DOI: 10.1126/science.1185468, 1512 (2010);327 Science

et al.Pia H. MoisanderFixation Domain

2Unicellular Cyanobacterial Distributions Broaden the Oceanic N

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Unicellular Cyanobacterial DistributionsBroaden the Oceanic N2 Fixation DomainPia H. Moisander,1* Roxanne A. Beinart,1† Ian Hewson,1‡ Angelicque E. White,2Kenneth S. Johnson,3 Craig A. Carlson,4 Joseph P. Montoya,5 Jonathan P. Zehr1

Nitrogen (N2)–fixing microorganisms (diazotrophs) are an important source of biologically availablefixed N in terrestrial and aquatic ecosystems and control the productivity of oligotrophic oceanecosystems. We found that two major groups of unicellular N2-fixing cyanobacteria (UCYN) have distinctspatial distributions that differ from those of Trichodesmium, the N2-fixing cyanobacterium previouslyconsidered to be the most important contributor to open-ocean N2 fixation. The distributions andactivity of the two UCYN groups were separated as a function of depth, temperature, and water columndensity structure along an 8000-kilometer transect in the South Pacific Ocean. UCYN group A can befound at high abundances at substantially higher latitudes and deeper in subsurface ocean watersthan Trichodesmium. These findings have implications for the geographic extent and magnitude ofbasin-scale oceanic N2 fixation rates.

Nitrogen (N2)–fixing microorganisms(diazotrophs) are an important sourceof fixed N in oligotrophic ocean ecosys-

tems (1, 2). Biological nitrogen (N2) fixation iscatalyzed by the enzyme nitrogenase and pro-vides about 100 to 150 Tg N per year to theopen ocean (3), about half of global biologicalN2 fixation (4). Large uncertainties remain inthe estimate, and N2 fixation rates derived fromgeochemical evidence have not been entirelyaccounted for by existing biological data (4). Theorganisms that fix N2 play a central role inproviding N to support primary productivity andthe vertical downward flux of organic matter(“export”) to the deep ocean that sequesterscarbon from the atmosphere (5). The filamentouscyanobacterium Trichodesmium was believed tobe the most abundant and active oceanic N2-fixing microorganism (6) until the discovery oftwounicellular diazotrophic cyanobacteria [UCYNgroup A (UCYN-A) and Crocosphaera watsonii(group B)], whose abundances and N2 fixationrates can be equal to or greater than those ofTrichodesmium (7–10). UCYN-A andC. watsoniiare abundant and widespread in tropical and sub-tropical oceans (7) but are more difficult to vi-sualize and quantify than Trichodesmium, andmuch less is known about their distributionsand growth requirements. UCYN-A is less than1 mm in diameter in size, has dim autofluo-

rescence (11), and has not been cultivated,whereasC. watsonii is a cultivated phycoerythrin-rich cyanobacterium 3 to 8 mm in size that canform small colonies (below we use Crocosphaerato include all C. watsonii–like cells) (12). UCYN-A is unusual in that it does not have genes forthe oxygen-evolving photosystem II or the car-bon fixation enzyme rubisco and thus appearsto be a photoheterotroph dependent on organiccarbon (13, 14), whereas Crocosphaera has thephotosynthetic machinery typical of cyanobac-teria (15).

The salient differences in the size andphysiology of the diazotroph groups (UCYN-A,Crocosphaera, and Trichodesmium) suggest thatthey occupy distinct niches and may differentiallyaffect primary productivity and the export of Cand N. Yet in basin-scale models of oceanic N2

fixation, it is generally assumed that a ratheruniform set of environmental conditions equallycontrols the abundance and distribution of alldiazotrophs (16, 17). N2 fixation is typicallyformulated as a function of some combination oftemperature, wind speed, water column stratifica-tion, photon fluxes, and the ratio of N:P, reflectingthe warm (≥~25°C), oligotrophic conditions gen-erally held to be required for Trichodesmiumblooms (6, 18). Under N-limiting open-ocean

