Journal of
Plankton Research
plankt.oxfordjournals.org
J. Plankton Res. (2016) 38(2): 380– 391. First published online February 16, 2016 doi:10.1093/plankt/fbw003
Costa Rica Dome: Flux and Zinc Experiments
Diazotroph community structure in the deep oxygen minimum zone of the Costa Rica Dome SHUNYAN CHEUNG, XIAOMIN XIA, CUI GUO AND HONGBIN LIU* DIVISION OF LIFE SCIENCE, THE HONG KONG UNIVERSITY OF SCIENCE AND TECHNOLOGY, CLEAR WATER BAY, KOWLOON, HONG KONG
*CORRESPONDING AUTHOR: [email protected]
Received April 28, 2015; accepted January 11, 2016 Corresponding editor: Pia Moisander
Oxygen minimum zones (OMZs), characterized by depleted dissolved oxygen concentration in the intermediate depth of the water column, are predicted to expand under the influence of global warming. Recent studies in the Eastern Tropical South Pacific Ocean and Arabian Sea have reported that heterotrophic nitrogen fixation is active in the OMZs. In this study, we investigated the community structure of diazotrophs in the OMZ of the Costa Rica Dome (CRD) upwelling region in the Eastern Tropical North Pacific Ocean, using 454-pyrosequencing of nifH gene amplicons. Comparing diazotroph assemblages in different depth strata of the OMZ (200–1000 m in depth), we found a unique diazotroph community in the OMZ core, which was mainly dominated by methanotroph-like diazotrophs, suggesting a potential coupling of nitrogen cycle and methane assimilation. In addition, some OTUs revealed in this study, especially those belonging to the large sub-cluster Vibrio diazotrophicus, were reported to be abundant and expressing the nifH gene in other OMZs. Our results suggest that the unique hydrographic conditions in OMZs may support similar assemblages of diazotrophs, and heterotrophic nitrogen fixation could also be occurring in our studied region. Our study provides the first insight into the composition and distribution of putative diazotrophs in the CRD OMZ. KEYWORDS: oxygen minimum zone; heterotrophic bacteria; nifH gene; methanotroph; 454-pyrosequencing
I N T RO D U C T I O N Biological nitrogen fixation, a process that converts dinitrogen gas to bioavailable ammonia, produces most of the ocean’s fixed nitrogen (Gruber, 2005). In marine
environments, biological nitrogen fixation has long been attributed to cyanobacterial diazotrophs. More recently, however, molecular-based studies have revealed the presence and expression of a nitrogenase gene in diverse
available online at www.plankt.oxfordjournals.org # The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]
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Table I: Basic information on DNA samples used in this study Station
Lat. (8N)
Lon. (8W)
Depth (m)
Sample name
No. of sequences
Good’s coverage index
Shannon diversity index
Remarks
St. 1
9.71
86.95
St. 3
9.95
92.92
St. 4
8.55
90.13
St. 5
9.35
87.66
350 500 300 400 600 700 1000 300 400 500 600 200 500 650 800
St. 1—350 m St. 1—500 m St. 3—300 m St. 3—400 m St. 3—600 m St. 3—700 m St. 3—1000 m St. 4—300 m St. 4—400 m St. 4—500 m St. 4—600 m St. 5—200 m St. 5—500 m St. 5—650 m St. 5—800 m
1191 2999 3000 2987 1981 2994 2993 3000 3000 2991 480 2993 2992 2998 3000
0.958 0.971 0.983 0.984 0.979 0.979 0.983 0.981 0.982 0.977 0.973 0.991 0.985 0.988 0.988
1.762 1.093 0.367 0.98 0.977 0.548 0.379 1.412 0.751 1.412 0.319 0.152 1.196 1.028 0.271
Core-OMZ Core-OMZ O-OMZ Core-OMZ Core-OMZ Core-OMZ O-OMZ O-OMZ Core-OMZ Core-OMZ O-OMZ O-OMZ Core-OMZ Core-OMZ O-OMZ
The sequence numbers, coverage and Shannon diversity indices of each sample were included. Samples were defined as ‘Core-OMZ’ or ‘Outside Core-OMZ’ in remarks.
heterotrophic bacteria, suggesting the potential importance of heterotrophic diazotrophs in a variety of marine environments (Zehr et al., 1998; Church et al., 2005; Farnelid et al., 2011; Bentzon-Tilia et al., 2014). Although these works have highlighted a new role of heterotrophic bacteria in nitrogen fixation, the distribution, diversity, physiology and nitrogen fixation contribution of heterotrophic diazotrophs are still unclear. Marine oxygen minimum zones (OMZs), defined by depletion of dissolved oxygen (DO) to ,2 mM and the presence of a secondary nitrite (NO2 2 ) maximum of .0.5 mM, are located mainly in the Eastern Tropical North Pacific (ETNP), the Eastern Tropical South Pacific (ETSP) and the Arabian Sea (Ulloa et al., 2012). Low-oxygen and nutrientrich water resulting from upwelling systems supports high primary productivity in surface waters and hence causes oxygen depletion (,2 mM) sub-surface (Ulloa et al., 2012), due to oxygen consumption associated with the degradation of sinking organic matter. Such conditions of oxygen depletion and high concentration of sinking organic matter facilitate denitrification and anammox in OMZ cores, which contribute 30–50% of total nitrogen loss to di-nitrogen in the global ocean (Codispoti et al., 2001). In addition, oxygen depletion, higher regeneration rate of bioavailable phosphate (Ingall and Jahnke, 1994) and iron in the oxygen-depleted water have been suggested to be ideal conditions for nitrogen fixation (Deutsch et al., 2001, 2007; Loescher et al., 2014). Recent studies in the ETSP (Fernandez et al., 2011; Loescher et al., 2014), the Arabian Sea (Jayakumar et al., 2012; Bird and Wyman, 2013) and the Baltic Sea (Farnelid et al., 2013) have demonstrated that nitrogenase gene expression or nitrogen fixation by heterotrophic bacteria occurs in OMZs and suboxic waters. In addition, the diazotroph communities in the OMZs studied were found to be distinct from those in oligotrophic marine waters (Loescher et al., 2014).
The Costa Rica Dome (CRD) is one of the permanent wind-driven upwelling systems in the ETNP and it overlies a stable OMZ from 350 to 700 m in depth (Wyrtki, 1964; Fiedler, 2002). Since heterotrophic bacterial nitrogen fixation rate or nitrogenase gene expression has been reported in the OMZs of the other two major OMZ regions (Fernandez et al., 2011; Jayakumar et al., 2012; Bird and Wyman, 2013; Farnelid et al., 2013; Loescher et al., 2014), we speculated that a functionally or phylogenetically similar assemblage may also be present in oxygen-deficient waters below the CRD. Moreover, since the OMZ of the ETNP is much deeper than that of the ETSP and the Arabian Sea, its diazotroph community may also differ from those of the other two regions in some respects, providing an opportunity to discover new genotypes with nitrogen fixation potential. In this study, we used 454-pyrosequencing of partial nifH gene fragments (Zehr and Turner, 2001) to investigate putative diazotroph community structure in the OMZ of the CRD.
METHOD Environmental conditions and sample collection This study was conducted at four stations (St. 1, 3, 4 and 5) in the OMZ off the coast of Costa Rica (98210 N–98570 N, 868580 W–928560 W) during the CRD-FLUZiE (FLUx and Zi Experiments) cruise (Landry et al., 2016) in June and July 2010 on board R/V Melville (Table I; Fig. 1). The Stations 1, 3, 4 and 5 were located in the Cycle 1, 3, 4 and 5 of the CRD-FLUZiE cruise, respectively. Basic hydrographic data (salinity, temperature, depth and DO concentration) and water samples were collected with a conductivity–temperature–depth rosette system, with dual Sea Bird oxygen
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Fig. 1. Map of station locations.
sensors (Sea Bird Electronics), and attached Niskin bottles. Depending on the concentration of DO, we defined the water layers with ,2 mM DO as the core-OMZs, and the waters outside the core-OMZs were defined as O-OMZs. In addition to the basic hydrographic data, NO2 2 , ammo) concentrations and total iron concentration nium (NHþ 4 profile were adopted from other studies of the CRDFLUZiE cruise (Buchwald, 2013; Vedamati, 2013). For molecular analyses, sea water samples of 500–1000 mL were size-fractionated with 2 and 0.2 mm polycarbonate membrane filters (47 mm, Millipore) under low vacuum. All of the molecular samples were flash frozen and stored at 2808C until DNA extraction.
