The Great Barrier Reef (GBR), situated within the tropical waters of north-eastern Australia, is the largest continuous coral reef in the world and a region of high biological productivity (Furnas, 2003). Forming a natural barrier between coastal waters and the oligotrophic Coral Sea, the GBR is a biologically and biogeochemically dynamic system that is influenced by both localized hydrodynamic features (e.g., riverine discharge) (Devlin and Brodie, 2005; Waterhouse et al., 2012) and large-scale oceanographic processes (e.g., Coral Sea inflow and upwelling events) (Furnas and Mitchell, 1996; Brinkman et al., 2002; Choukroun et al., 2010).

Concentrations and sources of particulate and dissolved nutrients in GBR waters vary across spatial gradients, such as between inshore and offshore regions (Furnas et al., 1995, 2011), but bioavailable forms of dissolved inorganic nutrients are generally low (<0.05 μM) (Furnas et al., 2011). Excess concentrations of phosphate compared to dissolved inorganic nitrogen (N:P, 1–3.5) indicate that nitrogen (N) could be a limiting nutrient for phytoplankton growth (Furnas et al., 2011). Indeed, NH₄⁺ and NO₃^- stocks are typically turned over by phytoplankton within a matter of hours (Furnas et al., 2005). However, while low dissolved inorganic N to chlorophyll a ratios indicate that phytoplankton growth cannot be supported for more than one doubling of biomass, the measured growth rates of phytoplankton populations across the reef are paradoxically high (Furnas et al., 2005). This discrepancy between bioavailable N and phytoplankton growth rate suggests that additional N sources play a significant role in supporting the phytoplankton assemblages within the pelagic waters of the GBR. One such N source could be provided by the activity of dinitrogen (N₂) fixing bacteria (diazotrophs).

Diazotroph activity is known to be an important source of bioavailable N within a number of discrete habitats in coral reefs systems. For example, N₂ fixing lineages of proteobacteria and cyanobacteria are known constituents of the coral holobiont (Lema et al., 2012, 2014; Santos et al., 2014; Zhang et al., 2016), supplying fixed N to symbiotic Symbiodinium (Lesser et al., 2007; Ceh et al., 2013). N₂ fixation has also been identified as an important process within coral reef sediments, contributing significantly toward NH₄⁺ pools within upper sediment layers (Capone et al., 1992; Alongi et al., 2006). In addition, particularly high rates of N₂ fixation, attributable to the cyanobacterium Calothrix, have been reported within microbial mats on intertidal reef flats (Wiebe et al., 1975; Larkum et al., 1988).

In addition to symbiotic and benthic N₂ fixation, pelagic diazotrophic cyanobacteria have also been shown to be abundant (Bell et al., 1999; Biegala and Raimbault, 2008) and active (Hewson et al., 2007) in the water column of coral reef lagoons. An earlier study of N₂ fixation by the photoautotrophic cyanobacterium Trichodesmium in northern GBR waters suggested its potential importance in the provision of fixed N to primary production (Bell et al., 1999). However, within the Heron Island Lagoon on the GBR, Hewson et al. (2007) demonstrated for bacterioplankton possessing the nifH gene, which encodes a subunit of the dinitrogenase reductase enzyme, diazotrophs were most similar to microbial mat and sediment-associated cyanobacteria and proteobacteria. While Trichodesmium and other typically planktonic phylotypes were only detected within nifH transcripts, suggesting they may be active but rare members of the pelagic diazotrophic assemblage in this region (Hewson et al., 2007).

While the significance of N₂ fixation for providing bioavailable N to coral reef ecosystems has been demonstrated by the quantitative incorporation of sedimentary and benthic reef flat N₂ fixation rates into a GBR N budget, the contribution of pelagic N₂ fixation to GBR N cycling is less well-understood (Furnas et al., 2011). The limited available information on the diversity, abundance and activity of diazotrophic bacteria within pelagic GBR environments has hindered efforts to develop a complete N budget for the GBR (Furnas et al., 2011). To address this gap, we measured N₂ fixation rates and determined diazotroph community composition and abundance within GBR surface waters. Further, we investigated relationships between the observed spatial patterns in diazotroph assemblage structure and the prevailing biotic and abiotic environmental characteristics across the GBR.