conditions, diazotrophs compete with each otherand other plankton for macro- and micronutrients,vitamins, and other growth factors, especially iron,needed for nitrogenase (19). Little is known abouthow the physiological differences of oceanicdiazotrophs are manifested in their global distribu-tions and activities. This information is essential tofill the large gaps in global N2 fixation estimates(4). We investigated distributions and activities ofthe major oceanic diazotrophs in the oligotrophicwestern South Pacific Ocean, in parallel withmeasurement of an extensive set of environmentalparameters (20).

Water samples were collected between 15°to 30°S and 155°E to 170°W (Fig. 1), wheresurface water temperatures decreased from thenorthernmost to the southernmost stations,consistent with latitudinal differences in radia-tive forcing. Diazotroph abundances werequantified by quantitative polymerase chainreaction (qPCR) of the nifH gene (encodingthe iron protein in the nitrogenase enzyme) (10),which corresponds to cell abundance becausethere is one copy of nifH per genome in all threegroups (UCYN-A, Crocosphaera, and Tricho-desmium) (13). We expected to see similargeographic distributions in abundance of alldiazotrophs, reflecting the N-limited character-istics of the oligotrophic ocean. Unexpectedly,there was a transition from Crocosphaera-dominated communities in warm surface watersin the north to UCYN-A–dominated communi-ties in cooler waters in the south (Figs. 1 and 2),and temperature was the most important factorcorrelating with these distributions (table S1).This evidence shows that there is vertical andhorizontal partitioning between populations ofUCYN-A and Crocosphaera. UCYN-A had amaximum abundance of 2.2 × 106 nifH genecopies per liter at the southernmost stations.UCYN-A was also detected at most otherstations but deeper in the water column (Fig.2). Crocosphaera was found at very highabundances in the northeastern part of the studyarea, with a surface maximum at station 21 andsubsurface maxima at stations 25 and 26. Peakabundance of Crocosphaera (8 × 106 nifHcopies per liter) at a depth of 37 m at station 25

1Department of Ocean Sciences, University of California SantaCruz, 1156 High Street, Santa Cruz, CA 95064, USA. 2Collegeof Oceanic and Atmospheric Sciences, Oregon State Uni-versity, 104 COAS Administration Building, Corvallis, OR97331, USA. 3Monterey Bay Aquarium Research Institute,7700 Sandholdt Road, Moss Landing, CA 95039, USA.4Department of Ecology, Evolution and Marine Biology, Uni-versity of California Santa Barbara, Santa Barbara, CA 93106,USA. 5School of Biology, Georgia Institute of Technology, 310Ferst Drive, Atlanta, GA 30332, USA.

*To whom correspondence should be addressed. E-mail:pmoisand@ucsc.edu†Present address: Department of Organismic and Evolution-ary Biology, Harvard University, Cambridge, MA 01238, USA.‡Present address: Department of Microbiology, 403 WingHall, Cornell University, Ithaca, NY 14853, USA.

Fig. 1. Sampling locations superimposed on a composite sea surface temperature plot (March to April2007) for the study area.

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is possibly the highest Crocosphaera cell densitypublished to date. The UCYN populations wereactive because the nifH gene was expressed(table S2) and N2 fixation was detected by using15N

2 uptake method. Maximum N2 fixation rateswere 0.026 nmol liter−1 hour−1 at station 4, dom-inated by Crocosphaera and with little or noUCYN-A, and 4.5 nmol liter−1 hour−1 at station10, which was dominated by UCYN-A withlittle Crocosphaera or Trichodesmium observed.The distributions of the unicellular N2-fixing cya-nobacteria differed from those of Trichodesmium.The latter had surface blooms of abundancesgreater than 106 nifH copies per liter at severalstations in the northeastern part of the study area(Fig. 1 and table S1). The distinct diazotroph dis-tributions indicate that UCYN-A, Crocosphaera,and Trichodesmium biomasses are differentiallyaffected by environmental conditions and controls.

