Significance Biological dinitrogen (N 2 ) fixation (BNF) is an important source of nitrogen in marine systems. Until recently, it was believed to be primarily limited to subtropical open oceans. Marine BNF is mainly attributed to cyanobacteria. However, recently an unusual N 2 -fixing unicellular cyanobacteria (UCYN-A)/haptophyte symbiosis was reported with a broader temperature range than other N 2 -fixing cyanobacteria. We report that the UCYN-A symbiosis is present and fixing N 2 in the Western Arctic and Bering Seas, further north than any previously reported N 2 -fixing marine cyanobacteria. Nanoscale secondary ion mass spectrometry enabled us to directly show that the symbiosis was fixing N 2 . These results show that N 2 -fixing cyanobacteria are not constrained to subtropical waters and challenge commonly held ideas about global marine N 2 fixation.

Abstract Biological dinitrogen (N 2 ) fixation is an important source of nitrogen (N) in low-latitude open oceans. The unusual N 2 -fixing unicellular cyanobacteria (UCYN-A)/haptophyte symbiosis has been found in an increasing number of unexpected environments, including northern waters of the Danish Straight and Bering and Chukchi Seas. We used nanoscale secondary ion mass spectrometry (nanoSIMS) to measure 15N 2 uptake into UCYN-A/haptophyte symbiosis and found that UCYN-A strains identical to low-latitude strains are fixing N 2 in the Bering and Chukchi Seas, at rates comparable to subtropical waters. These results show definitively that cyanobacterial N 2 fixation is not constrained to subtropical waters, challenging paradigms and models of global N 2 fixation. The Arctic is particularly sensitive to climate change, and N 2 fixation may increase in Arctic waters under future climate scenarios.

Biological N 2 fixation, the reduction of atmospheric N 2 to biologically available nitrogen, is an important source of nitrogen (N) in oligotrophic tropical and subtropical oceans (1). Historically, studies of marine N 2 fixation focused on the well-known cyanobacterium Trichodesmium and the diatom symbiont Richelia, which were reported primarily from warm (>20 °C) waters (2) with low concentrations of fixed N (nitrate and ammonium) (3). The discovery of a unicellular cyanobacterial symbiont (UCYN-A) of a haptophyte alga (4, 5) expanded the geographic distribution of marine N 2 -fixers to waters with lower temperatures and higher concentrations of fixed inorganic N (6, 7). These regions include the high-latitude waters of the Danish Strait (8) and Western Arctic (9, 10). The presence of N 2 -fixers does not confirm N 2 fixation activity because N 2 fixation is a highly regulated process inhibited by multiple environmental factors. Here, we demonstrate that the cyanobacterial symbiont UCYN-A fixes N 2 in the cold, high latitude waters of the Western Arctic.

UCYN-A is an unusual cyanobacterium lineage that has lost many of the typical cyanobacterial metabolic pathways, including the ability to fix CO 2 and evolve O 2 in photosynthesis (5). The organism is a symbiont with a small planktonic unicellular haptophyte alga, related to Braarudosphaera bigelowii (4, 11). UCYN-A is uncultivated and can only be detected by its DNA or through visualization with catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). UCYN-A comprises multiple closely related lineages with distinct haptophyte hosts that can be differentiated by distinct DNA sequences (12). Generally, haptophytes are geographically widespread including cold, high-latitude waters (13). The symbiosis between a N 2 -fixing cyanobacterium and a haptophyte may facilitate a unique adaptation to N 2 fixation in colder waters, such as the Arctic Ocean, where other N 2 -fixing marine cyanobacteria have not been found.

Little is known about marine N 2 fixation in polar regions, partially because low temperatures are believed to inhibit the growth and activity of N 2 -fixing cyanobacteria, such as in Trichodesmium and Crocosphaera (2, 14). Trichodesmium has occasionally been reported in high latitudes (62°N) (15) but does not appear to fix N 2 when advected into cold waters (16). However, temperature alone does not necessarily preclude N 2 fixation because microorganisms can fix N 2 in ice-covered Antarctic lakes to near boiling temperatures at hydrothermal vents (17, 18). A few studies have reported low but measurable N 2 fixation rates in the Arctic (0.02–7.7 nmol N L−1 d−1) (19, 20). Recent studies in the Bering and Chukchi Seas (9, 10) reported bulk water N 2 fixation rates of 2.3–3.6 nmol N L−1d−1 and detected DNA from UCYN-A and other Bacteria but could not link N 2 fixation to specific microorganisms.

