Highly heterogeneous diazotroph communities in the Kuroshio Current and the Tokara Strait, Japan.
ABSTRACT: In this study, we used 454-pyrosequencing to report the highly diverse diazotroph communities in the Kuroshio and its adjacent waters along a transect across the Tokara Strait, Japan. Terrestrial input from the islands resulted in a highly heterogeneous diazotroph community within a relatively small geographic region, which was presumably caused by the remarkably different responses of UCYN-A2, UCYN-C and Trichodesmium to the steep environmental gradient. On the other hand, most major cyanobacterial OTUs found in this study were also detected in an unpublished dataset from the upstream Kuroshio, which suggests transportation of diazotrophs by the Kuroshio in large geographic scale. A significant amount of UCYN-C was found in the Kuroshio and offshore stations, suggesting the importance of this potentially overlooked group in the western North Pacific Ocean (WNPO). Moreover, a novel sublineage of UCYN-B was defined, which was predominant in an oligotrophic water sample; and it was also found to be widely distributed in oceanic waters. In addition, the apparent increase in relative abundance of UCYN-A2 from offshore to near-shore water provides evidence for the earlier and under-debating view that UCYN-A2 prefers coastal conditions. Our report provides new knowledge for understanding the phylogeny and ecology of unicellular cyanobacterial diazotrophs in WNPO.
Project description:Australia's tropical waters represent predicted 'hotspots' for nitrogen (N2) fixation based on empirical and modelled data. However, the identity, activity and ecology of diazotrophs within this region are virtually unknown. By coupling DNA and cDNA sequencing of nitrogenase genes (nifH) with size-fractionated N2 fixation rate measurements, we elucidated diazotroph dynamics across the shelf region of the Arafura and Timor Seas (ATS) and oceanic Coral Sea during Austral spring and winter. During spring, Trichodesmium dominated ATS assemblages, comprising 60% of nifH DNA sequences, while Candidatus Atelocyanobacterium thalassa (UCYN-A) comprised 42% in the Coral Sea. In contrast, during winter the relative abundance of heterotrophic unicellular diazotrophs (?-proteobacteria and ?-24774A11) increased in both regions, concomitant with a marked decline in UCYN-A sequences, whereby this clade effectively disappeared in the Coral Sea. Conservative estimates of N2 fixation rates ranged from <1 to 91?nmol?l(-1)?day(-1), and size fractionation indicated that unicellular organisms dominated N2 fixation during both spring and winter, but average unicellular rates were up to 10-fold higher in winter than in spring. Relative abundances of UCYN-A1 and ?-24774A11 nifH transcripts negatively correlated to silicate and phosphate, suggesting an affinity for oligotrophy. Our results indicate that Australia's tropical waters are indeed hotspots for N2 fixation and that regional physicochemical characteristics drive differential contributions of cyanobacterial and heterotrophic phylotypes to N2 fixation.
Project description:Knowledge of the ecology of N2-fixing (diazotrophic) plankton is mainly limited to oligotrophic (sub)tropical oceans. However, diazotrophs are widely distributed and active throughout the global ocean. Likewise, relatively little is known about the temporal dynamics of diazotrophs in productive areas. Between February 2014 and December 2015, we carried out 9 one-day samplings in the temperate northwestern Iberian upwelling system to investigate the temporal and vertical variability of the diazotrophic community and its relationship with hydrodynamic forcing. In downwelling conditions, characterized by deeper mixed layers and a homogeneous water column, non-cyanobacterial diazotrophs belonging mainly to nifH clusters 1G (Gammaproteobacteria) and 3 (putative anaerobes) dominated the diazotrophic community. In upwelling and relaxation conditions, affected by enhanced vertical stratification and hydrographic variability, the community was more heterogeneous vertically but less diverse, with prevalence of UCYN-A (unicellular cyanobacteria, subcluster 1B) and non-cyanobacterial diazotrophs from clusters 1G and 3. Oligotyping analysis of UCYN-A phylotype showed that UCYN-A2 sublineage was the most abundant (74%), followed by UCYN-A1 (23%) and UCYN-A4 (2%). UCYN-A1 oligotypes exhibited relatively low frequencies during the three hydrographic conditions, whereas UCYN-A2 showed higher abundances during upwelling and relaxation. Our findings show the presence of a diverse and temporally variable diazotrophic community driven by hydrodynamic forcing in an upwelling system.