The abundances of UCYN-A and Croco-sphaera in the surface layers correlated withwater temperature (Figs. 1 and 3 and table S1),but the diazotroph abundances were inverselycorrelated with each other [r2 = 0.397, P = 0.000,number of samples (n) = 96, cubic regression].Abundances were weakly or not correlated withsalinity; oxygen saturation; dissolved organiccarbon (DOC); soluble reactive phosphorus(SRP); nitrate; nitrite; total dissolved nitrogen(TDN); chlorophyll a; fluorescence; and abun-

dances of picocyanobacteria, picoeukaryotes, andother diazotroph phylotypes (table S1). Maxi-mum abundances were observed at 24°C and29°C for UCYN-A and Crocosphaera, respec-tively (Fig. 3). Collectively, these data suggestthat UCYN-A has a lower temperature optimumthan Crocosphaera. This is consistent with datafrom the North Pacific, where highest UCYN-Aand Crocosphaera abundances were detected atabout 23.5°C and 26°C, respectively (21), and inthe eastern Atlantic, where high UCYN-Aabundances were observed at water temperaturesranging from 19° to 24°C (9). In addition,observations of nifH transcripts of UCYN-Ahave been made at 12° to 19°C (22, 23), whereasTrichodesmium is thought not to be active below20°C (6). Global distributions of UCYN-A andCrocosphaera from all known data indicate thatUCYN frequently grow in waters with lowertemperatures than are considered necessary forTrichodesmium blooms (Fig. 4). In particular,this report shows that the geographic range ofUCYN-A extends beyond the 25°C isotherm,indicating that previous regional or global N2

fixation estimates based on distributions ofTrichodesmium have been underestimated.

The different depth distributions of UCYN-Aand Crocosphaera indicate they might be adaptedto different light intensities, similar to the high-lightand low-light ecotypes of the nondiazotrophic

cyanobacterium Prochlorococcus. The peakabundances of UCYN-A occurred significantlydeeper than those of Crocosphaera or Trichodes-mium [P = 0.000, n = 23, one-way analysis ofvariance (ANOVA)] (Fig. 2). The abundancemaximumofUCYN-A, observed from the surfaceto 50- to 75-m depths at the southernmost stations,was centered on a weak and broad density frontthat characterized the entire study area and wasdriven by a latitudinal gradient in surface watertemperatures. Moving north, peak abundances ofUCYN-A at each station followed approximatelythe 23 and 23.5 isopycnals (kg m−3) (Fig. 1 andfig. S1); thus, peak abundances were found deeperin the water column in the northernmost stations.The depth of maximum abundance of UCYN-Awas therefore positively correlated with seasurface temperature (r2 = 0.390, P = 0.000, n =23, linear regression), suggesting UCYN-A maygrow deeper in areas with warm surface waters.The different depth preferences of the UCYNgroups may reflect their light, temperature, andnutritional requirements.

Intriguingly, the peak abundances of UCYN-Acoincided with elevated nitrate concentrations insurface waters (fig. S1) that appeared to be re-lated to shoaling isopycnal surfaces, indicatingthat there had been a vertical input of nutrientsand trace elements from deep water. AlthoughUCYN-A does not have genes for assimilatorynitrate or nitrite reduction (14), our data showthat UCYN-A is abundant in waters where nu-trients were recently entrained, an observationconsistent with recent reports of high abun-dances of UCYN-A in nutrient-enriched estua-rine and coastal waters (22–24) and eddies (25),unlike what is known about Trichodesmium (6).These UCYN-A–dominated waters may haveremnants of non–N2-fixing phytoplankton blooms,stimulated by vertical mixing. A large area withelevated surface water chlorophyll a concen-trations, indicative of increased phytoplanktonbiomass, was observed in ocean color satellitedata south of the study area, associated with thegeneral position of the Tasman Front (26).These observations support a link between highphytoplankton biomass and high UCYN-Aabundance (fig. S2), which is expected on thebasis of recent UCYN-A genome informationsuggesting that it requires a close associationwith other organisms (13, 14). DOC concen-trations followed meridional trends previouslydescribed for the South Pacific (27) and werelower in concentration in the surface whereUCYN-A abundance was the greatest (Fig. 1,fig. S1, and table S1). Although the reducedDOC concentrations at the location of UCYN-Amaxima are consistent with recent vertical mixingin the area, heterotrophic microbial activity maycontribute to this trend and requires furtherstudy. The differential distributions of UCYN-Aand Crocosphaera observed in the water massesmixing in the area, and traced by temperature,may have been affected by differences in thechemical composition of these water masses.