DNA extraction, nifH PCR and 454-pyrosequencing of DNA samples Genomic DNA was extracted from the 0.2-mm membrane filters with TRIzol plus Genomic DNA mini kit (Invitrogen, Carlsbad, CA, USA). All the DNA samples were eluted in 60-mL ultrapure water (Invitrogen) and stored at 2208C until further analysis. NifH gene fragments were amplified from the genomic DNA samples following the nested polymerase chain reaction (PCR) protocol (Zehr and Turner, 2001). The nested PCR reaction was done in duplicate with the Platinum Taq DNA polymerase PCR system (Invitrogen)
in a volume of 12.5 mL containing 1 rxn PCR buffer, 4 mM MgCl2, 400 mM dNTPs, 1 mM primers (nifH 3 and nifH 4 for the first round, nifH 1 and nifH 2 for the second round), 1 unit Platinum Taq polymerase and 1 mL of total genomic DNA. After the second round of the nested PCR, 1 mL of the PCR products was used to run 10 cycles of PCR with sample-specific multiplex identifiers (MIDs) and adaptor-attached primers (Farnelid et al., 2011). The PCR conditions were identical to those of the nested PCR. The duplicates of PCR products were pooled with the same amount and then gel purified with Quick gel purification kit (Invitrogen). For 454-pyrosequencing, MID-adaptor-labeled nifH gene amplicons of different samples were mixed with the same concentrations to construct an amplicon library according to Rapid Library construction protocols (Roche, 454 Life Science). Then, the DNA library attached beads were loaded onto a Pico TiterPlate and sequenced with a GS Junior System (Roche, 454 Life Science). Negative controls, in which no template DNA was added, were done in all the nested PCR. According to the results of DNA gel electrophoresis, no visible DNA bands were observed in the negative control lanes. A section of gel encompassing the size of nifH amplicons (439 bp) was sliced from the negative control lane and purified as mentioned. The elution of the negative control collected after the gel purification was processed
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as the samples, and sequenced with GS Junior System (Roche, 454 Life Science).
Sequence quality control and analysis Quality control of raw sequence data was conducted with Mothur (Schloss et al., 2009). Low-quality sequences (average quality score ,25), short sequences (,300 bases in length), ambiguous base containing sequences, homopolymers containing sequences (homopolymers .8 bases) and chimeric sequences were removed. Also, the MID sequences were trimmed. The trimmed sequences were de-noised with 0.01 sigma value to reduce the effects of PCR bias. After being aligned with the reference sequences from the online nifH gene database of Ribosomal Database Project (Wang et al., 2013), phylogenetic distances between these high-quality sequences were then calculated, and the sequences were clustered at 95% similarity using Mothur. On the basis of 95% similarity clustering in nucleotide basis (Fernandez et al., 2011), operational taxonomic units (OTUs), representative sequences for each OTU, rarefaction curve, Good’s coverage indices and Shannon diversity indices were generated or calculated with Mothur. For the similarity among the samples, the samples were clustered with Braycurtis calculator using the Unweighted Pair Group Method with the Arithmetic Mean (UPGMA) algorithm in Mothur. The OTUs contained 10 sequences individually, which are defined as top OTUs, were selected for the subsequent phylogenetic analysis. The remaining OTUs, which consisted of ,10 sequences per OTU, were grouped as rare species. To identify the top OTUs, representative sequences were first translated into amino acid (aa) sequences with the FRAMEBOT online pipeline (Wang et al., 2013). The aa sequences were used to search the protein sequences database on NCBI via protein BLAST (BLASTp) webpage (Mcginnis and Madden, 2004). The OTU representative sequences and the selected reference sequences from the NCBI protein sequences database were then aligned with ClustalW in MEGA 6.0 (Tamura et al., 2013) and used to construct a neighbor-joining phylogenetic tree ( p-distance) with MEGA 6.0 (Tamura et al., 2013). The phylogenetic tree was further edited using iTOL (Letunic and Bork, 2011). The OTUs that clustered together with the same reference sequences (.96% similarity in aa with the reference sequences) were merged into the same sub-clusters. Then, these subclusters were named with species of the corresponding reference sequences and used to display relative abundances of different diazotrophs in different samples. All raw sequences obtained in this study have been deposited in the NCBI Sequence Read Archive with accession number SRP057492.
Environmental variables and diazotroph community structure To reveal the relationships between diazotroph community variances and environmental variables, relative abundance of the sub-clusters in each samples was analyzed with detrended correspondence analysis (DCA) using Canoco 4.5 (Ter Braak and Smilauer, 2002). The data for two deep samples (St. 3—1000 m and St. 5—800 m) were þ excluded from the analysis, because NO2 2 and NH4 concentrations were not measured at these depths. The environmental variables (temperature, salinity, DO, NO2 2 and ) and the relative abundance of the sub-clusters were NHþ 4 square root transformed. On the basis of the result of DCA, the largest length of gradient value among the four axes was between 3 and 4; therefore, redundancy analysis was used to analyze relationships among diazotroph community variances and environmental variables in Canoco 4.5 (Ter Braak and Smilauer, 2002). The environmental variables with significant relationships (P , 0.05) were selected to explain the diazotroph community variances, which were assessed in permutation tests with 499 unrestricted Monte Carlo permutations.
R E S U LT S Hydrography and nutrients The hydrographic profiles for the four studied water columns were similar although the depth and thickness of the core-OMZ layers were slightly different among the stations. Sharp decreases of temperature and increases of salinity were detected at 30–40 m, which was the signal of upwelling of deep water (Fig. 2a and b). In general, the core-OMZs, with DO concentration ,2 mM, were found in the depth range of 350–700 m (Fig. 2c). Sub-surface 2 2 peaks of NHþ 4 and NO2 (secondary NO2 maxima) were found below the euphotic layers, and the highest concentration of NO2 2 was detected in the core-OMZs (0.9–1.5 mM) (Fig. 3). The largest core-OMZ (400–700 m) occurred at Station 3, with the largest vertical extent of the NO2 2 maximum. Conversely, the lowest concentration and depth range of NO2 2 were found at Station 4, coinciding with the smallest core-OMZ (350–500 m). In addition, comparing with euphotic zone and O-OMZs, concentration of iron increased sharply in the core-OMZs (Fig. 2d).
Diazotroph community diversity and similarity In total, 39 599 DNA sequences were included in this study and the sequence number of each sample is provided in Table I. The Good’s coverage indices of the samples
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Fig. 2. Vertical profiles of (a) temperature, (b) salinity, (c) dissolved oxygen (DO) and (d) total iron at the sampling stations.
