https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
1 Diversity and distribution of Nitrogen Fixation Genes in the Oxygen Minimum Zones of the
2 World Oceans
3 1Amal Jayakumar and 1Bess B. Ward
4 5 1Department of Geosciences 6 Princeton University 7 Princeton, NJ 08544 8
9 Abstract
10 Diversity and community composition of nitrogen fixing microbes in the three main oxygen
11 minimum zones (OMZs) of the world ocean were investigated using operational taxonomic unit
12 (OTU) analysis of nifH clone libraries. Representatives of the all four main clusters of nifH genes
13 were detected. Cluster I sequences were most diverse in the surface waters and the most abundant
14 OTUs were affiliated with Alpha- and Gammaproteobacteria. Cluster II, III, IV assemblages were
15 most diverse at oxygen depleted depths and none of the sequences were closely related to sequences
16 from cultivated organisms. The OTUs were biogeographically distinct for the most part – there was
17 little overlap among regions, between depths or between cDNA and DNA. Only a few
18 cyanobacterial sequences were detected. The prevalence and diversity of microbes that harbour nifH
19 genes in the OMZ regions, where low rates of N fixation are reported, remains an enigma.
20
21 Introduction
22 Nitrogen fixation is the biological process that introduces new biologically available
23 nitrogen into the ocean, and thus constrains the overall productivity of large regions of the ocean
24 where N is limiting to primary production. The most abundant and most important diazotrophs
25 in the ocean are members of the filamentous genus Trichodesmium and several unicellular
1 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
26 genera, including Chrocosphaera sp. and the symbiotic genus Candidatus Atelocyanobacterium
27 thalassa (UCYN-A). Although these cyanobacterial species are wide spread and have different
28 biogeographical distributions (Moisander et al. 2010), they are restricted to surface waters,
29 mainly in tropical or subtropical regions.
30 Because diazotrophs have an ecological advantage in N depleted waters, and because those
31 conditions occur in the vicinity of oxygen minimum zones, due to the loss of fixed N by
32 denitrification, it has been proposed that N fixation should be favoured in regions of the ocean
33 influenced by OMZs (Deutsch et al. 2007). The search for non cyanobacterial diazotrophs has
34 resulted in discovery of diverse nifH genes, but they have not been associated with significant rates
35 of N fixation (Moisander et al. 2017). It has also been suggested that the energetic constraints on N
36 fixation might be partially alleviated under reducing, i.e., anoxic, conditions (Großkopf and LaRoche
37 2012). In response to these ideas, the search for organisms with the capacity to fix nitrogen has been
38 focused recently in regions of the ocean that contain OMZs. That search usually takes the form of
39 characterizing and quantifying one of the genes involved in the fixation reaction, nifH, which
40 encodes the dinitrogenase reductase enzyme. Here we report on the distribution and diversity of
41 nifH genes in all three of the world ocean’s major OMZs, including samples from both surface and
42 anoxic depths, and both DNA and cDNA (i.e., both presence and expression of the nifH genes).
43
44 Materials and Methods:
45 Samples analysed for this study were collected from the three major OMZ regions of the
46 world oceans (Table1) from surface, oxycline and oxygen depleted zone (ODZ) depths. Particulate
47 material from water samples (5 – 10 L), collected using Niskin samplers, mounted on a CTD
48 (Conductivity-Temperature-Depth) rosette system (Sea-Bird Electronics), was filtered onto Sterivex
2 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
49 capsules (0.2 µm filter, Millipore, Inc., Bedford, MA) immediately after collection using peristaltic
50 pumps. The filters were flash frozen in liquid nitrogen and stored at -80°C until DNA and RNA
51 could be extracted. For samples from the Arabian Sea, DNA extraction was carried out using the
52 PUREGENETM Genomic DNA Isolation Kit (Qiagen, Germantown, MD) and the RNA was
53 extracted using the ALLPrep DNA/RNA Mini Kit (Qiagen, Germantown, MD). For samples
54 collected from ETNP and ETSP DNA and RNA were simultaneously extracted using the ALLPrep
55 DNA/RNA Mini Kit (Qiagen, Germantown, MD). SuperScript III First Strand Synthesis System
56 (Invitrogen, Carlsbad, CA, USA) was used to synthesise cDNA immediately after extraction
57 following purification of RNA using the procedure described by the manufacturer, including RT
58 controls. DNA was quantified using PicoGreen fluorescence (Molecular Probes, Eugene, OR)
59 calibrated with several dilutions of phage lambda standards.
60 PCR amplification of nifH genes from environmental sample DNA and cDNA was done on
61 an MJ100 Thermal Cycler (MJ Research) using Promega PCR kit following the nested reaction
62 (Zehr et al. 1998), with slight modification as in Jayakumar et al. (2017). Briefly, 25µl PCR
63 reactions containing 50 pmoles each of outer primer and 20-25ng of template DNA, were amplified
64 for 30 cycles (1 min at 98°C, 1 min at 57°C, 1 min at 72°C), followed by amplification with the
65 inner PCR primers 50 pmoles each (Zehr and McReynolds 1989). Water for negative controls and
66 PCR was freshly autoclaved and UV-irradiated every day. Negative controls were run with every
67 PCR experiment, to minimize the possibility of amplifying contaminants (Zehr et al. 2003). The
68 PCR preparation station was also UV irradiated for 1 hour before use each day and the number of
69 amplification cycles was limited to 30 for each reaction. Each reagent was tested separately for
70 amplification in negative controls. nifH bands were excised from PCR products after electrophoresis
71 on 1.2% agarose gel, and were cleaned using a QIAquick Nucleotide Removal Kit (Qiagen). Clean
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72 nifH products were inserted into a pCR®2.1-TOPO® vector using One Shot® TOP10 Chemically
73 Competent E. coli, TOPO TA Cloning® Kit (Invitrogen) according to manufacturer’s specifications.
74 Inserted fragments were amplified with M13 Forward (-20) and M13 Reverse primers from
75 randomly picked clones. PCR products were sequenced at Macrogen DNA Analysis Facility using
76 Big DyeTM terminator chemistry (Applied Biosystems, Carlsbad, CA, USA). Sequences were
77 edited using FinchTV ver. 1.4.0 (Geospiza Inc.), and checked for identity using BLAST. Consensus
78 nifH sequences (359 bp) were translated to amino acid (aa) sequences (108 aa after trimming the
79 primer region) and aligned using ClustalX (Thompson et al. 1997) along with published nifH
80 sequences from the NCBI database. Neighbor-joining trees were produced from the alignment using
81 distance matrix methods (PAUP 4.0, Sinauer Associates). Bootstrap analysis was used to estimate
82 the reliability of phylogenetic reconstruction (1000 iterations). The nifH sequence from
83 Methanosarcina lacustris (AAL02156) was used as an outgroup. The accession numbers from
84 GenBank for the nifH sequences in this study are Arabian Sea DNA sequences JF429940– JF429973
85 and cDNA sequences accession numbers JQ358610–JQ358707, ETNP DNA sequences KY967751-
86 KY967929 and cDNA sequence KY967930-KY968089, and ETSP DNA sequences MK408165–
87 MK408307 and cDNA sequences MK408308–MK408422.
88
89 Standardization and verification of specificity for Q-PCR assays was performed as described
90 previously (Jayakumar et al. 2009). Primers nifHfw and nifHrv (Mehta et al. 2003, Dang et al. 2013)
91 forward 5-GGHAARGGHGGHATHGGNAARTC-3 and reverse 5-
92 GGCATNGCRAANCCVCCRCANAC-3, which correspond to the amino acid positions 10 to 17
93 (GKGGIGKS) and 132 to 139 (VCGGFAMP) of Klebsiella pneumoniae numbering (Mehta et al.