Fig. 2. Horizontal and vertical distributions of diazotrophs in the study area. (A) UCYN-A, (B)Crocosphaera, and (C) Trichodesmium spp. abundances [log10 (nifH copies per liter)] with station numbersand sampling depths indicated. Maximum abundance (D) and depth of maximum abundance (E) ofUCYN-A and Crocosphaera and (F) mean depth (TSD) of maximum abundance of UCYN-A, Crocosphaera,and Trichodesmium spp. (one-way ANOVA).

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The magnitude and geographic extent ofoceanic N2 fixation determine the rate of primaryproductivity and vertical export of carbon in theoligotrophic ocean. Most previous estimates ofglobal N2 fixation are dependent on distributionsor factors that control the growth of Trichodes-mium (16, 17). The results of this study show thatactively N2-fixing populations of the two majoroceanic UCYN groups have broader latitudinaldistribution than Trichodesmium, analogous tothe global distributions of strains and ecotypes of

the non–N2-fixing oceanic cyanobacteria (28).Observations of distributions of UCYN-A at rel-atively high latitudes and in coastal waters geo-graphically extends the oceanic regions where N2

fixation can occur, which contrasts with previousparadigms based on the temperature range forTrichodesmium. The data show that temperatureis a major driver of the differential distributionsfor different taxa, although additional factors,such as non–N2-fixing phytoplankton blooms,may be important for the growth of UCYN-A.

Unicellular N2-fixing microorganisms are muchmore difficult to detect than larger diazotrophssuch as Trichodesmium and may thrive in differ-ent geographic regions. In order to better constrainestimates of the N inputs to the global ocean viaN2 fixation, it will be necessary to adequately rep-resent the activity of unicellular N2-fixing micro-organisms in ecosystem models.

References and Notes1. P. Falkowski, Nature 387, 272 (1997).2. T. Tyrrell, Nature 400, 525 (1999).3. N. Gruber, in Nitrogen in the Marine Environment,

D. G. Capone, E. J. Carpenter, D. A. Bronk,M. R. Mulholland, Eds. (Academic Press and Elsevier,Burlington, MA, 2008), pp. 1–49.

4. J. N. Galloway et al., Biogeochemistry 70, 153 (2004).5. D. Karl et al., Nature 388, 533 (1997).6. D. G. Capone, J. P. Zehr, H. W. Paerl, B. Bergman,

E. J. Carpenter, Science 276, 1221 (1997).7. J. P. Zehr et al., Nature 412, 635 (2001).8. J. P. Montoya et al., Nature 430, 1027 (2004).9. R. J. Langlois, D. Hümmer, J. LaRoche, Appl. Environ.

Microbiol. 74, 1922 (2008).10. M. J. Church, B. D. Jenkins, D. M. Karl, J. P. Zehr, Aquat.

