the detection limits and experimental and that from a depth of 123 cmbsf was 2%. The relative system of qPCR for subgroups Ba and Bb, the abundances of abundances of Bacteroidetes and Planctomycetes in the tag these groups in the surface sediments were less than 2.9×104 communities were also higher than those in the SSU rRNA and 5.7×102 copies g–1 sediment, respectively. This result is gene libraries. At several depths, a potentially methanogenic/ consistent with the results of the SSU rRNA gene community methanotrophic archaeal lineage was found to be a minor analyses that indicated the predominance of group α MGI in population in the tag community and clone library. the thaumarchaeal populations. Betaproteobacterial amoA was also detected in the sediments, with maximum abundance Amplification and diversity of functional genes; amoA, mcrA, being found at 45 cmbsf (8.4×104 copies g–1 sediment). and hzsA, and qPCR of amoA genes The amplification of the archaeal amoA and mcrA genes qPCR analyses for SSU rRNA of nitrite oxidizers and hzsA was examined in order to detect the presence of The abundance of nitrite oxidizers was examined using group- archaeal ammonia oxidizers, methanogens/anaerobic metha- specific SSU rRNA gene qPCR to targetNitrospira , Nitrospina, notrophs, and anaerobic ammonium oxidizers. As a result, and a potential nitrite-oxidizing SFNLG (40) (Fig. 3). The only the amplicons of archaeal amoA genes were obtained. results obtained indicated that Nitrospira members were the The archaeal amoA gene communities, which consisted of most abundant nitrite oxidizer, and its abundance peaks (1.5 five subgroups, were more diverse than those in the thaumar- and 1.6×105 copies g–1 sediment) were found in samples from chaeal communities observed in the SSU rRNA gene clone 5 and 45 cmbsf, respectively. The second most abundant analysis (Table S6). The grouping of archaeal amoA in the nitrite oxidizer was shown to be Nitrospina members, with present study was based on previous studies (2, 10, 41). The maximum abundance (3.7×104 copies g–1 sediment) being most abundant phylogroup was the Ba subgroup, with subgroups detected in a sample from 45 cmbsf. A potential nitrite- A and D as predominant populations. Coordination between oxidizing SFNLG was a minor population. 192 Nunoura et al.

Fig. 5. Matrices of UniFrac distances and Bray-Curtis and Jaccard dissimilarity indices of SSU rRNA gene communities obtained by tag sequencing for trench bottom sediments in the Challenger Deep, the Mariana Trench, shown by colored bars.

is not a major player in the nitrogen cycle in the trench bottom Gaps among PCR-dependent analyses for archaeal communities sediment. Based on the presence of sufficient amounts of – In qPCR analyses, we found a gap between the relative NO2 at depths at which denitrification outcompeted aerobic + abundances of total archaeal amoA and the archaeal SSU nitrification, low NH4 concentrations may be a limiting factor rRNA gene; total archaeal amoA was 27–45% of the archaeal for the development of the anammox bacterial population in SSU rRNA gene abundance above 65 cmbsf and decreased to this sedimentary environment (Fig. 1 and 3). 6% and 0.8% at 100 and 123 cmbsf, respectively (Fig. 3). This gap was accounted for by the results of SSU rRNA gene Comparison between hadal water and sediment microbial tag sequencing using a primer mixture of 505F/907R, but communities cannot be explained by the results of the classical SSU rRNA The relationship between microbial communities in bottom gene clone analysis, which revealed the predominance of the sediments and the overlying deep-sea waters has been rarely MGI thaumarchaeal population in all samples (Fig. 5, Table investigated in hadal environments. Cell density was two S3 and S5). In the tag analysis, the relative abundance of tags orders of magnitude larger in surface sediments than in hadal related to MGI thaumarchaeotes in archaeal SSU rRNA gene water (41) (Fig. 3). Based on a comparison of SSU rRNA tags ranged between 54 and 75% in sediments above 65 cmbsf, gene tag communities, MGI Thamumarchaeota, Planctomycetes, but was only 2% in sediments from 123 cmbsf, and the pre- Gemmatimonadetes, “Ca. Marinimicrobia” (SAR406), and dominance of the “Ca. Woesearchaeota” (DHVE6) population Bacteroidetes were commonly abundant in hadal water 50 m was observed in deeper sections. The possible underestimation above the trench bottom and in the bottom sediment at the of “Ca. Woesearchaeota” in the archaeal SSU rRNA gene same sampling station (41) (Fig. 4). The relative abundances communities using the archaea-specific primer set Arch21F/ of MGI, SAR324, and Chloroflexi SAR202 in the SSU rRNA Arch958R was also revealed in a similar comparison for another gene tag community were higher in hadal sediments than in subseafloor environment (43). Accordingly, the archaeal com- trench bottom water, whereas these groups are known to be munity structures dominated by “Ca. Woesearchaeota” shown the representative lineages of oceanic waters (52). In contrast, by SSU rRNA gene tag sequencing were likely to be less biased “Ca. Pelagibacterales” (SAR11), one of the predominant lineages than those shown by the SSU rRNA gene clone analysis. in oceanic waters, was absent in the benthic communities. Distance matrices were created using the UniFrac program Nitrogen cycle in Challenger Deep sediments to compare the tag communities from trench bottom water Molecular analyses revealed that the maximum abundance and sediment samples (Fig. 5). The results obtained indicated of ammonia-oxidizing archaea (AOA) and nitrite-oxidizing that the tag communities, including the trench bottom water bacteria (NOB) occurred in surface sediments (Fig. 3). The community, were similar to each other at higher taxonomic abundance peaks of ammonia oxidizers (AOA and ammonia- levels. Similarities among the tag communities were also oxidizing bacteria [AOB]) and NOB were also observed at tested by two dissimilarity indices: Bray-Curtis and Jaccard 45 cmbsf. The distribution pattern was consistent with the (Fig. 5). Overall, the Jaccard index for the SSU rRNA gene – stable isotopic signatures of NO3 that suggest the co-occurrence tags implied that tag populations in hadal water and sediments of nitrification and denitrification above 45 cmbsf (Fig. 2). In were distinct from each other; however, shared populations – contrast, the peak of NO2 at 65 cmbsf was likely to be the were found in some of the abundant taxa/divisions. Bray-Curtis result of partial denitrification (Fig. 1). On the other hand, the dissimilarity based on the abundance of each OTU also correlation between the decreasing population of Nitrospira showed similar results. These distance matrices were tested – and increasing concentration of NO2 suggests that the shallow for each of the dominant taxa/divisions (Fig. S1, S2, and S3). NO2– peak was the result of ammonia oxidation; however, it As an exception, the benthic thaumarchaeal population was currently remains unclear why the Nitrospira population similar to that in trench bottom water at the species or genus decreased at this depth (Fig. 1, 2, and 3). level. On the other hand, the UniFrac index indicated that the The unsuccessful amplification ofhzsA suggests that anammox abundant populations of Acidobacteria, Chloroflexi, and “Ca. Microbial Diversity in Hadal Sediments 193

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