The paradoxical nature of many coral reefs, whereby relatively high biological productivity occurs within marine waters where ambient concentrations of dissolved inorganic nutrients are low, has resulted in efforts to reconcile nutrient dynamics within coral reef systems (e.g., Furnas et al., 1995, 2005, 2011; Hearn et al., 2001; Falter et al., 2004; Schaffelke et al., 2012). Some early studies demonstrated that biological N₂ fixation might play an important role in supplying bioavailable N within benthic reef habitats (Webb et al., 1975; Wiebe et al., 1975; Larkum et al., 1988), and more recently, diazotrophic bacteria have been shown to be an important constituent of the coral holobiont, supplying N requirements for symbiotic Symbiodinium (Lesser et al., 2004, 2007; Lema et al., 2012, 2014; Olson and Lesser, 2013; Zhang et al., 2016). However, within the pelagic environment of the GBR the role of biological N₂ fixation is less well-understood (Furnas et al., 2011), despite evidence to suggest that large discrepancies exist between nutrient availability and phytoplankton growth (Furnas et al., 2005). Here, we found a diverse community of cyanobacterial and non-cyanobacterial diazotrophs inhabiting GBR surface waters during Austral winter, and relatively high qualitative rates of N₂ fixation within coastal, inner and outer reef habitats, indicating that diazotrophic bacterioplankton might act as a significant source of fixed N within the oligotrophic GBR.

Through our nifH amplicon sequencing analyses, we provide the first in-depth characterization of the potential for N₂ fixation within bacterioplankton assemblages across the GBR. Using this approach, Cluster 1B cyanobacterial diazotrophs were identified as the dominant phylotype distributed throughout GBR surface waters, specifically OTUs closely related to Trichodesmium spp. showed high relative and absolute abundances. Seven OTUs sharing >90% AAI to Trichodesmium erythraeum were detected in the amplicon sequencing dataset, three of which were targeted by the Trichodesmium spp. qPCR assay employed herein. Trichodesmium spp. are routinely observed in tropical, oligotrophic environments, including coral reef lagoons, where they can form large surface aggregations and contribute substantially to N₂ fixation (Bell et al., 1999; Campbell et al., 2005; McKinna et al., 2011; Bonnet et al., 2015b; Turk-Kubo et al., 2015). Although surface aggregations were not observed during the present study, qPCR analyses indicated that the abundance of Trichodesmium spp. were at times high, reaching up to 3.5 × 10⁵ nifH copies L^-1, consistent with observations from the neighboring Coral and Solomon Seas during Austral winter conditions (Bonnet et al., 2015b). Trichodesmium has previously been recognized as an important feature of pelagic GBR microbial communities, through microscopic enumeration and satellite remote sensing (Bell et al., 1999; McKinna et al., 2011), and it is estimated that N₂ fixation by Trichodesmium alone could contribute ∼0.7–3.0 t N km^-2 to GBR surface waters. At sites where qualitative N₂ fixation rates of ∼68 nmol L^-1 d^-1 were observed, we found Trichodesmium to be abundant (3.4–5.2 × 10⁴ nifH copies L^-1). By virtue of both its abundance, diversity and activity, Trichodesmium therefore potentially plays a very important role in supporting the growth and production of non-diazotrophic assemblages across the GBR pelagic zone.

Beyond Trichodesmium, a number of other OTUs affiliated with Cluster 1B cyanobacterial diazotrophs were present in GBR surface waters. These included OTUs related to the filamentous genus Leptolyngbya, which has previously been found to actively fix N₂ in benthic cyanobacterial mats (Woebken et al., 2014) including within coral reef systems (Charpy et al., 2010), and OTUs associated with the unicellular cyanobacterial symbiont UCYN-A, including ecotypes 1 and 2 which are widely distributed throughout the global ocean (Farnelid et al., 2016; Zehr et al., 2016). The majority of these Cluster 1B OTUs were present in relatively low abundances, contributing to <15 and 10% of nifH sequences at their maxima. The observed low relative abundance of UCYN-A1 and UCYN-A2 in GBR surface waters during Austral winter is consistent with our previous observations for the adjoining Coral Sea, whereby UCYN-A ecotypes effectively disappeared in Austral winter when compared to spring (Messer et al., 2015b). However, in the eastern Coral Sea, high UCYN-A abundances (determined by qPCR) have been reported during Austral winter (Bonnet et al., 2015b), and UCYN-A has been reported to be the dominant diazotrophic phylotype within the Noumea Lagoon, New Caledonia (Turk-Kubo et al., 2015). While maximum abundances of UCYN-A appear to occur at more southern latitudes in the western South Pacific during Austral autumn (Moisander et al., 2010).