Microb. Ecol. 38, 3 (2005).11. N. L. Goebel, C. A. Edwards, B. J. Carter, K. M. Achilles,

J. P. Zehr, J. Phycol. 44, 1212 (2008).12. E. A. Webb, I. M. Ehrenreich, S. L. Brown, F. W. Valois,

J. B. Waterbury, Environ. Microbiol. 11, 338 (2009).13. J. P. Zehr et al., Science 322, 1110 (2008).14. H. J. Tripp et al., Nature 464, 90 (2010).15. J. P. Zehr, S. R. Bench, E. A. Mondragon, J. McCarren,

E. F. DeLong, Proc. Natl. Acad. Sci. U.S.A. 104, 17807(2007).

16. R. R. Hood, V. J. Coles, D. G. Capone, J. Geophys. Res.109, C06006 (2004).

17. C. Deutsch, J. L. Sarmiento, D. M. Sigman, N. Gruber,J. P. Dunne, Nature 445, 163 (2007).

18. A. E. White, Y. H. Spitz, R. M. Letelier, J. Geophys. Res.112, C12006 (2007).

19. S. A. Sañudo-Wilhelmy et al., Nature 411, 66 (2001).20. Materials and methods are available as supporting

material on Science Online.21. M. J. Church, K. M. Björkman, D. M. Karl, M. A. Saito,

J. P. Zehr, Limnol. Oceanogr. 53, 63 (2008).22. S. M. Short, J. P. Zehr, Environ. Microbiol. 9, 1591 (2007).23. J. A. Needoba, R. A. Foster, C. Sakamoto, J. P. Zehr,

K. S. Johnson, Limnol. Oceanogr. 52, 1317 (2007).24. A. P. Rees, J. A. Gilbert, B. A. Kelly-Gerreyn, Mar. Ecol.

Prog. Ser. 374, 7 (2009).25. M. J. Church et al., Global Biogeochem. Cycles 23,

GB2020 (2009).26. M. E. Baird et al., Deep-Sea Res. I 55, 1438 (2008).27. D. A. Hansell, C. A. Carlson, D. J. Repeta, R. Schlitzer,

Oceanography 22, 202 (2009).28. Z. I. Johnson et al., Science 311, 1737 (2006).29. We thank R. Paerl, C. Sakamoto, T. Cote, N. Pereira,

M. Furnas, B. Carter, M. Ochiai, K. London, E. Preston,M. Hogan, and personnel onboard R/V Kilo Moana fortechnical assistance and C. Edwards for helpful discussions.This study was supported by NSF–Division of Ocean Sciences(OCE) (grant 0425363), NSF Emerging Frontiers Program(Center for Microbial Oceanography: Research andEducation) (grant 0424599), and the Gordon and BettyMoore Foundation for J.P.Z., and NSF-OCE (grant 0425583)for J.P.M. A.E.W. was supported by NSF-OCE(grant 0623596, R. Letelier, principal investigator).

Supporting Online Materialwww.sciencemag.org/cgi/content/full/science.1185468/DC1Materials and MethodsFigs. S1 and S2Tables S1 to S3References

3 December 2009; accepted 17 February 2010Published online 25 February 2010;10.1126/science.1185468Include this information when citing this paper.

Fig. 3. Relationship of unicellular diazotroph abundances [log10 (nifH copies per liter)] and temperature. (A)UCYN-A, (B) Crocosphaera. f = 5.13 – 0.1754x + 0.0638x2 – 0.0124x3 describes the nonlinear relationshipbetween UCYN-A and temperature and f= –5.16 + 0.361x the linear relationship between Crocosphaera andtemperature.

Fig. 4. Global distribution and abundances of (A) UCYN-A and (B) Crocosphaera unicellularcyanobacteria compiled from all known published literature. The maximum value is shown for siteswhere multiple depths were analyzed. Latitudinal 18°C and 25°C isotherms [average values of bimonthlymeans from July 2002 to July 2009 fromNASAModerate Resolution Imaging Spectroradiometer (MODIS)]are indicated with dotted and dashed lines, respectively. Literature sources for cyanobacterial distributionsare listed in the supporting online material. EQ indicates equator.

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