þ Fig. 3. Vertical profiles of concentrations of nitrite (NO2 2 ) and ammonium (NH4 ). Shadow layers indicate core-OMZs, where the DO concentration was lower than 2 mM.
ranged from 0.958 to 0.991 (Table I), showing that the sequencing was deep enough for the diazotroph community study. For the negative control, no sequence of nifH gene amplicon was recovered with the 454-pyrosequencing. The rarefaction curves of the samples did not plateau (Supplementary data, Fig. S1), which suggested that more sequences are needed for studying the rare species. Therefore, we focused on the top OTUs in this study. In general, diazotroph community composition and diversity
within the core-OMZs were different from those outside the core-OMZs although some exceptional cases were observed. The Shannon diversity index values for communities in the core-OMZs were generally greater than those outside the core-OMZs (Table I). In terms of community similarity (Fig. 4), communities within the core-OMZs clustered together; and the communities 100 m above and 100 m below the core-OMZs (St. 3—300 m; St. 5—200 m; St. 4—600 m) also clustered. The communities
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Fig. 4. UPGMA dendrogram showing the relationship between samples. Samples from the core-OMZs are labeled with asterisks.
in deeper waters (St. 3—1000 m and St. 5—800 m), where DO was higher than that in the core-OMZs, were different. The samples from St. 4—300 m were taken close to the core-OMZ (50 m apart), and the diazotroph community and diversity were similar to that of the core-OMZ. Two samples from Station 3 (St. 3—600 m and St. 3—700 m) did not cluster with the other seven core-OMZ samples although the DO concentration was ,2 mM. This suggests that there were other environmental variables, besides DO, influencing diazotroph community structure.
to the same reference sequences were grouped into subclusters (at least 96% similarity in aa). These sub-clusters were named with the species identities of the reference sequences. OTU 29 in Cluster III, which did not show high similarity (at least 96% similarity in aa) with any reference sequences, was assigned to unidentified Cluster III. As a result, 37 OTUs were grouped into 18 sub-clusters, among which Methylocella palustris and Vibrio diazotrophicus were the dominant diazotrophs (Fig. 6). The sub-cluster M. palustris was mainly found (50–90% in relative abundance) in the core-OMZs, while sub-cluster V. diazotrophicus dominated (.90% in relative abundance) in some water samples from both the core-OMZs and O-OMZs. The deeper-water communities were dominated by different sub-clusters, with Bradyrhizobium sp. IRBG 2 and Klebsiella sp. GG41E dominating in St. 3—1000 m and St. 5—800 m, respectively. Different sub-clusters showed different distribution patterns. For Alphaproteobacteria, most sub-clusters were unique to the core-OMZs, while Bradyrhizobium sp. IRBG 2 was unique to the O-OMZs. Besides that, Bradyrhizobium sp. Z15 and Azorhizobium caulinodans ORS 571 were found in both core-OMZs and O-OMZs. Deltaproteobacteria, sulfate-reducing bacteria, were unique to the core-OMZ. Most Gammaproteobacteria were widely distributed in different layers of the water column, except Pectobacterium atrosepticum and Tolumonas auensis, which were unique to the core-OMZ. As for the remaining sub-clusters, Cluster IV was unique to the core-OMZs, and unidentified Cluster III was detected in both core-OMZs and O-OMZs.
Diazotroph community structure According to the results of BLASTp, three cyanobacterialike OTUs were detected in St. 1—350 m, which contributed 58% of the total sequences of the sample (1656 of 2847 sequences). These OTUs were removed from the dataset during phylogenetic analysis, which is explained in the discussion part. A total of 37 OTUs were selected for the phylogenetic analysis. From the online protein database of NCBI, 49 reference sequences (mostly 96–100% similarity in aa) were selected and used in this study. These 49 sequences included reference sequences with known identities and some environmental DNA/cDNA sequences from OMZ-related studies (Fernandez et al., 2011; Jayakumar et al., 2012; Bird and Wyman, 2013; Farnelid et al., 2013; Bentzon-Tilia et al., 2014; Loescher et al., 2014) and other marine environments. Overall, the representative sequences of the top 37 OTUs fell within 3 defined clusters of nifH gene (Fig. 5) (Zehr et al., 2003b). The majority of OTUs (31/37 OTUs) belonged to the Cluster I proteobacteria. The remaining OTUs fell within Cluster III (4/37 OTUs) and Cluster IV (2/37 OTUs). The representative sequences that showed high similarity
Diazotroph community structure and environmental variables Among all the available environmental variables, DO, temperature and NO2 2 affected diazotroph community significantly (P , 0.05). These three variables explained 80% of the variance in community structure together. The most abundant sub-clusters, M. palustris and V. diazotrophicus, showed negative and positive correlations with DO, respectively (Fig. 7). Some sub-clusters unique to core-OMZs showed more significant correlations with NO2 2 , including Hyphomicrobium sp. MC1, Novosphingobium malaysiense and T. auensis. In addition, temperature influenced the distributions and relative abundances of sulfate-reducing bacteria (Deltaproteobacteria).
Cyanobacteria-like OTUs in sub-surface water The cyanobacteria-like OTUs, which were removed from the dataset, were phylogenetically close to Calothrix sp. PCC 7507 (accession number: WP015131644). Among
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Fig. 5. Neighbor-joining phylogenetic tree constructed with nifH gene amino acid sequences. The representative sequences of the OTUs detected in this study are labeled with OTU 1 to OTU 37; and the accession number of each reference sequence is shown in front of the sequence name. Bootstrap resampling was performed for 1000 times; and the values that are higher than 50% are labeled with gray circular symbols on the branches.
the 3 revealed cyanobacteria-like OTUs, 2 OTUs (contributed 97.2% of the removed sequences) showed 100 and 98% similarity in aa with Calothrix sp. PCC 7507. The third OTU only had 95% similarity with Calothrix sp. PCC 7507 and its closest marine sequence (accession no.: AAZ31164.1); hence, this OTU is relatively distant to all previously revealed nifH sequences.
DISCUSSION Hydrographic conditions and nitrogen fixation in OMZs Oxygen deficiency and the occurrence of the secondary NO2 2 maxima (Fig. 3) in the core-OMZs of CRD indicates
that there were active N-loss processes (e.g. anammox and nitrate reduction) occurring in the core-OMZs, which have also been observed and studied in the OMZs in the ETSP and Arabian Sea (Gala´n et al., 2009; Ward et al., 2009). Two other studies conducted during the CRDFLUZiE cruise reported active anammox activity (Kong et al., 2013) and deficiency of DIN in the middle of core-OMZs at Stations 3, 4 and 5 (Buchwald, 2013). Nitrogen fixation of heterotrophic diazotrophs has been reported in the OMZs of the ETSP and Arabian Sea (Fernandez et al., 2011; Jayakumar et al., 2012; Bird and Wyman, 2013; Loescher et al., 2014), where the hydrographic profiles were similar to those in the CRD. Moreover, iron concentration, which was suggested to be important for nitrogenase activity (Deutsch et al., 2007),
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Fig. 6. Relative abundance of diazotroph sub-clusters in different samples. The water depths within the core-OMZs are marked with pink shadows. Names of the sub-clusters that were also detected in other OMZs are labeled in red. The proteobacteria sub-clusters were grouped into the corresponding classes, including Alphaproteobacteria (a), Betaproteobacteria (b), Deltaproteobacteria (d) and Gammaproteobacteria ( g).
diazotroph community (Fernandez et al., 2011). Therefore, both hydrographic conditions and Shannon diversity indices imply the importance of measuring nitrogen fixation rate in future studies.
Contamination issue of nested PCR of nifH
Fig. 7. Correlation biplot based on a redundancy analysis (RDA), depicting the relationship between relative abundances of the diazotroph sub-clusters and the environmental factors.
also increased significantly in the core-OMZs. Hence, the overall hydrographic conditions in the CRD OMZ were suitable for nitrogen fixation. In addition, Shannon diversity indices of diazotroph communities were generally higher in the core-OMZs (Table I). A previous study in the Peruvian OMZ reported positive correlation between nitrogen fixation rate and Shannon diversity indices of the
It has been reported that some heterotrophic nifH sequences may be amplified from bacterial cells or DNA present in some PCR reagents, during nested PCR (Zehr et al., 2003a; Goto et al., 2005). However, the DNA contaminants in different PCR reagents are not necessarily the same, and the OTUs affiliated with the reported DNA contaminants may also actually inhabit in the marine environment (Farnelid et al., 2013). Therefore, the negative control of the nested PCR was sequenced in our study. The absence of nifH gene sequences in the negative control suggests that the recovered OTUs in this study originated from the studied area.