94 2003), were used (100 pmoles per 25 mL reaction) to amplify a ~400 bp region of the nifH gene for
4 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
95 nifH quantification. Assays were carried out with Qiagen master mix (Qiagen Sciences, Maryland,
96 USA) at an annealing temperature of 56 °C. Amplification conditions were chosen based on
97 amplification efficiency and reproducible results with a single product, after test assays on a
98 Stratagene MX3000P (Agilent Technologies, La Jolla, CA, USA). The amplified products were
99 visualized after electrophoresis in a 1.0% agarose gels stained with ethidium bromide. Standards for
100 quantification were prepared by amplifying a constructed plasmid containing the nifH gene
101 fragment, followed by quantification and serial dilution. Assays for all depths were carried out
102 within a single assay plate (Smith et al. 2006). Each assay included triplicates of the no template
103 controls (NTC), no primer control (NPR), four or more standards, and 20-25ng of template DNA of
104 the environmental DNA samples. A subset of samples from the previous run was included in
105 subsequent assays, as well as a new dilution series for standard curves on every assay. These new
106 dilution series were produced immediately following re-quantification of plasmid DNA
107 concentrations to verify gene abundance (because concentrations declined upon storage and freeze–
108 thaw cycles). Automatic analysis settings were used to determine the threshold cycle (Ct) values.
109 The copy numbers were calculated according to: Copy number = (ng * number/mole) / (bp * ng/g *
110 g/ mole of bp) and then converted to copy number per ml seawater filtered, assuming 100%
111 extraction efficiency.
112
113 The nifH nucleotide alignment (of 787 sequences) was used to define operational
114 taxonomic units (OTUs) on the basis of DNA sequence identity. Distance matrices based on this
115 nucleotide alignment were generated in MOTHUR (Schloss and Handlesman 2009). The
116 relative nifH richness within each clone library was evaluated using rarefaction analysis. OTUs
117 were defined as sequences which differed by ≤3% using the furthest neighbor method in the
5 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
118 MOTHUR program (Schloss and Handlesman 2009). The 3% OTU definition is similar to the
119 level at which species are conventionally defined using 16S rDNA sequences, so it may
120 overestimate the meaningful diversity of the functional gene.
121
122 Results and Discussion:
123 DNA and cDNA sequences (787 in total) derived from the OMZ regions of the Arabian
124 Sea (AS), Eastern Tropical North Pacific (ETNP) and Eastern Tropical South Pacific (ETSP)
125 were subjected to OTU and phylogenetic analyses to compare the diversity and community
126 composition, biogeography and gene expression, of nifH possessing microbes among the three
127 OMZ regions. Phylogenetic analysis of the sequences from the AS, ETNP and ETSP were
128 reported previously (Jayakumar et al. 2012, Jayakumar et al. 2017, Chang et al. 2019), but the
129 sequences have been combined for additional analyses here. We compared the threshold OTU
130 definitions at 3 and 10% and found that the number of OTUs decreased, as expected, as the
131 resolution decreased. Even at the 3% threshold, however, OTUs tended to separate by depth and
132 location, indicating a functionally useful distinction at this level. Thresholds of 3 – 5% as the
133 OTU definition correspond to within and between species level distinctions for nifH (Gaby et al.
134 2018). The sequences from the OMZ regions represented all four sequence clusters (I, II, III, IV)
135 described by Zehr et al. (1998).
136
137 Cluster I nifH OTU distributions: Diversity analysis of the nifH cluster 1 sequences
138 for the three OMZs based on OTUs using MOTHUR identified 41 OTUs at a distance threshold
139 of 3% (Supplemental Table 1A and B). The number of sequences and the number of OTUs
140 varied widely among depths and stations, so the results are grouped by region (AS, ETNP,
6 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
141 ETSP) or depth horizon (surface or OMZ, including upper oxycline depths) or cDNA vs DNA
142 (Table 1).
143 For all regions and depths combined, the number of OTUs detected (41) was less than the
144 sum of OTUs detected when each region was analyzed separately (45), indicating that there was
145 some overlap of OTUs among regions. The overlap was not large, however. Only three of the 12
146 most abundant OTUs contained sequences from more than one region and none contained
147 sequences from all three regions (Figure 1A). When sequences for all three regions were
148 combined, only four of the 12 most abundant OTUs contained sequences from both depth
149 horizons (Figure 1B). Most OTUs represented a single depth, and many a single sample.
150 The Arabian Sea was strikingly less diverse than other regions and sample subsets
151 (Figure 2). For example, when all DNA and cDNA sequences for all depths are grouped
152 together, the Arabian Sea (OTUs = 14, Chao = 21) contains less species richness than the
153 combined surface samples from all three regions (OTUs = 25, Chao = 52), despite having a
154 similar number of total sequences (178 for the Arabian Sea, 198 for all surface samples
155 combined). This lack of diversity in the AS data may be partly due to the preponderance of
156 cDNA sequences, which generally contained less diversity than a similar number of DNA
157 sequences (see below).
158 Although similar numbers of sequences were obtained for cDNA (255) vs DNA (257),
159 the OTU “density”, i.e., number of OTUs per number of sequences analyzed, was higher for
160 DNA (0.136 for DNA, 0.094 for cDNA). The Chao statistic verified this observation for the
161 combined data from each region in predicting higher total numbers of OTUs for DNA (Chao =
162 42) than for cDNA (Chao = 24). This difference could indicate that some of the nifH genes
163 present were not expressed at the time of sampling, but the cDNA sequences were not simply a
7 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
164 subset of the DNA community. Half of the 12 most abundant OTUs contained either cDNA or
165 DNA (Figure 1C), meaning that some genes were never expressed and some expressed genes
166 could not be detected in the DNA.
167 For all regions combined, similar numbers of OTUs were detected in surface waters
168 (OTUs = 25) and in OMZ samples (OTUs = 23), although a larger number of sequences was
169 analyzed for the OMZ environment (198 vs. 314 sequences for surface and OMZ depths,
170 respectively). It might be expected that the presence of phototrophic diazotrophs in the surface
171 water would lead to greater diversity there, but only one OTU representing a known
172 cyanobacterial phototroph (OTU-12 = Katagmynene spiralis or Trichodesmium) was identified,
173 so most of the additional diversity must be present in heterotrophic or unknown sequences.