Project description:Major advances in understanding the diversity, distribution, and activity of marine N2-fixing microorganisms (diazotrophs) have been made in the past decades, however, large gaps in knowledge remain about the environmental controls on growth and mortality rates. In order to measure diazotroph net growth rates and microzooplankton grazing rates on diazotrophs, nutrient perturbation experiments and dilution grazing experiments were conducted using free-floating in situ incubation arrays in the vicinity of Station ALOHA in March 2016. Net growth rates for targeted diazotroph taxa as well as Prochlorococcus, Synechococcus and photosynthetic picoeukaryotes were determined under high (H) and low (L) nitrate:phosphate (NP) ratio conditions at four depths in the photic zone (25, 45, 75, and 100 m) using quantitative PCR and flow cytometry. Changes in the prokaryote community composition in response to HNP and LNP treatments were characterized using 16S rRNA variable region tag sequencing. Microzooplankton grazing rates on diazotrophs were measured using a modified dilution technique at two depths in the photic zone (15 and 125 m). Net growth rates for most of the targeted diazotrophs after 48 h were not stimulated as expected by LNP conditions, rather enhanced growth rates were often measured in HNP treatments. Interestingly, net growth rates of the uncultivated prymnesiophyte symbiont UCYN-A1 were stimulated in HNP treatments at 75 and 100 m, suggesting that N used for growth was acquired through continuing to fix N2 in the presence of nitrate. Net growth rates for UCYN-A1, UCYN-C, Crocosphaera sp. (UCYN-B) and the diatom symbiont Richelia (associated with Rhizosolenia) were uniformly high at 45 m (up to 1.6 ± 0.5 d-1), implying that all were growing optimally at the onset of the experiment at that depth. Differences in microzooplankton grazing rates on UCYN-A1 and UCYN-C in 15 m waters indicate that the grazer assemblage preyed preferentially on UCYN-A1. Deeper in the water column (125 m), both diazotrophs were grazed at substantial rates, suggesting grazing pressure may increase with depth in the photic zone. Constraining in situ diazotroph growth and mortality rates are important steps for improving parameterization for diazotrophs in global ecosystem models.
Project description:In addition to the cyanobacterial N2-fixers (diazotrophs), there is a high nifH gene diversity of non-cyanobacterial groups present in marine environments, yet quantitative information about these groups is scarce. N2 fixation potential (nifH gene expression), diversity and distributions of the uncultivated diazotroph phylotype ?-24774A11, a putative gammaproteobacterium, were investigated in the western South Pacific Ocean. ?-24774A11 gene copies correlated positively with diazotrophic cyanobacteria, temperature, dissolved organic carbon and ambient O2 saturation, and negatively with depth, chlorophyll a and nutrients, suggesting that carbon supply, access to light or inhibitory effects of DIN may control ?-24774A11 abundances. Maximum nifH gene-copy abundance was 2 × 10(4)?l(-1), two orders of magnitude less than that for diazotrophic cyanobacteria, while the median ?-24774A11 abundance, 8 × 10(2)?l(-1), was greater than that for the UCYN-A cyanobacteria, suggesting a more homogeneous distribution in surface waters. The abundance of nifH transcripts by ?-24774A11 was greater during the night than during the day, and the transcripts generally ranged from 0-7%, but were up to 26% of all nifH transcripts at each station. The ubiquitous presence and low variability of ?-24774A11 abundances across tropical and subtropical oceans, combined with the consistent nifH expression reported in this study, suggest that ?-24774A11 could be one of the most important heterotrophic (or photoheterotrophic) diazotrophs and may need to be considered in future N budget estimates and models.
Project description:Marine planktonic cyanobacteria capable of fixing molecular nitrogen (termed 'diazotrophs') are key in biogeochemical cycling, and the nitrogen fixed is one of the major external sources of nitrogen to the open ocean. Candidatus Atelocyanobacterium thalassa (UCYN-A) is a diazotrophic cyanobacterium known for its widespread geographic distribution in tropical and subtropical oligotrophic oceans, unusually reduced genome and symbiosis with a single-celled prymnesiophyte alga. Recently a novel strain of this organism was also detected in coastal waters sampled from the Scripps Institute of Oceanography pier. We analyzed the metagenome of this UCYN-A2 population by concentrating cells by flow cytometry. Phylogenomic analysis provided strong bootstrap support for the monophyly of UCYN-A (here called UCYN-A1) and UCYN-A2 within the marine Crocosphaera sp. and Cyanothece sp. clade. UCYN-A2 shares 1159 of the 1200 UCYN-A1 protein-coding genes (96.6%) with high synteny, yet the average amino-acid sequence identity between these orthologs is only 86%. UCYN-A2 lacks the same major pathways and proteins that are absent in UCYN-A1, suggesting that both strains can be grouped at the same functional and ecological level. Our results suggest that UCYN-A1 and UCYN-A2 had a common ancestor and diverged after genome reduction. These two variants may reflect adaptation of the host to different niches, which could be coastal and open ocean habitats.