Feature species observed in the CRD OMZ Sub-cluster V. diazotrophicus was one of the most dominant sub-clusters in CRD OMZ; and it was detected in both O-OMZs and core-OMZs (Fig. 6). According to the result of BLASTp, this sub-cluster was also the most
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abundant Gammaproteobacterial diazotroph (named cluster P8, reached 106 nifH gene copies per L of water) in the Peruvian OMZs (100% similarity in aa, accession no.: AEA49228) and was observed to be actively expressing nifH genes (Fernandez et al., 2011; Loescher et al., 2014). Moreover, nifH gene transcripts of similar diazotrophs (96% similarity in aa, accession no.: AFM94362) were also detected in the OMZ of the Arabian Sea (Jayakumar et al., 2012). Therefore, sub-cluster V. diazotrophicus may also be actively fixing nitrogen in the CRD OMZ, where the overall hydrographic conditions were similar to the Peruvian and Arabian Sea OMZs. The high relative abundance and wide distribution of subcluster V. diazotrophicus in both core-OMZs and O-OMZs is consistent with the findings of Loescher et al. (Loescher et al., 2014). Vibrio diazotrophicus was reported to be a facultative anaerobe (Guerinot et al., 1982), and the nitrogenase protein of Vibrio natriegens (96% similarity in aa with sub-cluster V. diazotrophicus) was produced in high amounts and stored under anaerobic conditions (Coyer et al., 1996). These previous studies may explain why V. diazotrophicus dominated throughout the water column in the CRD OMZ and other OMZs. The most dominant sub-cluster (M. palustris) detected in the core-OMZs was closely related to Alphaproteobacterial methanotrophs (99% similarity in aa). A number of studies have reported the coupling between nitrogen fixation and methane assimilation (Murrell and Dalton, 1983; Khadem et al., 2010; Larmola et al., 2014). It has been reported that methane is released from submarine sediments, hydrothermal seeps and cold seeps (Karaca, 2011; Levin et al., 2012) into the water column off Costa Rica, including the core-OMZs (Schleicher and Wallmann, 2007; Pack et al., 2015). Previous studies have reported that Alphaproteobacterial methanotrophs can grow in microaerobic and even anaerobic conditions (Vecherskaya et al., 2009) and that methane oxidation can be nitrogen-limited (Bodelier et al., 2000a, b). High relative abundance of the methanotroph-like sub-cluster was also observed at St. 4— 300 m (Fig. 6), where DO was 4.5 mM, suggesting that this sub-cluster might be facultatively anaerobic. In addition, another methanotroph-like sub-cluster (Hyphomicrobium sp. MC1) was also detected in the core-OMZs. These methanotroph-like sub-clusters may benefit from the highmethane and low-oxygen conditions in the core-OMZs. Although sub-cluster M. palustris mostly dominated in the core-OMZs and sub-cluster V. diazotrophicus was generally associated with the O-OMZs, they were detected in both types of waters. It implies that other environmental factors besides DO might govern the distributions of these subclusters. At stations 3 and 5, sub-cluster V. diazotrophicus was detected in almost all water layers, while its relative abundance decreased in layers where sub-cluster M. palustris was
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dominant (Fig. 6). The succession between these two subclusters might be explained by other environmental factors, such as methane availability, which allowed the sub-cluster M. palustris to outgrow V. diazotrophicus in some layers. Desulfobulbus alkaliphilus (sulfate-reducing bacteria) and T. auensis dominated in St. 4—500 m and St. 3—600 m (Fig. 6), respectively. Their occurrence in the core-OMZs may be supported by the anaerobic conditions and sulfate availability (Canfield et al., 2010) and by the higher availability of organic matter (Fischer-Romero et al., 1996) in the core-OMZs. Previous studies have indicated that sulfate-reducing diazotrophs are abundant in the Peruvian OMZ (Loescher et al., 2014) and the suboxic water of the Baltic Sea (Farnelid et al., 2013). Although they were relatively abundant (40% in relative abundance) in only one sample (St. 4—500 m) in this study (Fig. 6), their existence provides evidence that sulfatereducing bacteria may be able to thrive in the core-OMZ of the ETNP. The cluster IV OTUs detected in this study were distant from the previously reported marine sequences (Fig. 5). In the Peruvian OMZ, distant cluster IV was the most abundant and active phylotype of nifH (Loescher et al., 2014). Traditionally, the Cluster IV was thought to be homologs of nitrogenase (Zehr et al., 2003b), of which nitrogen fixation activity was still unclear. Therefore, the nitrogen fixing function of the Cluster IV diazotrophs in the OMZs needs to be addressed in the future studies. Calothrix, detected in this study, is a symbiotic cyanobacterium in the diatom Chaetocoeros that has only been reported in the Equatorial Atlantic upwelling region (Foster et al., 2009), but not in any OMZs in ETNP or Arabian Sea. Previous studies have demonstrated that diatom blooms are supported in upwelling regions (Allen et al., 2005; Bruland et al., 2005) and that sinking eukaryotic chloroplasts can be detected at 200 and 500 m depths in the CRD OMZ (Jing et al., 2013). Hence, the source of these diatom symbiotic diazotrophs in the St. 1—350 m is believed to be sinking particles. Therefore, these cyanobacteria-like OTUs were removed from the dataset during the phylogenetic analysis. The distant cyanobacteria-like OTU discovered in this study supports the view that the unique hydrographic setting of the CRD OMZ provides niches for unique strains of diazotrophic cyanobacteria, similar to the findings of unique Synechococcus strains in the CRD (Ahlgren et al., 2014).
Influence of hydrographic conditions on diazotroph community composition Comparing with the sequences detected in the ETSP and Arabian Sea OMZs, 6 sub-clusters detected in CRD were also detected in other OMZs (Fig. 6), accounting for
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70% of the total sequences in this study. The presence of common diazotrophs in spatially separated OMZs could be due to the similar hydrographic conditions (DO depletion, secondary NO2 2 maxima and availability of iron) among different OMZs. The significant correlations between diazotroph community structure and DO, temperature and NO2 2 indicate that the large variations of these parameters through the water column played a major role in shaping the community structure of diazotrophs in this unique environment. Although M. palustris and V. diazotrophicus showed negative and positive correlations with DO (Fig. 7), respectively, the largely reciprocal distribution pattern of these two sub-clusters in CRD waters suggests the importance of other environmental variables (e.g. availability of methane) that were not measured in this study. Loescher et al. (Loescher et al., 2014) reported that subcluster V. diazotrophicus (named P8 in their paper) was negatively correlated with NO2 2 in the Peruvian OMZ, while such a pattern was not found in this study. This difference may be due to different analytical methods, as copy number of nifH gene was used in the Peruvian study and relative abundance of nifH gene was used in the present study. It appears that the diazotrophs in the CRD OMZ were influenced by a combination of different environmental factors. Hence, more environmental parameters (e.g. concentration of methane and sulfate) should be measured in the future studies of the CRD OMZ, to better explain the distributions of different sub-clusters of heterotrophic diazotrophs. In summary, our study provides the first insight into the composition and distribution of the putative diazotroph assemblages in the CRD OMZ of the ETNP. Diversity of diazotrophs was generally higher in the core-OMZs than at other depths in the water column. High similarity of the diazotroph community with other OMZs also indicated that heterotrophic nitrogen fixation could be a process potentially occurring in the CRD OMZ, especially for the existence of sub-cluster V. diazotrophicus. The finding of a dominant methanotroph-like sub-cluster implies a potential coupling between nitrogen fixation and methane metabolism in the CRD OMZ although factors controlling the distribution of the methanotroph-like sub-cluster and sub-cluster V. diazotrophicus are still unclear. Lastly, phylogenetically distant cluster IV and cyanobacteria-like diazotrophs were discovered in this study. These findings highlight the importance of further studies of the nitrogen fixation process in the CRD OMZ, including identifying active nifH phylotypes, quantifying particular nifH gene abundance and expression of the dominant sub-clusters and understanding the coupling between methanotrophy and nitrogen cycling.
S U P P L E M E N TA RY DATA Supplementary data can be found online at http://plankt. oxfordjournals.org.
DATA A RC H I V I N G All raw sequences obtained in this study have been deposited in the NCBI Sequence Read Archive with accession number SRP057492.
AC K N OW L E D G E M E N T S We thank chief scientist Prof. Michael R. Landry of the Scripps Institution of Oceanography for giving us the opportunity to participate on the FLUZiE cruise, Dr Takafumi Kataoka for taking the samples, and the captain and crew of the R/V Melville for their professional assistance.
FUNDING This study was supported by Hong Kong Grants Council GRF (661911, 661912, 661813).