Materials and Methods Samples were taken in the Bering Sea, Chukchi Sea, on the Chukchi Shelf, and in the Beaufort Sea in September 2016. DNA Extraction, nifH Amplification, and qPCR. Samples (2–4 L) were filtered by peristaltic pump onto sequential 3 and 0.2 µm polyphenylene ether filters (0.2 µm, 25 mm; Supor-200; Pall Life Sciences) in Swinnex filter holders. DNA was extracted using a modified DNeasy Plant Mini Kit (Qiagen) protocol, described in detail in ref. 33. PCR amplification of the nifH gene used degenerate universal nifH primers YANNI/450 and up/down in a nested reaction (34), with the second round primers (up/down) modified to contain common sequence linkers (35). Library preparation was carried out by the DNA Sequencing Core Facility at the University of Illinois at Chicago (rrc.uic.edu/cores/genome-research/sequencing-core/). Amplicons were sequenced using Illumina MiSeq, to a sequencing depth of 40,000 sequences per sample. UCYN-A1 and UCYN-A2 abundances were estimated using TaqMan qPCR chemistry and primers and probes specific for UCYN-A1 (36) and UCYN-A2 (11) and their respective haptophyte partners, UCYN-A1 host (SI Appendix, Materials and Methods) and UCYN-A2 host (11), in samples positive for nifH amplification. 15N 2 Rate Measurement Incubations. N 2 fixation was assessed using a modified version of the 15N-bubble method (39). Water samples for rate-measurement incubations were collected from Niskin bottles into gas-tight 1-L glass media bottles (KIMAX model no. 611001000) capped with black open-top caps with gray butyl septa (model no. 240680). The caps and septa were preconditioned in saltwater brine for 60 d before use. The media bottles and caps were acid-washed (10% HCl) and rinsed with copious amounts of high-purity water (18.2 MΩ cm−1). The glass media bottles were also combusted at 500 °C for 4 h before use. Measuring Cell-Specific N 2 Fixation Rates Using NanoSIMS. To visualize and map both strains and their respective hosts (UCYN-A1/UCYN-A1 host and UCYN-A2/UCYN-A2 host), a double CARD-FISH protocol was used according to the protocols detailed in refs. 22 and 23. The full suite of HRP probes, competitor oligonucleotides, and helper probes are given in SI Appendix, Table S1. Before nanoSIMS analysis, cells were transferred to a gridded silicon chip (1.2 cm × 1.2 cm with a 1 mm × 1 mm raster; Pelcotec SFG12 Finder Grid Substrate) and imaged and mapped under epifluorescence on a Zeiss Axioplan epifluorescence microscope equipped with digital imaging at the University of California, Santa Cruz (UCSC). 15N measurements of individual cells were determined by NanoSIMS analyses performed at Stanford Nano Shared Facilities (https://snsf.stanford.edu) on a Cameca NanoSIMS 50L at Stanford University. Image planes were accumulated after first being aligned. Isotope data were taken as a sum of counts in each plane per pixel. Cell outlines and regions of interest (ROIs) were determined as the best fit based on original CARD-FISH image, electron microscopy image, and accumulated images in 12C14N− and 12C−. Cell size was determined based on ROIs of the defined haptophyte or UCYN-A cell. Cell-specific N 2 fixation rates were determined by calculating the carbon content per cell based on a spherical cell volume (V) from the measured cell diameter determined by the ROI following the calculations of ref. 27. The C:N ratio of 6.3 was measured in UCYN-A from the tropical North Atlantic (28) and was used in our calculation to estimate N content of the cell. The limit of detection (LOD) was determined to be three times the SD of 15N in unenriched samples (0.02 At%), similar to the LOD determination described by Jayakumar et al. (37). More detailed methods and calculations can be found in SI Appendix. Bulk N 2 Fixation Rate Measurements. Bottles were filled in triplicate and capped with ambient air bubbles removed. All bottles were immediately placed in mesh bags to mimic the light intensity at collection depth. Different depths received different levels of screening. The bottles were then amended with 1.1 or 2.5 mL of enriched (>99%) 15N 2 gas purchased from Cambridge Isotope Laboratories, Inc. (lot no. I-199168A). Higher volumes of 15N 2 gas were used in all samples after Station 1 to obtain enrichment levels closer to 10% (average 15N 2 enrichment of 5.8 ± 2.1%). Samples were incubated for 24 h in flow-through incubators on deck (surface samples) or environmental chambers (deep samples) set to 0 °C ± 1 °C. Before use in the incubations, subsamples of the 15N 2 gas stocks were assessed for 15NH 4 +, 15NO 3 −, and 15NO 2 − contamination according to the methods described in ref. 38. No contamination was measured. Incubations were terminated after 24 h. A membrane inlet mass spectrometer (MIMS) was used to assess the level of 15N enrichment in each sample immediately upon incubation termination. The MIMS data for each individual bottle were used to calculate uptake rates. Size-fractionated bulk N 2 fixation rates were determined by filtering in series through 3.0-µm silver filters and then precombusted (450 °C for 2 h) glass fiber filter (GF-75) with a nominal pore size of 0.3 µm. Filters were stored frozen at −20 °C in sterile microcentrifuge tubes until analysis. Filters were thawed and dried overnight at 40 °C and analyzed on a Sercon Integra2 SL isotope ratio mass spectrometer tuned to low mass samples. The mass range of calibration standards was 1–10 µg N (low range) or 5–20 µg N (high range) of Sigma-Aldrich ammonium sulfate salt (0.366022/−0.77), which was calibrated against the NIST RM 8573, USGS40 (0.36465/−4.52) with a precision of 0.315 parts per thousand. The LOD for the mass of N was 0.51 µg N. The mass range of samples analyzed was 2.66–19.91 µg N. The LOD (i.e., 3x mass of the 15N blank) was 0.103 At%, and the average minimum quantifiable rate (MQR) of the bulk N 2 fixation rates was 0.4 ± 0.7 nmol N L−1 d−1. The LOD and MQRs were calculated according to Montoya et al. (39) and Gradoville et al. (40) for each size fraction and propagated as error to represent total N 2 fixation. Controls (natural abundance) samples were collected from the Niskin in dedicated, acid-washed (10% HCl), high-density polyethylene bottles and filtered in a separate laboratory on a filtration unit designated for no isotope use. Blank natural abundance samples were analyzed on an Integra2 combined Isotope ratio mass spectrometer with an SL autosampler that had not been exposed to enriched samples. The average δ15N was 7.8 ± 4.2 (0.37 At%) for the >3-µm size fraction and 7.2 ± 2.9 (0.37 At%) for the 0.3- to 3-µm size fraction. Data and Materials Availability. All data are provided in this article and SI Appendix, with the exception of the raw UCYN-A nifH sequences, which have been deposited in the Sequence Read Archive (www.ncbi.nlm.nih.gov/sra) under Bioproject ID PRJNA476143.