174 Rarefaction curves (Figure 2) indicate that sampling did not approach saturation either for
175 region or depth. The Chao statistic also indicated that much diversity remains to be explored,
176 despite the great uncertainty in these estimates. The total number of OTUs detected, the shape of
177 the rarefaction curve and the diversity indicators (Figure 2, Table 1) all indicate that the greatest
178 nifH diversity occurred in surface waters, and much of that diversity was in singletons, i.e., not
179 represented in the 12 most abundant OTUs, which represented 441 (86 %) of the total 512 nifH
180 Cluster 1 sequences analyzed. Most of that diversity was contained in the ETNP, not solely a
181 function of number of sequences analyzed (Figure 2).
182 Cluster I nifH Phylogeny: Phylogenetic affiliations at both DNA and protein level are
183 shown for the 12 most abundant OTUs in Table 2. The most abundant OTU (129 sequences),
184 OTU-1, contained Gammaproteobacterial DNA and cDNA sequences from both surface and
185 OMZ depths of the ETNP and cDNA sequences from oxycline and OMZ depths in the Arabian
186 Sea (Figure 3). Although very similar to each other, none of these sequences had higher than
8 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
187 91% identity at the DNA level (96% at AA level) with cultivated strains and were most closely
188 related to Pseudomonas stutzeri. P. stutzeri is a commonly isolated marine denitrifier, but it is
189 also known to possess the capacity for N fixation (Krotzky and Werner 1987). OTU-4, OTU-6
190 and OTU-8 also contained Gammaproteobacterial sequences. All had high identity with
191 cultivated strains at the protein level but none were >91% identical to cultivated strains at the
192 DNA level.
193 Gammaproteobacterial sequences with very close identities to Azotobacter vinelandii have
194 been reported from the Arabian Sea ODZ and also from the ETSP (Turk-Kubo et al. 2014). This
195 group of nifH sequences with close identities to A. vinelandii was also retrieved from the English
196 Channel, Himalayan soil, South Pacific gyre, Gulf of Mexico, mangrove soil and many other
197 environments (Figure 3). Azotobacter- like sequences were included in OTU-6 but were not closest
198 identity at the DNA level. Although a large number of clones were analyzed here, no sequence that
199 was closely associated with A. vinelandii was retrieved from the three regions. None of the g-
200 244774A11 sequences, Gammaproteobacterial relatives that were abundant in the South Pacific
201 (Moisander et al. 2014), were detected in this study.
202 OTUs-2, 3, 5, 10, and 11 all represented Alphaproteobacterial sequences, with closest
203 identities to various Bradyrhizobium, Sphingomonas and Methylosinus species. Thus,
204 Alphaproteobacterial sequences (206 sequences) were the most abundant in the clone library. OTU-2
205 contained almost exclusively ETSP ODZ DNA and cDNA sequences (plus one AS ODZ DNA
206 sequence). OTU-3 contained DNA sequences from ETNP surface waters. OTU-5 contained
207 exclusively Arabian Sea DNA sequences from Station 3, while OTU-10 contained only surface
208 samples from the ETNP. An OTU threshold of 11% grouped all (179 sequences in five OTUs) of
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209 these Alphaproteobacterial sequences together, but the 3% threshold is consistent with the
210 phylogenetic tree, which shows small scale biogeographical separation of sequence groups.
211 OTUs-7 and -9 were identified as Betaproteobacteria with closest identities to Rubrivivax
212 gelatinosum and Burkholderia, 91 and 90% respectively at the DNA level. However, at the AA
213 level, these sequences were 99 and 100% identical to Novosphingobium malaysiense and S.
214 azotifigens, both Alphaproteobacteria, and again were biogeographically distinct. OTU-7 contained
215 25 DNA sequences from the ODZ depths in the Arabian Sea, and OTU-9 contained 17
216 Burkholderia-like sequences from the oxycline at Station 1 in the Arabian Sea. No
217 Betaproteobacterial nifH sequences were detected in the ETNP or ETSP, but sequences similar to
218 Burkholderia phymatum, Cupriavidus sp. and Sinorhizobium meliloti were reported from ETSP
219 previously (Fernandez et al. 2015). Consistent with our previous report, however, there is no clear
220 separation between the alfa and the beta groups in nifH phylogeny (Jayakumar et al 2017).
221 Most of the Cluster I ETSP sequences from this study were contained in two OTUs (2 and 4).
222 OTU-2 contained 89 Alphaproteobacterial sequences with >98% identity to nifH sequences from
223 Bradyrhizobium sp. Uncultured bacterial sequences retrieved from the South China Sea, English
224 Channel, mangrove sediment, wastewater treatment and grassland soil were related to these ETSP
225 sequences. OTU-4 contained 29 Gammaproteobacterial sequences retrieved from both surface and
226 ODZ depths. Four of the remaining ETSP Cluster I sequences were grouped together as OTU-17
227 (Alphaproteobacteria, 89 and 96% identities with Methyloceanibacter sp. and Bradyrhizobium sp. at
228 the DNA and AA level respectively), three were in OTU-23 (Bradyrhizobium 100% identity) and
229 two were singletons. One of the singletons was most closely related to uncultured soil and sediment
230 sequences and to Azorhizobium sp. (86%) and one had 97% identity with Bradyrhizobium
231 denitrificans and many sequences from marine sediments.
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232 OTU-22 represents the Deltaproteobacterial group. This novel group was reported
233 previously from the ETNP (Jayakumar et al. 2017) and has three sequences from Arabian sea (OTU-
234 22) and two singletons from ETNP surface waters. nifH possessing Deltaproteobacteria have been
235 reported not only from all the three ODZs but also in several other marine environments including
236 Chesapeake Bay water column, microbial mats from intertidal sandy beach in a Dutch barrier island,
237 Jiaozhou Bay sediment, Rongcheng Bay sediment, Bohai Sea, Mediterranean Sea, Narragansett Bay,
238 and the south Pacific gyre.
239 Although Trichodesmium like clones have been retrieved from the surface waters of the
240 Arabian Sea and the ETNP OMZs, only ten clones (OTU-12) in the combined clone library analyzed
241 here were related to Trichodesmium (98% identity), including both cDNA and DNA from the
242 Arabian Sea and cDNA from the ETNP. These sequences were actually 100% identical to
243 Katagnymene spiralis, a close relative of Trichodesmium isolated from the South Pacific Ocean.
244 Turk-Kubo et al. (2014) also retrieved only a few cyanobacterial sequences from the ETSP. No other
245 cyanobacterial nifH sequences were identified.
246 Clusters II, III, IV nifH OTU distributions: The other three nifH clusters were combined
247 for OTU analysis due to the limited number of sequences and OTUs obtained. A total of 18 OTUs
248 were identified in the combined set of 275 sequences with a 3% distance threshold (Table 2). Most
249 of the Cluster II, III, IV sequences were from the ETNP and ETSP. As with the Cluster I sequences,
250 there was very little geographic and depth overlap among these OTUs (Figure 4A, 4B). Only OTU-
251 1 contained sequences from more than one site, the ETNP and the ETSP. OTU-2 contained only
252 cDNA sequences representing ODZ depths at both ETNP stations. OTU-3 contained exclusively
253 ETSP DNA sequences from surface and cDNA sequences from ODZ depths. Only 10 of the Cluster
254 II, III, IV sequences were from the Arabian Sea, and they formed three separate OTUs, a greater
11 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
255 “OTU density” than was present at either of the Pacific sites. As observed for Cluster I, most of the
256 OTUs that were detected in the DNA were not being expressed, and those that were expressed were
257 not detected in the DNA (Figure 4C).