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J. Plankton Res. (2016) 38(2): 380– 391. First published online February 16, 2016 doi:10.1093/plankt/fbw003
Costa Rica Dome: Flux and Zinc Experiments
Diazotroph community structure in the deep oxygen minimum zone of the Costa Rica Dome SHUNYAN CHEUNG, XIAOMIN XIA, CUI GUO AND HONGBIN LIU* DIVISION OF LIFE SCIENCE, THE HONG KONG UNIVERSITY OF SCIENCE AND TECHNOLOGY, CLEAR WATER BAY, KOWLOON, HONG KONG
*CORRESPONDING AUTHOR: [email protected]
Received April 28, 2015; accepted January 11, 2016 Corresponding editor: Pia Moisander
Oxygen minimum zones (OMZs), characterized by depleted dissolved oxygen concentration in the intermediate depth of the water column, are predicted to expand under the influence of global warming. Recent studies in the Eastern Tropical South Pacific Ocean and Arabian Sea have reported that heterotrophic nitrogen fixation is active in the OMZs. In this study, we investigated the community structure of diazotrophs in the OMZ of the Costa Rica Dome (CRD) upwelling region in the Eastern Tropical North Pacific Ocean, using 454-pyrosequencing of nifH gene amplicons. Comparing diazotroph assemblages in different depth strata of the OMZ (200–1000 m in depth), we found a unique diazotroph community in the OMZ core, which was mainly dominated by methanotroph-like diazotrophs, suggesting a potential coupling of nitrogen cycle and methane assimilation. In addition, some OTUs revealed in this study, especially those belonging to the large sub-cluster Vibrio diazotrophicus, were reported to be abundant and expressing the nifH gene in other OMZs. Our results suggest that the unique hydrographic conditions in OMZs may support similar assemblages of diazotrophs, and heterotrophic nitrogen fixation could also be occurring in our studied region. Our study provides the first insight into the composition and distribution of putative diazotrophs in the CRD OMZ. KEYWORDS: oxygen minimum zone; heterotrophic bacteria; nifH gene; methanotroph; 454-pyrosequencing
I N T RO D U C T I O N Biological nitrogen fixation, a process that converts dinitrogen gas to bioavailable ammonia, produces most of the ocean’s fixed nitrogen (Gruber, 2005). In marine
environments, biological nitrogen fixation has long been attributed to cyanobacterial diazotrophs. More recently, however, molecular-based studies have revealed the presence and expression of a nitrogenase gene in diverse
available online at www.plankt.oxfordjournals.org # The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]
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Table I: Basic information on DNA samples used in this study Station
Lat. (8N)
Lon. (8W)
Depth (m)
Sample name
No. of sequences
Good’s coverage index
Shannon diversity index
Remarks
St. 1
9.71
86.95
St. 3
9.95
92.92
St. 4
8.55
90.13
St. 5
9.35
87.66
350 500 300 400 600 700 1000 300 400 500 600 200 500 650 800
St. 1—350 m St. 1—500 m St. 3—300 m St. 3—400 m St. 3—600 m St. 3—700 m St. 3—1000 m St. 4—300 m St. 4—400 m St. 4—500 m St. 4—600 m St. 5—200 m St. 5—500 m St. 5—650 m St. 5—800 m
1191 2999 3000 2987 1981 2994 2993 3000 3000 2991 480 2993 2992 2998 3000
0.958 0.971 0.983 0.984 0.979 0.979 0.983 0.981 0.982 0.977 0.973 0.991 0.985 0.988 0.988
1.762 1.093 0.367 0.98 0.977 0.548 0.379 1.412 0.751 1.412 0.319 0.152 1.196 1.028 0.271
Core-OMZ Core-OMZ O-OMZ Core-OMZ Core-OMZ Core-OMZ O-OMZ O-OMZ Core-OMZ Core-OMZ O-OMZ O-OMZ Core-OMZ Core-OMZ O-OMZ
The sequence numbers, coverage and Shannon diversity indices of each sample were included. Samples were defined as ‘Core-OMZ’ or ‘Outside Core-OMZ’ in remarks.
heterotrophic bacteria, suggesting the potential importance of heterotrophic diazotrophs in a variety of marine environments (Zehr et al., 1998; Church et al., 2005; Farnelid et al., 2011; Bentzon-Tilia et al., 2014). Although these works have highlighted a new role of heterotrophic bacteria in nitrogen fixation, the distribution, diversity, physiology and nitrogen fixation contribution of heterotrophic diazotrophs are still unclear. Marine oxygen minimum zones (OMZs), defined by depletion of dissolved oxygen (DO) to ,2 mM and the presence of a secondary nitrite (NO2 2 ) maximum of .0.5 mM, are located mainly in the Eastern Tropical North Pacific (ETNP), the Eastern Tropical South Pacific (ETSP) and the Arabian Sea (Ulloa et al., 2012). Low-oxygen and nutrientrich water resulting from upwelling systems supports high primary productivity in surface waters and hence causes oxygen depletion (,2 mM) sub-surface (Ulloa et al., 2012), due to oxygen consumption associated with the degradation of sinking organic matter. Such conditions of oxygen depletion and high concentration of sinking organic matter facilitate denitrification and anammox in OMZ cores, which contribute 30–50% of total nitrogen loss to di-nitrogen in the global ocean (Codispoti et al., 2001). In addition, oxygen depletion, higher regeneration rate of bioavailable phosphate (Ingall and Jahnke, 1994) and iron in the oxygen-depleted water have been suggested to be ideal conditions for nitrogen fixation (Deutsch et al., 2001, 2007; Loescher et al., 2014). Recent studies in the ETSP (Fernandez et al., 2011; Loescher et al., 2014), the Arabian Sea (Jayakumar et al., 2012; Bird and Wyman, 2013) and the Baltic Sea (Farnelid et al., 2013) have demonstrated that nitrogenase gene expression or nitrogen fixation by heterotrophic bacteria occurs in OMZs and suboxic waters. In addition, the diazotroph communities in the OMZs studied were found to be distinct from those in oligotrophic marine waters (Loescher et al., 2014).
The Costa Rica Dome (CRD) is one of the permanent wind-driven upwelling systems in the ETNP and it overlies a stable OMZ from 350 to 700 m in depth (Wyrtki, 1964; Fiedler, 2002). Since heterotrophic bacterial nitrogen fixation rate or nitrogenase gene expression has been reported in the OMZs of the other two major OMZ regions (Fernandez et al., 2011; Jayakumar et al., 2012; Bird and Wyman, 2013; Farnelid et al., 2013; Loescher et al., 2014), we speculated that a functionally or phylogenetically similar assemblage may also be present in oxygen-deficient waters below the CRD. Moreover, since the OMZ of the ETNP is much deeper than that of the ETSP and the Arabian Sea, its diazotroph community may also differ from those of the other two regions in some respects, providing an opportunity to discover new genotypes with nitrogen fixation potential. In this study, we used 454-pyrosequencing of partial nifH gene fragments (Zehr and Turner, 2001) to investigate putative diazotroph community structure in the OMZ of the CRD.
METHOD Environmental conditions and sample collection This study was conducted at four stations (St. 1, 3, 4 and 5) in the OMZ off the coast of Costa Rica (98210 N–98570 N, 868580 W–928560 W) during the CRD-FLUZiE (FLUx and Zi Experiments) cruise (Landry et al., 2016) in June and July 2010 on board R/V Melville (Table I; Fig. 1). The Stations 1, 3, 4 and 5 were located in the Cycle 1, 3, 4 and 5 of the CRD-FLUZiE cruise, respectively. Basic hydrographic data (salinity, temperature, depth and DO concentration) and water samples were collected with a conductivity–temperature–depth rosette system, with dual Sea Bird oxygen
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Fig. 1. Map of station locations.
sensors (Sea Bird Electronics), and attached Niskin bottles. Depending on the concentration of DO, we defined the water layers with ,2 mM DO as the core-OMZs, and the waters outside the core-OMZs were defined as O-OMZs. In addition to the basic hydrographic data, NO2 2 , ammo) concentrations and total iron concentration nium (NHþ 4 profile were adopted from other studies of the CRDFLUZiE cruise (Buchwald, 2013; Vedamati, 2013). For molecular analyses, sea water samples of 500–1000 mL were size-fractionated with 2 and 0.2 mm polycarbonate membrane filters (47 mm, Millipore) under low vacuum. All of the molecular samples were flash frozen and stored at 2808C until DNA extraction.