Project description:Growth limitation of phytoplankton and unicellular nitrogen (N(2)) fixers (diazotrophs) were investigated in the oligotrophic Western South Pacific Ocean. Based on change in abundances of nifH or 23S rRNA gene copies during nutrient-enrichment experiments, the factors limiting net growth of the unicellular diazotrophs UCYN-A (Group A), Crocosphaera watsonii, ?-Proteobacterium 24774A11, and the non-diazotrophic picocyanobacterium Prochlorococcus, varied within the region. At the westernmost stations, numbers were enhanced by organic carbon added as simple sugars, a combination of iron and an organic chelator, or iron added with phosphate. At stations nearest the equator, the nutrient-limiting growth was not apparent. Maximum net growth rates for UCYN-A, C. watsonii and ?-24774A11 were 0.19, 0.61 and 0.52 d(-1), respectively, which are the first known empirical growth rates reported for the uncultivated UCYN-A and the ?-24774A11. The addition of N enhanced total phytoplankton biomass up to 5-fold, and the non-N(2)-fixing Synechococcus was among the groups that responded favorably to N addition. Nitrogen was the major nutrient-limiting phytoplankton biomass in the Western South Pacific Ocean, while availability of organic carbon or iron and organic chelator appear to limit abundances of unicellular diazotrophs. Lack of phytoplankton response to nutrient additions in the Pacific warm pool waters suggests diazotroph growth in this area is controlled by different factors than in the higher latitudes, which may partially explain previously observed variability in community composition in the region.
Project description:Diazotrophic cyanobacteria, those capable of fixing di-nitrogen (N2), are considered one of the major sources of new nitrogen (N) in the oligotrophic tropical ocean, but direct incorporation of diazotrophic N into food webs has not been fully examined. In the Amazon River-influenced western tropical North Atlantic (WTNA), diatom diazotroph associations (DDAs) and the filamentous colonial diazotrophs Trichodesmium have seasonally high abundances. We sampled epipelagic mesozooplankton in the Amazon River plume and WTNA in May-June 2010 to investigate direct grazing by mesozooplankton on two DDA populations: Richelia associated with Rhizosolenia diatoms (het-1) and Hemiaulus diatoms (het-2), and on Trichodesmium using highly specific qPCR assays targeting nitrogenase genes (nifH). Both DDAs and Trichodesmium occurred in zooplankton gut contents, with higher detection of het-2 predominantly in calanoid copepods (2.33-16.76 nifH copies organism-1). Abundance of Trichodesmium was low (2.21-4.03 nifH copies organism-1), but they were consistently detected at high salinity stations (>35) in calanoid copepods. This suggests direct grazing on DDAs, Trichodesmium filaments and colonies, or consumption as part of sinking aggregates, is common. In parallel with the qPCR approach, a next generation sequencing analysis of 16S rRNA genes identified that cyanobacterial assemblage associated with zooplankton guts was dominated by the non-diazotrophic unicellular phylotypes Synechococcus (56%) and Prochlorococcus (26%). However, in two separate calanoid copepod samples, two unicellular diazotrophs Candidatus Atelocyanobacterium thalassa (UCYN-A) and Crocosphaera watsonii (UCYN-B) were present, respectively, as a small component of cyanobacterial assemblages (<2%). This study represents the first evidence of consumption of DDAs, Trichodesmium, and unicellular cyanobacteria by calanoid copepods in an area of the WTNA known for high carbon export. These diazotroph populations are quantitatively important in the global N budget, widespread and hence, the next step is to accurately quantify grazing. Nonetheless, these results highlight a direct pathway of diazotrophic N into the food web and have important implications for biogeochemical cycles, particularly oligotrophic regions where N2 fixation is the main source of new nitrogen.