258 Rarefaction curves (Figure 5) indicate that sampling for Cluster II, III, IV did not
259 approach saturation. The Chao statistic also indicated that much diversity remains to be
260 explored, despite the great uncertainty in these estimates. Unlike the Cluster I analysis, there
261 were relatively few singletons in the Cluster II, III, IV data and the assemblages were dominated
262 by a few types.
263 Clusters II, III, IV nifH phylogeny: Three large OTUs (OTU-1, -4 and -6) in Clusters II,
264 III, IV belonged to nifH Cluster IV and Alphaproteobacteria/Spirochaeta and Deltaproteobacteria
265 were the dominant phylogenies (Table 2, Figure 6). The largest OTU, OTU-1, contained 88 DNA
266 sequences from the ETNP ODZ depths from both stations and from both depths in the ETSP. This
267 OTU had no similarity to any cultured microbe. OTU-4 contained 30 sequences from the ETSP, all
268 cDNA from one surface station, in nifH Cluster IV.
269 OTU-2 (75 sequences) in Cluster II contained only cDNA sequences, all from ODZ
270 samples in the ETNP (both stations), and had no close relatives among cultivated species. Turk-
271 Kubo et al. (2014) also retrieved a few clones identified as belonging to Cluster II from the
272 euphotic zone of the ETSP. OTU-3 contained 35 sequences in Cluster III and was dominated by
273 DNA sequences from surface depths of the ETSP. OTU-5 represented Deltaproteobacteria in
274 nifH Cluster III and contained 18 identical DNA sequences from 90 m at Station BB1 in the
275 ETNP. Thus, of the five most common OTUs (89% of the total Cluster II, III, IV sequences
276 analyzed), only one could be identified to a closely related genus (i.e., OTU-4 with 90% identity
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277 with R. palustris) and there was no overlap between DNA and cDNA OTUs from the same
278 depths.
279 The other 13 OTUs in the Cluster II, III, IV sequences represented either Cluster III or IV.
280 None of these were very closely related to any cultivated sequences. OTU-6 contained both DNA
281 and cDNA from the OMZ at one ETSP station. OTU-7 contained four sequences from ETNP
282 surface waters with close identities with a sequence retrieved from Bohai sea. OTU-11, had one
283 DNA and one cDNA sequences from the ETSP. All of the other sequences were less than 84%
284 identical to any sequence in the database and could only be loosely identified as Firmicutes or
285 Proteobacteria.
286
287 Conclusions
288 The OMZ regions of the world ocean contain substantial nifH diversity, both in surface
289 waters and oxygen depleted intermediate depths. Surface waters contained greater diversity for
290 Cluster I, but the ODZ held the highest diversity for Clusters II, III, IV. Cyanobacterial sequences
291 were rare and were not detected in the ETSP. The ETSP contained the least diversity of Cluster I
292 sequences, while Cluster II, III, IV were least abundant and least diverse in the Arabian Sea. Most
293 of the sequences in all four Clusters of the conventional nifH phylogeny were not closely related to
294 any sequences from cultivated Bacteria or Archaea. The most abundant OTUs in Cluster I and in
295 Clusters II, III, and IV could be assigned to the Alphaproteobacteria, followed by the
296 Gammaproteobacteria for Cluster I and Deltaproteobacteria accounted for Clusters II, III, IV
297 sequences. Most of the OTUs were not shared among regions, depths or DNA vs cDNA and
298 sometimes were restricted to individual samples. Some Cluster I sequences had high identity to
299 known species (e.g., Bradyrhizobium, Trichodesmium) but most of the Cluster II, III, IV sequences
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300 were only distantly related to any cultured species. While measurements of N2 fixation rates are not
301 reported here, the abundance of cDNA sequences suggests that the cells harboring these genes are
302 active. Low, but analytically significant, rates have been detected in ODZ depths in the ETNP
303 (Jayakumar et al. 2017) and ETSP (Chang et al. 2019), which suggests that non-cyanobacterial N
304 fixation could make a minor contribution to the nitrogen budget of the ocean. It is therefore
305 important in future work to determine how the diversity described here actually contributes to
306 biogeochemically significant reactions and what environmental and biotic factors might influence or
307 control the activity of diazotrophs in the dark ocean.
308
309
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310 Figure Legends
311 Figure 1. Histogram of the 12 most common OTUs from Cluster I nifH clone libraries from the
312 three OMZ regions. OTUs were considered common if the total number of sequences in an
313 OTU was ≥2% of the total number of nifH clones analyzed (The common OTUs contained 441
314 of the 512 Cluster I sequences). OTUs were defined according to 3% nucleotide sequence
315 difference using the furthest neighbor method. OTU designation is from most common (OTU-1)
316 to least. A) OTU distribution among regions. B) OTU distribution between OMZ (including
317 core of the ODZ and the upper oxycline depths) and surface depths (oxygenated water). C)
318 OTU distribution of cDNA vs DNA clones.
319
320
321 Figure 2. Rarefaction curve displaying observed OTU richness versus the number of clones
322 sequenced for Cluster I nifH sequences (cDNA and DNA). OTUs were defined and designated as
323 in Figure 1. Chao estimators (individual symbols) are shown for each of the same subsets
324 represented in the rarefaction curves.
325
326 Figure 3. Phylogenetic tree of Cluster 1 based on amino acid sequences. Positions of the OTUs
327 are shown relative to their nearest neighbors from the database. Individual sequence identities
328 comprising each OTU are listed in Supplemental Table 2.
329
330 Figure 4. Histogram of the 6 most common OTUs from Cluster I nifH clone libraries from the
331 three OMZ regions. OTUs were considered common if the total number of sequences in an
332 OTU was ≥2% of the total number of nifH clones analyzed (the common OTUs contained 252 of
15 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
333 the 275 Cluster II, III, IV sequences). OTUs were defined according to 3% nucleotide sequence
334 difference using the furthest neighbor method. OTU designation is from most common (OTU-1)
335 to least. A) OTU distribution among regions. B) OTU distribution between OMZ (including
336 core of the ODZ and the upper oxycline depths) and surface depths (oxygenated water). C)
337 OTU distribution of cDNA vs DNA clones.
338
339 Figure 5. Rarefaction curve displaying observed OTU richness versus the number of clones
340 sequenced for Cluster II, III, IV nifH sequences (cDNA and DNA). OTUs were defined and
341 designated as in Figure 4. Chao estimators (individual symbols) are shown for each of the same
342 subsets represented in the rarefaction curves.