DNA extraction, nifH PCR and 454-pyrosequencing of DNA samples Genomic DNA was extracted from the 0.2-mm membrane filters with TRIzol plus Genomic DNA mini kit (Invitrogen, Carlsbad, CA, USA). All the DNA samples were eluted in 60-mL ultrapure water (Invitrogen) and stored at 2208C until further analysis. NifH gene fragments were amplified from the genomic DNA samples following the nested polymerase chain reaction (PCR) protocol (Zehr and Turner, 2001). The nested PCR reaction was done in duplicate with the Platinum Taq DNA polymerase PCR system (Invitrogen)
in a volume of 12.5 mL containing 1 rxn PCR buffer, 4 mM MgCl2, 400 mM dNTPs, 1 mM primers (nifH 3 and nifH 4 for the first round, nifH 1 and nifH 2 for the second round), 1 unit Platinum Taq polymerase and 1 mL of total genomic DNA. After the second round of the nested PCR, 1 mL of the PCR products was used to run 10 cycles of PCR with sample-specific multiplex identifiers (MIDs) and adaptor-attached primers (Farnelid et al., 2011). The PCR conditions were identical to those of the nested PCR. The duplicates of PCR products were pooled with the same amount and then gel purified with Quick gel purification kit (Invitrogen). For 454-pyrosequencing, MID-adaptor-labeled nifH gene amplicons of different samples were mixed with the same concentrations to construct an amplicon library according to Rapid Library construction protocols (Roche, 454 Life Science). Then, the DNA library attached beads were loaded onto a Pico TiterPlate and sequenced with a GS Junior System (Roche, 454 Life Science). Negative controls, in which no template DNA was added, were done in all the nested PCR. According to the results of DNA gel electrophoresis, no visible DNA bands were observed in the negative control lanes. A section of gel encompassing the size of nifH amplicons (439 bp) was sliced from the negative control lane and purified as mentioned. The elution of the negative control collected after the gel purification was processed
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as the samples, and sequenced with GS Junior System (Roche, 454 Life Science).
Sequence quality control and analysis Quality control of raw sequence data was conducted with Mothur (Schloss et al., 2009). Low-quality sequences (average quality score ,25), short sequences (,300 bases in length), ambiguous base containing sequences, homopolymers containing sequences (homopolymers .8 bases) and chimeric sequences were removed. Also, the MID sequences were trimmed. The trimmed sequences were de-noised with 0.01 sigma value to reduce the effects of PCR bias. After being aligned with the reference sequences from the online nifH gene database of Ribosomal Database Project (Wang et al., 2013), phylogenetic distances between these high-quality sequences were then calculated, and the sequences were clustered at 95% similarity using Mothur. On the basis of 95% similarity clustering in nucleotide basis (Fernandez et al., 2011), operational taxonomic units (OTUs), representative sequences for each OTU, rarefaction curve, Good’s coverage indices and Shannon diversity indices were generated or calculated with Mothur. For the similarity among the samples, the samples were clustered with Braycurtis calculator using the Unweighted Pair Group Method with the Arithmetic Mean (UPGMA) algorithm in Mothur. The OTUs contained 10 sequences individually, which are defined as top OTUs, were selected for the subsequent phylogenetic analysis. The remaining OTUs, which consisted of ,10 sequences per OTU, were grouped as rare species. To identify the top OTUs, representative sequences were first translated into amino acid (aa) sequences with the FRAMEBOT online pipeline (Wang et al., 2013). The aa sequences were used to search the protein sequences database on NCBI via protein BLAST (BLASTp) webpage (Mcginnis and Madden, 2004). The OTU representative sequences and the selected reference sequences from the NCBI protein sequences database were then aligned with ClustalW in MEGA 6.0 (Tamura et al., 2013) and used to construct a neighbor-joining phylogenetic tree ( p-distance) with MEGA 6.0 (Tamura et al., 2013). The phylogenetic tree was further edited using iTOL (Letunic and Bork, 2011). The OTUs that clustered together with the same reference sequences (.96% similarity in aa with the reference sequences) were merged into the same sub-clusters. Then, these subclusters were named with species of the corresponding reference sequences and used to display relative abundances of different diazotrophs in different samples. All raw sequences obtained in this study have been deposited in the NCBI Sequence Read Archive with accession number SRP057492.
Environmental variables and diazotroph community structure To reveal the relationships between diazotroph community variances and environmental variables, relative abundance of the sub-clusters in each samples was analyzed with detrended correspondence analysis (DCA) using Canoco 4.5 (Ter Braak and Smilauer, 2002). The data for two deep samples (St. 3—1000 m and St. 5—800 m) were þ excluded from the analysis, because NO2 2 and NH4 concentrations were not measured at these depths. The environmental variables (temperature, salinity, DO, NO2 2 and ) and the relative abundance of the sub-clusters were NHþ 4 square root transformed. On the basis of the result of DCA, the largest length of gradient value among the four axes was between 3 and 4; therefore, redundancy analysis was used to analyze relationships among diazotroph community variances and environmental variables in Canoco 4.5 (Ter Braak and Smilauer, 2002). The environmental variables with significant relationships (P , 0.05) were selected to explain the diazotroph community variances, which were assessed in permutation tests with 499 unrestricted Monte Carlo permutations.
R E S U LT S Hydrography and nutrients The hydrographic profiles for the four studied water columns were similar although the depth and thickness of the core-OMZ layers were slightly different among the stations. Sharp decreases of temperature and increases of salinity were detected at 30–40 m, which was the signal of upwelling of deep water (Fig. 2a and b). In general, the core-OMZs, with DO concentration ,2 mM, were found in the depth range of 350–700 m (Fig. 2c). Sub-surface 2 2 peaks of NHþ 4 and NO2 (secondary NO2 maxima) were found below the euphotic layers, and the highest concentration of NO2 2 was detected in the core-OMZs (0.9–1.5 mM) (Fig. 3). The largest core-OMZ (400–700 m) occurred at Station 3, with the largest vertical extent of the NO2 2 maximum. Conversely, the lowest concentration and depth range of NO2 2 were found at Station 4, coinciding with the smallest core-OMZ (350–500 m). In addition, comparing with euphotic zone and O-OMZs, concentration of iron increased sharply in the core-OMZs (Fig. 2d).
Diazotroph community diversity and similarity In total, 39 599 DNA sequences were included in this study and the sequence number of each sample is provided in Table I. The Good’s coverage indices of the samples
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Fig. 2. Vertical profiles of (a) temperature, (b) salinity, (c) dissolved oxygen (DO) and (d) total iron at the sampling stations.