In addition to the cyanobacterial diazotrophs, our data provide the first estimates of the diversity and abundance of heterotrophic diazotrophs in GBR surface waters. Of particular importance were the Gamma A OTUs affiliated with Cluster 1G, which were the second most prevalent diazotrophic phylotype in our amplicon sequencing analyses. Specifically, OTUs clustering with the Gamma A group at times comprised >50% of sequences at a given site, although the abundances of these organisms as determined by qPCR were generally low, with peaks of only 5.9 × 10² nifH copies L^-1. This could reflect the possible preferential amplification of this phylotype by the PCR primers used in this study (Turk-Kubo et al., 2012), and highlights the importance of complementary qPCR analyses to verify amplicon sequencing based approaches. The abundance of Gamma A throughout GBR surface waters are in-line with previous studies utilizing Gamma A qPCR assays. For instance, Bonnet et al. (2015b) reported similar Gamma A abundances in the eastern Coral Sea and Solomon Sea, and Moisander et al. (2014) reported median Gamma A abundances of 8 × 10² nifH copies L^-1 throughout the western South Pacific Ocean. Indeed, low abundances of Gamma A have been reported for much of the major ocean basins, indicating that they are a ubiquitous component of diazotrophic bacterioplankton in tropical, oligotrophic ecosystems (Langlois et al., 2015). Our data show the Gamma A clade to also be widespread throughout GBR surface waters, and it is particularly notable that members of this clade comprised a significant proportion of the diazotroph community at sites where relatively high qualitative rates of community N₂ fixation were observed while Trichodesmium abundances were low.

Compared to the few other studies reporting water column N₂ fixation rates in coral reef environments, the qualitative rates we observed in GBR waters were relatively high. For example, the highest rates observed here, between 31 and 68 nmol L^-1 d^-1, were greater than those observed in a New Caledonian coral lagoon (<10 nmol L^-1 d^-1; Biegala and Raimbault, 2008), substantially greater than those reported for the eastern Coral Sea (≤2 nmol L^-1 d^-1; Bonnet et al., 2015b), and in-line with those measured in the western Coral Sea, adjacent to the GBR (56 nmol L^-1 d^-1; Messer et al., 2015b). Moreover, the rates observed in the present study are within the higher range of N₂ fixation rates compiled within a global database of marine N₂ fixation (Luo et al., 2012), indicating that N₂ fixation within GBR waters indeed represents a significant source of N at the local scale, and a potentially significant region of N₂ fixation activity at the global scale.

It must be noted, however, that the “bubble” method used to measure N₂ fixation (Montoya et al., 1996) in this study, has previously been shown to underestimate N₂ fixation by 50% or more, and will also depend on the composition of the underlying diazotroph community, as well as the time of sampling relative to the diurnal cycle of N₂ fixation within specific clades, and the physical properties of the sampling site (e.g., temperature and salinity) which all influence the solubility of N₂ gas in seawater (Mohr et al., 2010; Großkopf et al., 2012; Wilson et al., 2012; Benavides et al., 2013). Despite these caveats, the Montoya et al. (1996) method was applied in this study because it was decided that it was favorable to underestimate the significance of N₂ fixation on the GBR, rather than potentially overestimate it by inadvertently introducing additional particulates, nutrients, or trace metals through pre-preparing ¹⁵N enriched natural or artificial seawater. Indeed, a recent study demonstrated that the preparation of ¹⁵N₂ enriched seawater could result in the enrichment of trace metals by up to 0.1 nmol L^-1, due to contact with standard laboratory ware used to prepare the solution (glass, rubber, and plastic) (Klawonn et al., 2015). Given that samples collected for each incubation experiment had distinct physical and chemical properties, such as variability in salinity and dissolved inorganic nutrient concentrations (Table 2), introducing enriched seawater that did not match the properties of the coastal, inner and outer GBR seawater sampled, could have influenced nutrient dynamics within our incubations. Therefore, our rate calculations were corrected according to Mohr et al. (2010) to account for the incomplete dissolution of ¹⁵N₂ in seawater.