Project description:Nitrogen (N2) fixation is a major source of nitrogen that supports primary production in the vast oligotrophic areas of the world's oceans. The Western Tropical South Pacific has recently been identified as a hotspot for N2 fixation. In the Noumea lagoon (New Caledonia), high abundances of the unicellular N2-fixing cyanobacteria group A (UCYN-A), coupled with daytime N2 fixation rates associated with the <10 ?m size fraction, suggest UCYN-A may be an important diazotroph (N2-fixer) in this region. However, little is known about the seasonal variability and diversity of UCYN-A there. To assess this, surface waters from a 12 km transect from the mouth of the Dumbea River to the Dumbea Pass were sampled monthly between July 2012 and March 2014. UCYN-A abundances for two of the defined sublineages, UCYN-A1 and UCYN-A2, were quantified using qPCR targeting the nifH gene, and the nifH-based diversity of UCYN-A was characterized by identifying oligotypes, alternative taxonomic units defined by nucleotide positions with high variability. UCYN-A abundances were dominated by the UCYN-A1 sublineage, peaked in September and October and could be predicted by a suite of nine environmental parameters. At the sublineage level, UCYN-A1 abundances could be predicted based on lower temperatures (<23°C), nitrate concentrations, precipitation, wind speed, while UCYN-A2 abundances could be predicted based on silica, and chlorophyll a concentrations, wind direction, precipitation, and wind speed. Using UCYN-A nifH oligotyping, similar environmental variables explained the relative abundances of sublineages and their associated oligotypes, with the notable exception of the UCYN-A2 oligotype (oligo43) which had relative abundance patterns distinct from the dominant UCYN-A2 oligotype (oligo3). The results support an emerging pattern that UCYN-A is comprised of a diverse group of strains, with sublineages that may have different ecological niches. By identifying environmental factors that influence the composition and abundance of UCYN-A sublineages, this study helps to explain global UCYN-A abundance patterns, and is important for understanding the significance of N2 fixation at local and global scales.
Project description:The Tropical North Atlantic (TNAtl) plays a critical role in the marine nitrogen cycle, as it supports high rates of biological nitrogen (N(2)) fixation, yet it is unclear whether this process is limited by the availability of iron (Fe), phosphate (P) or is co-limited by both. In order to investigate the impact of nutrient limitation on the N(2)-fixing microorganisms (diazotrophs) in the TNAtl, trace metal clean nutrient amendment experiments were conducted, and the expression of nitrogenase (nifH) in cyanobacterial diazotrophs in response to the addition of Fe, P, or Fe+P was measured using quantitative PCR. To provide context, N(2) fixation rates associated with the <10 ?m community and diel nifH expression in natural cyanobacterial populations were measured. In the western TNAtl, nifH expression in Crocosphaera, Trichodesmium, and Richelia was stimulated by Fe and Fe+P additions, but not by P, implying that diazotrophs may be Fe-limited in this region. In the eastern TNAtl, nifH expression in unicellular cyanobacteria UCYN-A and Crocosphaera was stimulated by P, implying P-limitation. In equatorial waters, nifH expression in Trichodesmium was highest in Fe+P treatments, implying co-limitation in this region. Nutrient additions did not measurably stimulate N(2) fixation rates in the <10 ?m fraction in most of the experiments, even when upregulation of nifH expression was evident. These results demonstrate the utility of using gene expression to investigate the physiological state of natural populations of microorganisms, while underscoring the complexity of nutrient limitation on diazotrophy, and providing evidence that diazotroph populations are slow to respond to the addition of limiting nutrients and may be limited by different nutrients on basin-wide spatial scales. This has important implications for our current understanding of controls on N(2) fixation in the TNAtl and may partially explain why it appears to be intermittently limited by Fe, P, or both.
Project description:Expression of nifH in 28 surface water samples collected during fall 2007 from six stations in the vicinity of the Cape Verde Islands (north-east Atlantic) was examined using reverse transcription-polymerase chain reaction (RT-PCR)-based clone libraries and quantitative RT-PCR (RT-qPCR) analysis of seven diazotrophic phylotypes. Biological nitrogen fixation (BNF) rates and nutrient concentrations were determined for these stations, which were selected based on a range in surface chlorophyll concentrations to target a gradient of primary productivity. BNF rates greater than 6?nmolN?l(-1)?h(-1) were measured at two of the near-shore stations where high concentrations of Fe and PO(4)(3-) were also measured. Six hundred and five nifH transcripts were amplified by RT-PCR, of which 76% are described by six operational taxonomic units, including Trichodesmium and the uncultivated UCYN-A, and four non-cyanobacterial diazotrophs that clustered with uncultivated Proteobacteria. Although all five cyanobacterial phylotypes quantified in RT-qPCR assays were detected at different stations in this study, UCYN-A contributed most significantly to the pool of nifH transcripts in both coastal and oligotrophic waters. A comparison of results from RT-PCR clone libraries and RT-qPCR indicated that a ?-proteobacterial phylotype was preferentially amplified in clone libraries, which underscores the need to use caution interpreting clone-library-based nifH studies, especially when considering the importance of uncultivated proteobacterial diazotrophs.