þ Fig. 3. Vertical profiles of concentrations of nitrite (NO2 2 ) and ammonium (NH4 ). Shadow layers indicate core-OMZs, where the DO concentration was lower than 2 mM.
ranged from 0.958 to 0.991 (Table I), showing that the sequencing was deep enough for the diazotroph community study. For the negative control, no sequence of nifH gene amplicon was recovered with the 454-pyrosequencing. The rarefaction curves of the samples did not plateau (Supplementary data, Fig. S1), which suggested that more sequences are needed for studying the rare species. Therefore, we focused on the top OTUs in this study. In general, diazotroph community composition and diversity
within the core-OMZs were different from those outside the core-OMZs although some exceptional cases were observed. The Shannon diversity index values for communities in the core-OMZs were generally greater than those outside the core-OMZs (Table I). In terms of community similarity (Fig. 4), communities within the core-OMZs clustered together; and the communities 100 m above and 100 m below the core-OMZs (St. 3—300 m; St. 5—200 m; St. 4—600 m) also clustered. The communities
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Fig. 4. UPGMA dendrogram showing the relationship between samples. Samples from the core-OMZs are labeled with asterisks.
in deeper waters (St. 3—1000 m and St. 5—800 m), where DO was higher than that in the core-OMZs, were different. The samples from St. 4—300 m were taken close to the core-OMZ (50 m apart), and the diazotroph community and diversity were similar to that of the core-OMZ. Two samples from Station 3 (St. 3—600 m and St. 3—700 m) did not cluster with the other seven core-OMZ samples although the DO concentration was ,2 mM. This suggests that there were other environmental variables, besides DO, influencing diazotroph community structure.
to the same reference sequences were grouped into subclusters (at least 96% similarity in aa). These sub-clusters were named with the species identities of the reference sequences. OTU 29 in Cluster III, which did not show high similarity (at least 96% similarity in aa) with any reference sequences, was assigned to unidentified Cluster III. As a result, 37 OTUs were grouped into 18 sub-clusters, among which Methylocella palustris and Vibrio diazotrophicus were the dominant diazotrophs (Fig. 6). The sub-cluster M. palustris was mainly found (50–90% in relative abundance) in the core-OMZs, while sub-cluster V. diazotrophicus dominated (.90% in relative abundance) in some water samples from both the core-OMZs and O-OMZs. The deeper-water communities were dominated by different sub-clusters, with Bradyrhizobium sp. IRBG 2 and Klebsiella sp. GG41E dominating in St. 3—1000 m and St. 5—800 m, respectively. Different sub-clusters showed different distribution patterns. For Alphaproteobacteria, most sub-clusters were unique to the core-OMZs, while Bradyrhizobium sp. IRBG 2 was unique to the O-OMZs. Besides that, Bradyrhizobium sp. Z15 and Azorhizobium caulinodans ORS 571 were found in both core-OMZs and O-OMZs. Deltaproteobacteria, sulfate-reducing bacteria, were unique to the core-OMZ. Most Gammaproteobacteria were widely distributed in different layers of the water column, except Pectobacterium atrosepticum and Tolumonas auensis, which were unique to the core-OMZ. As for the remaining sub-clusters, Cluster IV was unique to the core-OMZs, and unidentified Cluster III was detected in both core-OMZs and O-OMZs.
Diazotroph community structure According to the results of BLASTp, three cyanobacterialike OTUs were detected in St. 1—350 m, which contributed 58% of the total sequences of the sample (1656 of 2847 sequences). These OTUs were removed from the dataset during phylogenetic analysis, which is explained in the discussion part. A total of 37 OTUs were selected for the phylogenetic analysis. From the online protein database of NCBI, 49 reference sequences (mostly 96–100% similarity in aa) were selected and used in this study. These 49 sequences included reference sequences with known identities and some environmental DNA/cDNA sequences from OMZ-related studies (Fernandez et al., 2011; Jayakumar et al., 2012; Bird and Wyman, 2013; Farnelid et al., 2013; Bentzon-Tilia et al., 2014; Loescher et al., 2014) and other marine environments. Overall, the representative sequences of the top 37 OTUs fell within 3 defined clusters of nifH gene (Fig. 5) (Zehr et al., 2003b). The majority of OTUs (31/37 OTUs) belonged to the Cluster I proteobacteria. The remaining OTUs fell within Cluster III (4/37 OTUs) and Cluster IV (2/37 OTUs). The representative sequences that showed high similarity
Diazotroph community structure and environmental variables Among all the available environmental variables, DO, temperature and NO2 2 affected diazotroph community significantly (P , 0.05). These three variables explained 80% of the variance in community structure together. The most abundant sub-clusters, M. palustris and V. diazotrophicus, showed negative and positive correlations with DO, respectively (Fig. 7). Some sub-clusters unique to core-OMZs showed more significant correlations with NO2 2 , including Hyphomicrobium sp. MC1, Novosphingobium malaysiense and T. auensis. In addition, temperature influenced the distributions and relative abundances of sulfate-reducing bacteria (Deltaproteobacteria).
Cyanobacteria-like OTUs in sub-surface water The cyanobacteria-like OTUs, which were removed from the dataset, were phylogenetically close to Calothrix sp. PCC 7507 (accession number: WP015131644). Among
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Fig. 5. Neighbor-joining phylogenetic tree constructed with nifH gene amino acid sequences. The representative sequences of the OTUs detected in this study are labeled with OTU 1 to OTU 37; and the accession number of each reference sequence is shown in front of the sequence name. Bootstrap resampling was performed for 1000 times; and the values that are higher than 50% are labeled with gray circular symbols on the branches.
the 3 revealed cyanobacteria-like OTUs, 2 OTUs (contributed 97.2% of the removed sequences) showed 100 and 98% similarity in aa with Calothrix sp. PCC 7507. The third OTU only had 95% similarity with Calothrix sp. PCC 7507 and its closest marine sequence (accession no.: AAZ31164.1); hence, this OTU is relatively distant to all previously revealed nifH sequences.
DISCUSSION Hydrographic conditions and nitrogen fixation in OMZs Oxygen deficiency and the occurrence of the secondary NO2 2 maxima (Fig. 3) in the core-OMZs of CRD indicates
that there were active N-loss processes (e.g. anammox and nitrate reduction) occurring in the core-OMZs, which have also been observed and studied in the OMZs in the ETSP and Arabian Sea (Gala´n et al., 2009; Ward et al., 2009). Two other studies conducted during the CRDFLUZiE cruise reported active anammox activity (Kong et al., 2013) and deficiency of DIN in the middle of core-OMZs at Stations 3, 4 and 5 (Buchwald, 2013). Nitrogen fixation of heterotrophic diazotrophs has been reported in the OMZs of the ETSP and Arabian Sea (Fernandez et al., 2011; Jayakumar et al., 2012; Bird and Wyman, 2013; Loescher et al., 2014), where the hydrographic profiles were similar to those in the CRD. Moreover, iron concentration, which was suggested to be important for nitrogenase activity (Deutsch et al., 2007),
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Fig. 6. Relative abundance of diazotroph sub-clusters in different samples. The water depths within the core-OMZs are marked with pink shadows. Names of the sub-clusters that were also detected in other OMZs are labeled in red. The proteobacteria sub-clusters were grouped into the corresponding classes, including Alphaproteobacteria (a), Betaproteobacteria (b), Deltaproteobacteria (d) and Gammaproteobacteria ( g).
diazotroph community (Fernandez et al., 2011). Therefore, both hydrographic conditions and Shannon diversity indices imply the importance of measuring nitrogen fixation rate in future studies.
Contamination issue of nested PCR of nifH
Fig. 7. Correlation biplot based on a redundancy analysis (RDA), depicting the relationship between relative abundances of the diazotroph sub-clusters and the environmental factors.
also increased significantly in the core-OMZs. Hence, the overall hydrographic conditions in the CRD OMZ were suitable for nitrogen fixation. In addition, Shannon diversity indices of diazotroph communities were generally higher in the core-OMZs (Table I). A previous study in the Peruvian OMZ reported positive correlation between nitrogen fixation rate and Shannon diversity indices of the
It has been reported that some heterotrophic nifH sequences may be amplified from bacterial cells or DNA present in some PCR reagents, during nested PCR (Zehr et al., 2003a; Goto et al., 2005). However, the DNA contaminants in different PCR reagents are not necessarily the same, and the OTUs affiliated with the reported DNA contaminants may also actually inhabit in the marine environment (Farnelid et al., 2013). Therefore, the negative control of the nested PCR was sequenced in our study. The absence of nifH gene sequences in the negative control suggests that the recovered OTUs in this study originated from the studied area.