In addition, some commercially available ¹⁵N₂ gas stocks have recently been found to be contaminated with ¹⁵NO₃, ¹⁵NH₄, and ¹⁵N₂O (Dabundo et al., 2014). Our study was performed prior to the publication of the Dabundo et al. (2014) study which reported the contamination of two batches of Sigma–Aldrich ¹⁵N₂ gas stocks, and Sigma–Aldrich gas lot SZ1670V (2013 batch) was used in this study. We cannot explicitly rule out that there was not contamination in the batch of ¹⁵N₂ that we used, therefore using the average concentration of ¹⁵NO₃, ¹⁵NH₄, and ¹⁵N₂O contamination in Sigma–Aldrich stocks reported by Dabundo et al. (2014) (298, 818, and 61 μmol/mole ¹⁵N respectively) we calculated that only an additional 3.2 × 10^-7 moles of ¹⁵N could have been added to our incubations during our trace additions (2.7 × 10^-4 moles) of ¹⁵N₂ gas (Supplementary Table 3). Therefore, we found that any potential ¹⁵N contamination would have had a negligible effect on our measured rates of N₂ fixation. Consequently, the rates of N₂ fixation reported herein represent qualitative estimates of N₂ fixation by a diverse population of diazotrophic bacterioplankton, and indicate that relatively high N₂ fixation activity can occur in GBR waters.

Previous studies investigating diazotrophy within the water column of the GBR have either not measured N₂ fixation rates (e.g., Hewson et al., 2007) or have measured N₂ fixation rates by individual Trichodesmium trichomes using acetylene reduction (e.g., Bell et al., 1999). We propose that N₂ fixation by the whole diazotroph community will significantly increase this estimate, and could theoretically support carbon fixation rates (assuming Redfield C:N ratios of phytoplankton) of between 0.2 and 4 μg C L^-1 d^-1. Although it is unlikely that all fixed N will be available to support C fixation, some autotrophic diazotrophs will directly contribute to primary production, while others may support primary production through the release of recently fixed N into the surrounding water column (Garcia et al., 2007; Lee Chen et al., 2011; Berthelot et al., 2015). For instance, the most abundant diazotroph observed in our study, the cyanobacterium Trichodesmium erythraeum, has been estimated to release between 50 and 90% of the N₂ that it fixes into the surrounding environment (Glibert and Bronk, 1994; Mulholland et al., 2004), where it is potentially transferred to associated bacteria, non-diazotrophic filaments, or phytoplankton (Glibert and Bronk, 1994; Mulholland et al., 2004, 2006; Mulholland and Bernhardt, 2005).

While the fate of N fixed by heterotrophic diazotrophs remains unknown, dissolved N release from mixed, natural communities of diazotrophic bacterioplankton is in the range of 16–30% of gross whole community N₂ fixation (Benavides et al., 2013; Bonnet et al., 2015a). Based on these numbers, we calculated potential dissolved N release, based on bulk qualitative N₂ fixation rate measurements, in GBR waters to be between 0.4 and 20 nmol L^-1 d^-1. Hence diazotroph-derived dissolved N could considerably increase the potential for N₂ fixation to support primary production within GBR waters, where ambient concentrations of DIN are considered limiting.