343
344 Figure 6. Phylogenetic tree of Clusters II, III, IV based on amino acid sequences. Positions of
345 the OTUs are shown relative to their nearest neighbors from the database. Individual sequence
346 identities comprising each OTU are listed in Supplemental Table 2.
347
348
349 Tables
350 Table 1. OTU summary for both clusters
351 Richness and diversity statistics for nifH clone libraries from three OMZ regions. ACE and
352 Chao are non-parametric estimators that predict the total number of OTUs in the original sample.
353
354 Table 2. OTU identities for both clusters
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355 Cultivated species with closest nucleotide identity to the OTUs identified in the nifH clone
356 libraries from three OMZ regions. Only the 12 most common OTUs (out of 41 total) are listed
357 for Cluster 1 sequences, and the six most common (out of 18 total) for the Clusters II, III, IV
358 libraries.
359
360 Supplemental
361
362 S Table 1A and B. List of sequences in each OTU for both clusters
363 S Table 2
364 365
366
367
17 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
368 References
369
370 1. Chang, B. X., Jayakumar, A., Widner, B., Bernhardt, P., Mordy, C. M., Mulholland, M. R. and 371 Ward, B. B., 2019. Low rates of dinitrogen fixation in the eastern tropical South Pacific. 372 Limnology And Oceanography. 64, 1913-1923. 373 2. Dang, H. Y., Yang, J. Y., Li, J., Luan, X. W., Zhang, Y. B., Gu, G. Z., Xue, R. R., Zong, M. Y. and 374 Klotz, M. G., 2013. Environment-Dependent Distribution of the Sediment nifH-Harboring 375 Microbiota in the Northern South China Sea. Applied and Environmental Microbiology. 376 79, 121-132. 377 3. Deutsch, C., Sarmiento, J. L., Sigman, D. M., Gruber, N. and Dunne, J. P., 2007. Spatial 378 coupling of nitrogen inputs and losses in the ocean. Nature. 445, 163-167. 379 4. Fernandez, C., Lorena Gonzalez, M., Munoz, C., Molina, V. and Farias, L., 2015. Temporal and 380 spatial variability of biological nitrogen fixation off the upwelling system of central Chile 381 (35-38.5 degrees S). Journal of Geophysical Research-Oceans. 120, 3330-3349. 382 5. Gaby, J. C., Rishishwar, L., Valderrama-Aguirre, L. C., Green, S. J., Valderrama-Aguirre, A., 383 Jordan, I. L. and Kostka, J. E., 2018. Diazotroph community characterization via a high- 384 throughput nifH amplicon sequencing and analysis pipeline. Applied And Environmental 385 Microbiology. 84, eO1512-01517. 386 6. Großkopf, T. and LaRoche, J., 2012. Direct and indirect costs of dinitrogen fixation in 387 Crocosphaera watsonii WH8501 and possible implications for the nitrogen cycle. 388 Frontiers in Microbiology. 3. 389 7. Jayakumar, A., Al-Rshaidat, M. M. D., Ward, B. B. and Mulholland, M. R., 2012. Diversity, 390 distribution, and expression of diazotroph nifH genes in oxygen-deficient waters of the 391 Arabian Sea. Fems Microbiology Ecology. 82, 597-606. 392 8. Jayakumar, A., Chang, B. N. X., Widner, B., Bernhardt, P., Mulholland, M. R. and Ward, B. B., 393 2017. Biological nitrogen fixation in the oxygen-minimum region of the eastern tropical 394 North Pacific ocean. Isme Journal. 11, 2356-2367. 395 9. Jayakumar, A., Naqvi, S. W. A. and Ward, B. B., 2009. Distribution and relative quantification 396 of key genes involved in fixed nitrogen loss from the Arabian Sea oxygen minimum zone. 397 Indian Ocean Biogeochemical Processes and Ecological Variability. (J. D. Wiggert and R. 398 R. Hood). American Geophysical Union, Washington, D. C.: 187-203. 399 10. Krotzky, A. and Werner, D., 1987. NITROGEN-FIXATION IN PSEUDOMONAS-STUTZERI. 400 Archives of Microbiology. 147, 48-57. 401 11. Mehta, M. P., Butterfield, D. A. and Baross, J. A., 2003. Phylogenetic diversity of nitrogenase 402 (nifH) genes in deep-sea and hydrothermal vent environments of the Juan de Fuca ridge. 403 Applied and Environmental Microbiology. 69, 960-970. 404 12. Moisander, P. H., Beinart, R. A., Hewson, I., White, A. E., Johnson, K. S., Carlson, C. A., 405 Montoya, J. P. and Zehr, J. P., 2010. Unicellular Cyanobacterial Distributions Broaden the 406 Oceanic N-2 Fixation Domain. Science. 327, 1512-1514. 407 13. Moisander, P. H., Benavides, M., Bonnet, S., Berman-Frank, I., White, A. E. and Riemann, L., 408 2017. Chasing after Non-cyanobacterial Nitrogen Fixation in Marine Pelagic 409 Environments. Frontiers in Microbiology. 8.