Acknowledgments We gratefully acknowledge Mary-Kate Rogener (University of Georgia) for providing MIMS analysis, Quinn Roberts (The Virginia Institute of Marine Science) for providing IMRS analysis, Rosie Gradoville (UCSC) for discussions about N 2 fixation rate measurements, as well as Laurie Juranek (Oregon State University) and the captain and crew of the Research Vessel Sikuliaq for field logistical support. We also thank Chuck Hitzman (Stanford Nano Shared Facility) for nanoSIMS consultation, Lubos Polerecky for look@nanoSIMS consulting, and Stefan Green and his staff (DNA Services Facility and the University of Illinois, Chicago) for sequencing consultation. We greatly appreciate Mick Follows (Massachusetts Institute of Technology) and Kevin Arrigo (Stanford University) for helpful discussions. This research was funded by National Science Foundation Award from the Office of Polar Programs (OPP) 1503614 (to J.P.Z.); Division of Ocean Sciences Awards 1241093 and 1559152 (to J.P.Z.) and OPP-1504307 (to R.E.S.); and Simons Foundation Simons Collaboration on Ocean Processes and Ecology (SCOPE) Award ID 329108 (to J.P.Z.). Part of this work was performed at the Stanford Nano Shared Facility under Award ECCS-1542152.

Footnotes Author contributions: K.A.T.-K., R.E.S., D.A.B., and J.P.Z. designed research; K.H., R.E.S., M.M.M., and D.A.B. performed research; K.H., K.A.T.-K., R.E.S., and M.M.M. analyzed data; and K.H., K.A.T.-K., and J.P.Z. wrote the paper with assistance from all authors.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The raw UCYN-A nifH sequences reported in this paper have been deposited in the Sequence Read Archive, www.ncbi.nlm.nih.gov/sra (Bioproject ID PRJNA476143).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1813658115/-/DCSupplemental.