Feature species observed in the CRD OMZ Sub-cluster V. diazotrophicus was one of the most dominant sub-clusters in CRD OMZ; and it was detected in both O-OMZs and core-OMZs (Fig. 6). According to the result of BLASTp, this sub-cluster was also the most
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abundant Gammaproteobacterial diazotroph (named cluster P8, reached 106 nifH gene copies per L of water) in the Peruvian OMZs (100% similarity in aa, accession no.: AEA49228) and was observed to be actively expressing nifH genes (Fernandez et al., 2011; Loescher et al., 2014). Moreover, nifH gene transcripts of similar diazotrophs (96% similarity in aa, accession no.: AFM94362) were also detected in the OMZ of the Arabian Sea (Jayakumar et al., 2012). Therefore, sub-cluster V. diazotrophicus may also be actively fixing nitrogen in the CRD OMZ, where the overall hydrographic conditions were similar to the Peruvian and Arabian Sea OMZs. The high relative abundance and wide distribution of subcluster V. diazotrophicus in both core-OMZs and O-OMZs is consistent with the findings of Loescher et al. (Loescher et al., 2014). Vibrio diazotrophicus was reported to be a facultative anaerobe (Guerinot et al., 1982), and the nitrogenase protein of Vibrio natriegens (96% similarity in aa with sub-cluster V. diazotrophicus) was produced in high amounts and stored under anaerobic conditions (Coyer et al., 1996). These previous studies may explain why V. diazotrophicus dominated throughout the water column in the CRD OMZ and other OMZs. The most dominant sub-cluster (M. palustris) detected in the core-OMZs was closely related to Alphaproteobacterial methanotrophs (99% similarity in aa). A number of studies have reported the coupling between nitrogen fixation and methane assimilation (Murrell and Dalton, 1983; Khadem et al., 2010; Larmola et al., 2014). It has been reported that methane is released from submarine sediments, hydrothermal seeps and cold seeps (Karaca, 2011; Levin et al., 2012) into the water column off Costa Rica, including the core-OMZs (Schleicher and Wallmann, 2007; Pack et al., 2015). Previous studies have reported that Alphaproteobacterial methanotrophs can grow in microaerobic and even anaerobic conditions (Vecherskaya et al., 2009) and that methane oxidation can be nitrogen-limited (Bodelier et al., 2000a, b). High relative abundance of the methanotroph-like sub-cluster was also observed at St. 4— 300 m (Fig. 6), where DO was 4.5 mM, suggesting that this sub-cluster might be facultatively anaerobic. In addition, another methanotroph-like sub-cluster (Hyphomicrobium sp. MC1) was also detected in the core-OMZs. These methanotroph-like sub-clusters may benefit from the highmethane and low-oxygen conditions in the core-OMZs. Although sub-cluster M. palustris mostly dominated in the core-OMZs and sub-cluster V. diazotrophicus was generally associated with the O-OMZs, they were detected in both types of waters. It implies that other environmental factors besides DO might govern the distributions of these subclusters. At stations 3 and 5, sub-cluster V. diazotrophicus was detected in almost all water layers, while its relative abundance decreased in layers where sub-cluster M. palustris was
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dominant (Fig. 6). The succession between these two subclusters might be explained by other environmental factors, such as methane availability, which allowed the sub-cluster M. palustris to outgrow V. diazotrophicus in some layers. Desulfobulbus alkaliphilus (sulfate-reducing bacteria) and T. auensis dominated in St. 4—500 m and St. 3—600 m (Fig. 6), respectively. Their occurrence in the core-OMZs may be supported by the anaerobic conditions and sulfate availability (Canfield et al., 2010) and by the higher availability of organic matter (Fischer-Romero et al., 1996) in the core-OMZs. Previous studies have indicated that sulfate-reducing diazotrophs are abundant in the Peruvian OMZ (Loescher et al., 2014) and the suboxic water of the Baltic Sea (Farnelid et al., 2013). Although they were relatively abundant (40% in relative abundance) in only one sample (St. 4—500 m) in this study (Fig. 6), their existence provides evidence that sulfatereducing bacteria may be able to thrive in the core-OMZ of the ETNP. The cluster IV OTUs detected in this study were distant from the previously reported marine sequences (Fig. 5). In the Peruvian OMZ, distant cluster IV was the most abundant and active phylotype of nifH (Loescher et al., 2014). Traditionally, the Cluster IV was thought to be homologs of nitrogenase (Zehr et al., 2003b), of which nitrogen fixation activity was still unclear. Therefore, the nitrogen fixing function of the Cluster IV diazotrophs in the OMZs needs to be addressed in the future studies. Calothrix, detected in this study, is a symbiotic cyanobacterium in the diatom Chaetocoeros that has only been reported in the Equatorial Atlantic upwelling region (Foster et al., 2009), but not in any OMZs in ETNP or Arabian Sea. Previous studies have demonstrated that diatom blooms are supported in upwelling regions (Allen et al., 2005; Bruland et al., 2005) and that sinking eukaryotic chloroplasts can be detected at 200 and 500 m depths in the CRD OMZ (Jing et al., 2013). Hence, the source of these diatom symbiotic diazotrophs in the St. 1—350 m is believed to be sinking particles. Therefore, these cyanobacteria-like OTUs were removed from the dataset during the phylogenetic analysis. The distant cyanobacteria-like OTU discovered in this study supports the view that the unique hydrographic setting of the CRD OMZ provides niches for unique strains of diazotrophic cyanobacteria, similar to the findings of unique Synechococcus strains in the CRD (Ahlgren et al., 2014).
Influence of hydrographic conditions on diazotroph community composition Comparing with the sequences detected in the ETSP and Arabian Sea OMZs, 6 sub-clusters detected in CRD were also detected in other OMZs (Fig. 6), accounting for
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70% of the total sequences in this study. The presence of common diazotrophs in spatially separated OMZs could be due to the similar hydrographic conditions (DO depletion, secondary NO2 2 maxima and availability of iron) among different OMZs. The significant correlations between diazotroph community structure and DO, temperature and NO2 2 indicate that the large variations of these parameters through the water column played a major role in shaping the community structure of diazotrophs in this unique environment. Although M. palustris and V. diazotrophicus showed negative and positive correlations with DO (Fig. 7), respectively, the largely reciprocal distribution pattern of these two sub-clusters in CRD waters suggests the importance of other environmental variables (e.g. availability of methane) that were not measured in this study. Loescher et al. (Loescher et al., 2014) reported that subcluster V. diazotrophicus (named P8 in their paper) was negatively correlated with NO2 2 in the Peruvian OMZ, while such a pattern was not found in this study. This difference may be due to different analytical methods, as copy number of nifH gene was used in the Peruvian study and relative abundance of nifH gene was used in the present study. It appears that the diazotrophs in the CRD OMZ were influenced by a combination of different environmental factors. Hence, more environmental parameters (e.g. concentration of methane and sulfate) should be measured in the future studies of the CRD OMZ, to better explain the distributions of different sub-clusters of heterotrophic diazotrophs. In summary, our study provides the first insight into the composition and distribution of the putative diazotroph assemblages in the CRD OMZ of the ETNP. Diversity of diazotrophs was generally higher in the core-OMZs than at other depths in the water column. High similarity of the diazotroph community with other OMZs also indicated that heterotrophic nitrogen fixation could be a process potentially occurring in the CRD OMZ, especially for the existence of sub-cluster V. diazotrophicus. The finding of a dominant methanotroph-like sub-cluster implies a potential coupling between nitrogen fixation and methane metabolism in the CRD OMZ although factors controlling the distribution of the methanotroph-like sub-cluster and sub-cluster V. diazotrophicus are still unclear. Lastly, phylogenetically distant cluster IV and cyanobacteria-like diazotrophs were discovered in this study. These findings highlight the importance of further studies of the nitrogen fixation process in the CRD OMZ, including identifying active nifH phylotypes, quantifying particular nifH gene abundance and expression of the dominant sub-clusters and understanding the coupling between methanotrophy and nitrogen cycling.
S U P P L E M E N TA RY DATA Supplementary data can be found online at http://plankt. oxfordjournals.org.
DATA A RC H I V I N G All raw sequences obtained in this study have been deposited in the NCBI Sequence Read Archive with accession number SRP057492.
AC K N OW L E D G E M E N T S We thank chief scientist Prof. Michael R. Landry of the Scripps Institution of Oceanography for giving us the opportunity to participate on the FLUZiE cruise, Dr Takafumi Kataoka for taking the samples, and the captain and crew of the R/V Melville for their professional assistance.
FUNDING This study was supported by Hong Kong Grants Council GRF (661911, 661912, 661813).
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