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410 14. Moisander, P. H., Serros, T., Paerl, R. W., Beinart, R. A. and Zehr, J. P., 2014. 411 Gammaproteobacterial diazotrophs and nifH gene expression in surface waters of the 412 South Pacific Ocean. The ISME Journal 8, 1962–1973. 413 15. Schloss, P. D. and Handlesman, J., 2009. Introducing DOTUR, a computer program for 414 defining operational taxonomic units and estimating species richness. Applied and 415 Environmental Microbiology. 71, 1501-1506. 416 16. Smith, C. J., Nedwell, D. B., Dong, L. F. and Osborn, A. M., 2006. Evaluation of quantitative 417 polymerase chain reaction-based approaches for determining gene copy and gene 418 transcript numbers in environmental samples. Environmental Microbiology. 8, 804-815. 419 17. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. and Higgins, D. G., 1997. The 420 CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided 421 by quality analysis tools. Nucleic Acids Research. 25, 4876-4882. 422 18. Turk-Kubo, K. A., Karamchandani, M., Capone, D. G. and Zehr, J. P., 2014. The paradox of 423 marine heterotrophic nitrogen fixation: abundances of heterotrophic diazotrophs do not 424 account for nitrogen fixation rates in the Eastern Tropical South Pacific. Environmental 425 Microbiology. 16, 3095-3114. 426 19. Zehr, J. P., Crumbliss, L. L., Church, M. J., Omoregie, E. O. and Jenkins, B. D., 2003. 427 Nitrogenase genes in PCR and RT-PCR reagents: implications for studies of diversity of 428 functional genes. Biotechniques. 35, 996-1005. 429 20. Zehr, J. P. and McReynolds, L. A., 1989. Use of degenerate oligonucleotides for amplification 430 of the nifH gene from the marine cyanobacterium Trichodesmium theiebautii. Applied 431 and Environmental Microbiology. 55, 2522-2526. 432 21. Zehr, J. P., Mellon, M. T. and Zani, S., 1998. New nitrogen-fixing microorganisms detected in 433 oligotrophic oceans by amplification of nitrogenase (nifH) genes. Applied and 434 Environmental Microbiology. 6, 3444-3450. 435
436
19 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
437 438 Figure.1
100
90 AS 80 ETNP 70 ETSP 60
50
40 OTU Abundance OTU
30
20
10
0 1 2 3 4 5 6 7 8 9 10 11 12 140 OTU Number
120 Surface OMZ 100
80
60 OTU Abundance OTU
40
20
0 1 2 3 4 5 6 7 8 9 10 11 12 120 OTU Number
100 DNA
cDNA
80
60 OTU Abundance OTU 40
20
0 1 2 3 4 5 6 7 8 9 10 11 12 OTU Number
439
20 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
440 Figure. 2
60 Surface ETNP 50 ODZ AS ETSP 40 Surface ETNP ODZ AS 30 ETSP Number of OTUs 20
10
0 0 50 100 150 200 250 300 350 400 Number of Clones Sampled
441 442
21 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
443 Figure. 3
UC Neuse River Estuary AAS22073 p UB Pacific Ocean AAZ31163
Katagnymene spiralis AAL36032 p UB tropical/subtropical Atlantic AAY59933 UC Arabian Sea surface AAV51281 s
Trichodesmium erythraeum IMS101 YP723617 UB North Pacific AAS98181 m OTU-12 u UB Eastern Tropical Atlantic ABV00643 a i UB South China Sea ABX39913 i
UB Gulf of Aqaba ABH02967 r
UB Western Tropical North Atlantic ABD62927 m 100 UB South China Sea surface ACV95413 Trichodesmium thiebautii AAA27368
UB Pacific Ocean-ETSP DNA AHY22772 es 100 Cyanothece sp. WH8902 AAV28035 acte Gloeothece sp. SK40 BAF51659 d
100 Cyanobacterium UCYN-A YP003421696 Group A b UB Pacific Ocean-ETSP DNA AHY23112
UB Pacific Ocean-ETSP DNA AHY22750 ho Lyngbya lagerheimii UTEX1930 AAA19029 no c Nostoc commune AAA21838 i
Richelia sp. SC01 ABB00036 a 52 r 50 UC Arabian Sea 10m AAV51306 60 UC Arabian Sea 25m AAV51296 T
Lyngbya sp. CCAP1446/10 ABQ65208 Cy Anabaena sp. A2 AAD30985 Aphanizomenon sp. KAC15 ABB96256 Synechococcus sp. JA-3-3Ab YP_475238 Calothrix sp. MCC-3A ABF81863 Crocosphaera watsonii WH850 AAP48976 Group B Bradyrhizobium sp. BTAi 1 BAC07281 Bradyrhizobium sp.AFQ33589 OTU-20 Bradyrhizobium denitrificans ADJ18188 OTU-2 OTU-10 OTU-14 OTU-26 Methylocystis rosea AAY89023 UB field soil Ithaca NY ABQ01506 Takara Reagent AAO73607 Arabian Sea UB JX064482 Bradyrhizobium sp. BBC54938 OTU-21 OTU-3 OTU-18 UB Pacific Ocean-ETSP DNA AHY22743 Bradyrhizobium japonicum ACT67975 Bradyrhizobium sp. CCBAU 33011 ABV44667 UB South Pacific Upwelling ABW80585 OTU-7 52 UB Pacific Ocean-ETSP DNA AHY22754 UB South China Sea ABX39742 Novosphingobium malaysiense WP039285924 53 UB Western English Channel ABP37994 UBSanya Mangrove sediment ABM67093 OTU-5 UB paper wastewater AAV52938 Marine Prot. bacterium Sippewissett AAD23112 UB Chazy NY grassland soil ACI26161 UB pulp and paper wastewater AAV52969 Sphingomonas azotifigens BAE71134 α turfgrass established soil ABY55141 OTU-9 OTU-23 OTU-17 Xanthobacter autotrophicus Py2 ABS65347 r I OTU-11 Bradyrhizobium sp. NPK+ROM8 AMX27928 e Bradyrhizobium diazoefficiens AIF73149 OTU-15
PCR Promega Rgnt AY225106 st OTU-19
PCR Reagent EU16668 u
UB South Pacific Upwelling AJC52151 l PCR Reagent ACI14248
UB South Pacific Upwelling AJC52156 C 96 UB South Pacific Upwelling AJC52161 UB South Pacific Upwelling AJC52149 UB South Pacific Upwelling AJC52154 UB South Pacific Upwelling AJC52150 OTU-25
58 Burkholderia phymatum STM815 CAD43950 a
UB South Pacific Upwelling AJC52155 i 50 Cupriavidus sp. mpp1.6 ADM25248 r Cupriavidus taiwanensis AAU85621 Paraburkholderia ferrariae ABO64209 61 Burkholderia sp. CACua-88 ADM13690 UB Pacific Ocean-ETSP DNA AHY22726
60 97 Methylosinus trichosporium OB3b ATQ67965 acte Methylosinus trichosporium AAO49392 β b UB South Pacific Upwelling AJC52153 PCRRgntContACI13947 o
76 UB South Pacific Upwelling AJC52158 e
