Diversity and activity of nitrogen-fixing communities across ocean basins

Recent observations of N2 fixation rates (NFR) and the presence of nitrogenase (nifH) genes from heterotrophic N2-fixing (diazotrophic) prokaryotes in unusual habitats challenge the paradigm that pelagic marine N2 fixation is constrained to cyanobacteria in warm, oligotrophic, surface waters. Here, we compare NFR and diazotrophic diversity (assessed via high-throughput nifH sequencing) from a region known to be dominated by cyanobacterial diazotrophs (the North Pacific Subtropical Gyre, NPSG) to two regions dominated by heterotrophic diazotrophs: the Eastern South Pacific (ESP, from the Chilean upwelling system to the subtropical gyre) and the Pacific Northwest coastal upwelling system (PNW). We observed distinct biogeographical patterns among the three regions. Diazotrophic community structure differed strongly between the NPSG, dominated by cyanobacterium UCYN-A, and the ESP, dominated by heterotrophic nifH group 1J/1K, yet surface NFR were similar in magnitude (up to 5.1 nmol N L d). However, while diverse, predominantly heterotrophic nifH genes were recovered from the PNW and the mesopelagic of the NPSG, NFR were undetectable in both of these environments (although glucose amendments stimulated low rates in the deep NPSG). Our work suggests that while diazotrophs may be nearly omnipresent in marine waters, the activity of this functional group is regionally restricted. Further, we show that the detection limits of the N2 fixation assay suggest that many of the low NFR reported for the mesopelagic (often<0.1 nmol N L d in the literature) are not indicative of active diazotrophy, highlighting the challenges of assessing the ecosystem significance of heterotrophic diazotrophs. Nitrogen (N) availability limits phytoplankton production in most marine ecosystems (Gruber 2004), and thus partially determines the drawdown of inorganic carbon from the atmosphere. The oceanic reservoir of bioavailable N is largely controlled by the balance of specific microbial processes: dinitrogen (N2) fixation adds fixed N and denitrification and anaerobic ammonia oxidation remove fixed N. Whether the global oceanic N budget is balanced or not is a topic of longstanding controversy (see Galloway et al. 2004; Gruber 2004). Nevertheless, multiple lines of evidence suggest historical underestimations of both marine N2 fixation rates (NFR) (Codispoti 2007; Deutsch et al. 2007; Großkopf et al. 2012) and the diversity and abundance of marine N2-fixers (diazotrophs) (Riemann et al. 2010). These findings have driven a reassessment of N2-fixing habitats and diazotrophic diversity (reviewed in Bombar et al. 2016). N2 fixation was traditionally thought to be dominated by cyanobacteria inhabiting warm, oligotrophic, surface ocean waters (Karl et al. 2002), but this paradigm is being challenged. Sequencing the nifH gene, which encodes the ironprotein component of the enzyme nitrogenase that catalyzes N2 fixation, has revealed potential marine diazotrophs spanning diverse prokaryotic lineages (Zehr et al. 1998), and nifH amplicons from putative heterotrophs have been reported to outnumber cyanobacterial amplicons in global surface ocean waters (Riemann et al. 2010; Farnelid et al. 2011). Furthermore, heterotrophic nifH genes and transcripts have been recovered from environments previously unexplored for N2 *Correspondence: [email protected] Present address: Ocean Sciences Department, University of California Santa Cruz, Santa Cruz, California Present address: Marine Biological Section, Department of Biology, University of Copenhagen, Helsingør, Denmark Additional Supporting Information may be found in the online version of this article. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1 LIMNOLOGY and OCEANOGRAPHY Limnol. Oceanogr. 00, 2017, 1–15 VC 2017 The Authors Limnology and Oceanography published by Wiley Periodicals, Inc. on behalf of Association for the Sciences of Limnology and Oceanography doi: 10.1002/lno.10542 fixation, including coastal upwelling regimes, oxygen minimum zones, and the deep sea (Hewson et al. 2007; Sohm et al. 2011; Jayakumar et al. 2012). Active N2 fixation in these environments, even at low rates, would increase overall marine N2 fixation estimates and expand our understanding of the biogeographical patterns of an ecologically important functional group, the diazotrophs (e.g., Bonnet et al. 2013; Rahav et al. 2013). The Eastern South Pacific (ESP), in particular, is a region that has garnered considerable attention as a potentially underappreciated habitat for marine N2 fixation. Though historically understudied as an environment for diazotrophs (Luo et al. 2012), focus on the ESP increased after a numerical model predicted high NFR in the region (Deutsch et al. 2007). Despite model predictions, expeditions to the ESP have revealed low NFR in the oligotrophic gyre (0.08–0.88 nmol L d, Halm et al. 2011; Dekaezemacker et al. 2013), albeit highly variable rates in the Chilean upwelling system (0.1–127 nmol L d, Fernandez et al. 2011, 2015). The presence of inorganic N can suppress N2 fixation by cyanobacterial diazotrophs (Knapp 2012; Knapp et al. 2012), so it is unsurprising that diazotrophic communities in the N-rich upwelling zone of the ESP appear to be heavily dominated by heterotrophic bacteria, particularly gamma-proteobacteria (Farnelid et al. 2011; Fernandez et al. 2011; Turk-Kubo et al. 2014). The quantitative Polymerase Chain Reaction (qPCR)derived nifH abundances of select heterotrophic phylotypes, however, appear too low to account for even the low NFR observed to date (Turk-Kubo et al. 2014). This mismatch between proxies for cell abundances and rates implies an underestimation of diazotrophic abundance or cell-specific rates, and/or an overestimation of NFR. These findings point to significant concerns regarding the inaccuracy of historical NFR measurements (Mohr et al. 2010; Dabundo et al. 2014) that have since been partially addressed (Wilson et al. 2012; B€ ottjer et al. 2016) and, more importantly, highlight our incomplete knowledge of the physiology, biogeography, and ecology of heterotrophic diazotrophs. In order to understand the linkage between the assembly of diazotrophic communities and the magnitude of NFR, we have developed a cross-ecosystem study spanning oligotrophic gyres to upwelling regimes. Specifically, we present the first pairing of NFR with fine-scale diazotrophic diversity assessments (via high-throughput nifH sequencing) in three oceanic regions: the ESP (from the upwelling zone to the gyre), the North Pacific Subtropical Gyre (NPSG), and the Pacific Northwest coast of the United States (PNW). While previous observations in the ESP indicate low NFR and cryptic heterotrophic diazotrophs, the NPSG fosters moderate NFR presumably driven by highly abundant cyanobacterial diazotrophs (0.6–3.2 nmol N L d, B€ ottjer et al. 2016). Meanwhile, N2 fixation has not been reported in the PNW, where the upwelling of cold, nitrate-rich waters would theoretically select against the growth of diazotrophs. Comparisons of these three potential habitats enable a global biogeographical perspective of marine diazotrophy. Here, we also address discontinuities between the genetic potential for diazotrophy and the presence of detectable NFR.