In the present study, ambient concentrations of DIN (NO_x + NH₃) across the GBR were relatively low at <0.20 μM, but DIN concentration significantly contributed to the observed spatial heterogeneity in diazotroph community composition. Due to the reduced energy requirements associated with assimilating DIN (NO_x + NH₃) compared with fixing N₂, biological N₂ fixation is considered to be influenced by concentrations of DIN (Karl et al., 2002; Knapp, 2012). In our study, where DIN concentrations were ≥0.11 μM we observed relatively low rates of N₂ fixation (between 2.6 and 5.9 nmol L^-1 d^-1) and more diverse diazotroph communities (H′ > 2.5). Conversely, when DIN concentrations were ≤0.09 μM we observed the highest rates of N₂ fixation (∼68 nmol L^-1 d^-1), associated with less diverse diazotroph communities (H′ = ∼1.5), typically dominated by Trichodesmium (at 30–50% of the diazotroph assemblage). In culture, cyanobacterial and proteobacterial diazotrophs have been shown to significantly decrease N₂ fixation with increasing DIN concentrations (Knapp et al., 2012; Bentzon-Tilia et al., 2015), indicating a switch to DIN supported growth (Kustka et al., 2003; Masuda et al., 2013; Bentzon-Tilia et al., 2015), which may reduce the demand for dissolved iron (Kustka et al., 2003). However, in the environment N₂ fixation is increasingly being found to occur outside of the classical ecological niche of low DIN waters (Fernandez et al., 2011; Knapp, 2012; Farnelid et al., 2013), and can even increase in response to simulated (mesocosm) and natural (mesoscale processes) co-additions of N with other nutrients (Dekaezemacker et al., 2013; Loscher et al., 2016). While the relationship between the availability of DIN and N₂ fixation in the environment is more complex than perhaps previously thought, the patterns we observed suggest a significant role for DIN in structuring spatial heterogeneity in diazotroph community composition, which in turn could impact biological N₂ fixation in GBR waters.

In addition to the influences of DIN, spatial heterogeneity in diazotrophic bacterioplankton was also significantly associated with the availability of the macro-nutrient phosphate. While ambient concentrations were generally low (<0.05 μM), sites with higher phosphate concentrations (0.024–0.049 μM) contained diazotroph communities dominated by γ-proteobacterial OTUs, while lower phosphate concentrations (0.014–0.018 μM) coincided with higher relative abundances of Trichodesmium. These observations may suggest differences in phosphate demand between the proteobacterial and cyanobacterial diazotrophs. Although previous phosphate enrichment experiments within GBR waters (Heron Island Lagoon) demonstrated no significant influence of phosphate on diazotroph abundance or nifH expression (Hewson et al., 2007), it is likely that a more complex relationship between phosphate concentration and N₂ fixation exists within natural populations. Other sources of phosphorous, such as phosphonates (Dyhrman et al., 2006) and phosphites (Polyviou et al., 2015), may be utilized by diazotrophs in situ. Indeed, a recent ecosystem model that considers the availability of labile dissolved organic phosphorous (DOP) as a factor influencing diazotrophic activity, increased the estimated global N₂ fixation budget by 30 Tg N yr^-1 (Somes and Oschlies, 2015). Moreover, in situ mesocosm experiments in the tropical North Atlantic have provided direct evidence for the stimulation of N₂ fixation after DOP addition, coinciding with a shift in diazotroph community composition (Meyer et al., 2016). Within the GBR, DOP estimates suggest concentrations similar to that of phosphate concentrations (Furnas et al., 2005), indicating that phosphate demand in GBR diazotroph communities could be met through labile DOP. Thus, the composition and abundance of GBR diazotroph assemblages are likely influenced by the availability of phosphate as well as other phosphorous sources, which, as our qualitative data indicates, may in turn significantly influence the activity of N₂ fixation.

Overall, the findings of this study demonstrate that biological N₂ fixation may be an important process within the pelagic realm of the GBR, where it has the potential to significantly support primary production. While we found that Trichodesmium dominates over spatially extensive areas of the GBR, heterotrophic N₂-fixing bacteria may also be an important component of GBR diazotroph assemblages. Our findings indicate that diazotroph community composition is driven by the concentration of key dissolved inorganic nutrients, and in regions where DIN concentrations are low, high rates of N₂ fixation can occur. These data highlight the need to re-evaluate N cycling dynamics within oligotrophic coral reef systems to include biological N₂ fixation as a potentially significant source of dissolved N within the water column.

LM, MB, and JS designed the study. LM collected and processed samples, performed experiments and laboratory assays, and analyzed and interpreted the data. AM and MF provided field support, CTD data, and collected samples. RC assisted with flow cytometry and dissolved nutrient analyses. LM, MB, and JS wrote the manuscript, with input from all authors. All authors approved the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.