Rubrivivax gelatinosus AHB60523 t mRNA eastern Mediterranean Sea ABQ50643 75 UB South Pacific Upwelling AJC52152 o 88 OTU-16 r 73 OTU-24 Sinorhizobium meliloti AAP48966 P UB copepod assoc. AAC36031 UB Copepod assoc. AF016602 UB Dutch barrier island-sandy intertidal beach ADD26542 OTU-27 Pseudomonas stutzeri CBL85101 bacterium CZ152S ADQ38905 64 Pseudomonas stutzeri AVX12447 Pseudomonas sp. CEG62552 Pseudomonas azotifigens BAD90116 Marinobacterium lutimaris DSM 22012 AHN13828 OTU-1 UB English Channel ABP37981 95 UB Pacific Ocean-ETSP DNA AHY22856 OTU-8 Pseudomonas oryzae SDS70900 UB Indian Ocean ADA57502 59 Azotobacter vinelandii 1NIP_B 51 UB Damma glacier soil ABU86698 Agrobacterium tumefaciens ACN88695 56 UB Himalayayan soil ACZ57832 Azotobacter chroococcum AYE20514 UB Pacific Ocean-ETSP DNA AHY23148 Arabian Sea UB JX064474 OTU-4 UB South Pacific Gyre ADO20650 UB Bahama Islands AAC36088 Oleibacter sp. MAK91178 Arabian Sea UB JX064473 88 UB South Pacific Gyre ADO20653 UB South Pacific Gyre ADO20643 UB South China Sea ABX39720 UB Chesapeake Bay AAO67664 68 UB Arabian Sea AAV68764 90 UB Arabian Sea AAV68768 70 Arabian Sea UB AY800134 UB South Pacific Gyre ADO20634 γ UB North Pacific ALOHA DNA ABI 18637 79 UB South Pacific Gyre ADO20632 61 OTU-6 UB Pacific Ocean-ETSP DNA AHY22799 UB South Pacific Gyre ADO20629 UB Eastern Tropical Atlantic ABV00665 UB Subtropical North Pacific AAP42775 83 Vibrio diazotrophicus AAA65435 Marine Prot. bacterium Sippewissett AAD23115 63 UB South Pacific Gyre ADO20645 Arabian Sea UB JX064475 UB Guangzhou mangrove sediment ADF32036 UB Gulf of Mexico ABF21178 Klebsiella sp. AAA16804 Teredinibacter turnerae AT7901 ARC12580 Magnetococcus marinus ABK43713 UB Pacific Ocean-ETSP DNA AHY23015 Arabian Sea UB JX064470 82 OTU-13 100 56 Arabian Sea UB JX064467 Marichromatium purpuratum AAC36094 Marichromatium purpuratum AAC36093 85 Methylomonas methanica AAK97417 Methylomonas sp. LW13 QBC29047 75 UB South Pacific Gyre ADO20614 96 UB South Pacific Gyre ADO20616 UB Pacific Ocean-ETSP DNA AHY22786 UB Intertidal microbial mat ADD26204 100 UB Chesapeake Bay Water AAZ06765 96 80 OTU-22 Geobacter sp. ABR01121 Geobacter pickeringii AJE04162 UB Jiaozhou Bay sediment ACN77120 UB Huanghai Sea and Bohai Sea AKS11123 86 UB Chesapeakwater AAZ06675 UB Dutch barrier island-sandy intertidal beach ADD26540 UB Pacific Ocean-ETSP DNA AHY22894 UB MD soil AHN50541 UB oil contaminated marine sediment AAY85432 100 δ UB Mediterranean Sea ABQ50817 UBNarragansett Bay AIL28609 63 UB Rongcheng Bay sediment AKV16181 UB Intertidal microbial mat ACY26274 UB copepod assoc. AF016608 Cluster II 96 UB copepod assoc. AF016603 Methanosarcina lacustris AAL02156 Cluster III YP002505948CcelH10 Methanosarcina lacustris AAL02156
0.01 changes Cluster IV
444 445
22 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
446 Figure. 4
80
70 AS 60 ETNP ETSP 50
40
OTU Abundance OTU 30
20
10
0 1 2 3 4 5 6 80 OTU Number
70 Surface OMZ 60
50
40
OTU Abundance OTU 30
20
10
0 1 2 3 4 5 6 100 OTU Number
90 DNA 80 cDNA 70
60
50
40 OTU Abundance OTU
30
20
10
0 1 2 3 4 5 6 OTU Number
447 448
23 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
449 Figure. 5.
40
35 All Cluster234 ODZ ETNP 30 Surface ETSP 25 AS All Cluster234 ODZ 20 ETNP Surface ETSP
Number of OTUs 15 AS
10
5
0 0 50 100 150 200 250 300 350 Number of Clones Sampled
450 451
24 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
452 Figure 6.
91 Rhodoplanes elegans WP111356811 100 OTU-4 Rhodopseudomonas palustris DX-1 ADU44011 Lachnospiraceae bacterium ND2006]WP035638048 Methanopyrus kandleri AV19 AAM02629 Ruminococcus sp. NK3A76 WP028510298 Clostridium intestinale WP021804255 V
Clostridium aerotolerans WP026891450 I Pelosinus fermentans JBW45 AJQ28834 Catabacter hongkongensis KKI51118 ster lu
Termite gut UB BAF52265 C Termite gut BAA28487 UB PNN32 100 UB Soil AHN51251 85 UB Soil AHN51375 OTU-1 70 Enhygromyxa salina PRP94280 54 OTU-6 Leptolyngbya boryana BAA00565 Coraliomargarita akajimensis WP013044574 UB marine anoxic enrichment AAC36034 100 OTU-3 UB ETSP denitrified waters AEA49118 66 UB MD Soil AHN50444 57 Verrucomicrobiae bacterium DG1235 EDY82020 UB South China Seawater ADT89936 UB GBR Seawater AEU12186 UB South China Seawater ADT89913 52 UB MD Soil AHN50902 61 99 Opitutaceae bacterium TAV5 WP_009512762 Didymococcus colitermitum WP043581909 UB Chesapeake Bay water AAZ06728 UB Dongkemadi Glacier foreland AJP08283 Desulfovibrio salexigens DSM2638 ACS78514 58 Pseudodesulfovibrio piezophilus C1TLV30 CCH50130 Desulfovibrio vulgaris str. Hildenborough YP_009055 Lentisphaerae bacterium GWF2_57_35 OGV45350 OTU-9 100 UB Pacific Ocean-ETSP DNA AHY22887 UB Chesapeake Bay water AAZ06701 Desulfatitalea tepidiphila WP054032742 64 Desulfobacteraceae bacterium RJP78566 UB MD Soil AHN50858 51 Desulfobacter curvatus AAD21801 Bact. Isolate Cyano.mat assoc. AAA65421 100 UB Marginal Sea China AKS11464 UB Marginal Sea China AKS11076 UB Forest soil ACI32145 100 OTU-7 76 UB Huanghai Sea and Bohai Sea AKS11225 Desulfovibrio inopinatus WP027183856 Desulfonatronum thioautotrophicum WP045221084 97 Desulfonatronum thiodismutans WP031386911 Desulfonatronum lacustre WP028571559 UB soil Mexico AER93044 Desulfovibrio gigas AAB09059 87 UB Tallgrass prairie Oklahoma AIE38864 Spirochaeta aurantia AAK01220 UB Pacific Ocean-ETSP DNA AHY22891 100 Desulfovibrio magneticus RS-1 BAH75547 57 Desulfovibrio carbinolicus QAZ67507 UB Pacific Ocean-ETSP DNA AHY22914 63 UB Marginal Sea China AKS11333 UB copepod assoc. GM214 AAC36022 96 78 OTU-11 UB Mediterranean Sponge -Spain: Montgri Coast AKP55777 I 52 Verrucomicrobiae bacterium S94 QBG48103 II
Kiritimatiellales bacterium WP 136081448 ster UB South China Sea ADT89822 lu UB copepod assoc. GM29 AAC36028 C 100 Prosthecochloris s sp. V1 WP110023904 64 UB Sanya Bay Coral AHI71637 75 Chlorobaculum macestae ABQ42889 Chloroherpeton thalassium ATCC 35110 ACF134991 82 OTU-5 UB Mediterranean Seawater-Spain: Montgri Coast AKP55968 70 UB Florida Keys Reef water ADV35081 73 UB MD Soil AHN51089 UB Mediterranean Seawater AKP55957 93 UB copepod assoc. GM212 AAC36023 UB marine anoxic enrichment AAC36036 UB Pacific Ocean-ETSP DNA AHY22827 UB marine anoxic enrichment AAC36035 63 mRNA Termite intestine assoc. NKNRT16 BAA86270 91 92 Treponema azotonutricium AAK01231 94 Treponema primitia ZAS-2 AF325801 Spirochaeta zuelzerae AAK01223 86 mRNA Termite intestine assoc. NKNRT6 BAA86276 UB Pacific Ocean ETSP DNA AHY23003 UB Termite intestine assoc. TKG6 BAA11789 Treponema bryantii AAK01224 100 Spirochaeta stenostrepta AAK01222 58 64 UB copepod assoc. GM216 AAC36027 Clostridium pasteurianum AAT37643 UB marine anoxic enrichment AAC36033 96 Sporobacter termitidis WP073078008 60 OTU-8 86 Massilibacillus massiliensis WP110955331 Desulfovibrio desulfuricans QCC85382 I 95 Proteobacteria
95 Cyanobacteria ster lu
Treponema denticola AAK01225 C Treponema primitia ZAS-1 AAK01227 88 Treponema pectinovorum AAK01226 100 Spirochaeta stenostrepta AAK01221 UB Termite intestine assoc. BAA28442 90 Rhodopseudomonas palustris CGA009 CAE26881 Clostridium pasteurianum CAA30361 94 UB Pacific Ocean-ETSP DNA AHY22721 I I Methanosarcina barkeri CAA39552 Methanoregula boonei 6A8 ABS565971 ster lu
70 Desulfitobacterium hafniense Y51 BAE858001 C OTU-2 77 Clostridium cellulolyticum str. H10 YP_002505948 83 OTU-10 Marine Surface water UB ADI24166 Methanocaldococcus villosus WP00459175 Methanobacterium ivanovii CAA30384 Methanococcus voltae CAA27407 100 Treponema primitia ZAS-2 AAK01230 77 Treponema primitia ZAS-1 AAK01228 97 Termite gut UB BAC210431 Termite gut UB BAF522131 Methanosarcina barkeri BAA34069 Methanosarcina lacustri AAL02156 0.05 changes
453 454
25 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
455 Table 1 OTU Summary Depths, No. of No. of regions No. of Unique OTUs OTU Sample subset included Sequences Sequences (cutoff ~3) /seq Shannon Simpson Chao Ace Cluster I AS Arabian Sea, 178 36 14 0.079 1.8 0.22 21 45 all depths
ETNP ETNP, all 207 80 25 0.121 2.37 0.14 37 34 depths ETSP ETSP, OMZ 127 51 6 0.047 0.87 0.53 7 8 depths
All ClusterI Three 512 165 41 0.080 2.7 0.11 59 67 regions, all depths All ClusterI DNA Three 257 97 35 0.136 2.8 0.08 42 45 regions, all depths All ClusterI cDNA Three 255 75 24 0.094 1.7 0.25 24 27 regions, all depths All ClusterI Surface Three 198 73 25 0.126 2.5 0.10 52 75 regions, surface depths All ClusterI OMZ Three 314 98 23 0.073 0.9 0.23 30 37 regions, all depths
Clusters II, III, IV AS Arabian Sea, 10 6 3 0.300 1.09 0.27 3 3 all depths ETNP ETNP, all 134 49 8 0.060 1.19 0.39 14 38 depths ETSP ETSP, all 131 64 8 0.061 1.37 0.30 9 19 depths All Clusters II,III,IV Three 275 117 18 0.065 1.88 0.21 28 26 regions, all depths All Clusters II,III,IV Three 155 65 12 0.077 1.20 0.37 22 17 DNA regions, all depths All Clusters II,III,IV Three 120 56 9 0.075 1.11 0.45 12 15 cDNA regions, all depths All Clusters II,III,IV Three 86 46 6 0.070 1.32 0.29 7 13 Surface regions, surface depths All Clusters II,III IV Three 189 76 15 0.079 1.57 0.29 46 24 OMZ regions, OMZ depths 456 457 458 459
26 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
460 Table 2 461
No. of Phylogenetic Closest cultured Identity Coverage Closest cultured Identity Coverage Sequences Affiliation relative (DNA) DNA % % relative (Protein) AA % % Cluster I OTU-1 129 Gamma Psuedomonas 91 98 Pseudomonas 95.8 99 stutzeri stutzeri strain SGAir0442 OTU-2 89 Alpha Bradyrhizobium sp 99 100 Bradyrhizobium 99 99 denitrificans strain LMG 8443 OTU-3 40 Alpha Bradyrhizobium 94 98 Bradyrhizobium sp. 99 98 sp. TM124 MAFF 210318 OTU-4 29 Gamma Marinobacterium 87 100 Oleibacter sp 100 99 lutimaris OTU-5 29 Alpha Methylosinus 92 99 Sphingomonas 99 100 trichosporium azotifigens OTU-6 25 Gamma Azotobacter 81 99 Psuedomonas 94 99 chroococcum stutzeri strain B3 OTU-7 25 Beta/Alpha Rubrivivax 91 99 Novosphingobium 99 100 gelatinosus malasiense OTU-8 17 Gamma Psuedomonas 91 98 Azotobacter 97 100 stutzeri chroococcum strain B3 OTU-9 17 Beta/Alfa Burkholderia 90 100 Sphingomonas 100 100 azotifigens OTU-10 16 Alpha Bradyrhizobium 97 98 Bradyrhizobium sp. 99 99 ORS 285 OTU-11 15 Alpha Bradyrhizobium 97 98 Bradyrhizobium 98 99 diazoefficiens OTU-12 10 Cyanobacterium Katagnymene 100 99 Trichodesmium 100 99 spiralis erythraeum
Clusters II, III IV
OTU-1 88 Alpha/Spirochaet Rhizobium sp 74 59 Treponema primitia 55 98 aceae ZAS-1] OTU-2 75 Delta/Firmicutes Geobacter 73 43 Desulfitobacterium 98 61 hafniense OTU-3 35 Verrumicrobia Opitutaceae 82 99 Coraliomargarita 95 99 bacterium akajimensis OTU-4 30 Alpha Rhodopseudomon 90 98 Rhodoplanes 96 99 as palustris elegans OTU-5 18 Delta/Chlorobi Desulfovibrio 79 99 Prosthecochloris 92 99 piezophilus sp. V1, Chloroherpeton thalassium, Chloroherpeton thalassium OTU-6 6 Beta/Delta Azoarcus 70 88 Enhygromyxa salina 70 74 communis OTU-7 4 Delta Desulfovibrio 81 99 Desulfovibrio 90 99 carbinolicus strain DSM 3852 inopinatus OTU-8 4 Delta/Firmicutes Desulfovibrio 77 100 Sporobacter 99 99 desulfuricans termitidis strain IC1
27 https://doi.org/10.5194/bg-2019-445 Preprint. Discussion started: 13 January 2020 c Author(s) 2020. CC BY 4.0 License.
OTU-9 3 Delta/Lentisphae Desulfovibrio 84 100 Lentisphaerae 84 100 rae magneticus RS-1 bacterium DNA GWF2_57_35, Desulfatitalea tepidiphila, Desulfobacteraceae bacterium OTU-10 3 Delta/Methanoc Desulfovibrio 77 100 Methanocaldococcus 65 99 occi desulfuricans villosus strain IC1 OTU-11 2 Verrucomicrobi Verrucomicrobia 87 100 Verrucomicrobia 97 99 bacterium S94 bacterium S94 a 462 463 464
28