MOLECULAR DIVERSITY OF MARINE PHYTOPLANKTON … · Punyasloke Bhadury2,3 and Bess B. Ward Department of Geosciences, Guyot Hall, Princeton University, Princeton, New Jersey 08544,

MOLECULAR DIVERSITY OF MARINE PHYTOPLANKTON COMMUNITIESBASED ON KEY FUNCTIONAL GENES1

Punyasloke Bhadury2,3 and Bess B. Ward

Department of Geosciences, Guyot Hall, Princeton University, Princeton, New Jersey 08544, USA

The phylogeny and diversity of two key functionalgenes were investigated as the basis for improvedunderstanding of the community structure of natu-ral phytoplankton assemblages in marine environ-ments. New partial NR (encoding eukaryoticassimilatory nitrate reductase) and rbcL (encodingLSU of RUBISCO) sequences from 10 culturedphytoplankton strains are reported. Phytoplanktoncommunity composition from Monterey Bay (MB), acoastal upwelling site on the California coast, andthe Western English Channel (EC), a North Atlanticspring bloom environment, was elucidated based onNR and rbcL sequences. Diatoms were by far themost frequently detected group in both environ-ments, consistent with their importance as a majorbloom-forming group. Both NR and rbcL librariescontained sequences representing cosmopolitantypes such as Emiliania huxleyi (Lohmann) W. W.Hay et H. P. Mohler, Phaeocystis, and Pseudo-nitzs-chia. The NR and rbcL libraries also containedsequences from other chromophytic algal groupsand the Dinophyceae (alveolates). Sequences show-ing identity with key bloom-forming organismsincluding E. huxleyi, Phaeocystis pouchetii (Har.) Lag-erh., Pseudo-nitzschia sp., and Thalassiosira sp. in therbcL libraries confirm previous studies from theseenvironments based on traditional approaches.Diversity ⁄ pattern analyses detected significant com-positional differences among the libraries, whichwere consistent with patterns identified by phyloge-netic analysis, but these patterns were not stronglycorrelated with obvious environmental variablessuch as temperature and nitrate concentration. Manynew and divergent NR and rbcL sequences arereported, but the extent to which they representunknown types cannot be determined until greatereffort is made to sequence the existing culturecollections.

Key index words: assimilatory nitrate reductase;community composition; diversity; marine phyto-plankton; rbcL

Abbreviations: NR, nitrate reductase; rbcL, LSU ofthe RUBISCO gene

Diatoms are an important component of the mar-ine contribution to global primary production(Nelson et al. 1995, Field et al. 1998). The domi-nance by large phytoplankton species, including dia-toms, at high biomass levels (i.e., during blooms)was confirmed in a metadata analysis (Irigoien et al.2004) that revealed patterns in diversity and abun-dance of phytoplankton communities in the marineenvironment. Diatoms are particularly important innitrate-driven environments, such as coastal upwell-ing and subpolar spring blooms; diatom-dominatedcoastal upwelling regimes may account for up to10% of global new production (Chavez and Toggwe-iler 1995). To understand the basis of their successin these environments, we undertook to exploretheir diversity and to investigate the compositionand biogeography of diatom assemblages. The twostudy sites represent two different kinds of nitrate(NO3

))–driven high-productivity environments:Monterey Bay represents a coastal upwelling systemwhere seasonal and episodic supply of nitrate to thesurface water supports a highly productive pelagicfishery. The Western English Channel represents asubpolar spring bloom environment where the sea-sonal signal of nitrate supply by mixing dominatesthe annual productivity pattern.

Modern study of natural phytoplankton assem-blages in upwelling or bloom environments hasemployed flow cytometry (Urbach and Chisholm1998, Collier 2000), photosynthetic pigment analysis(Bidigare and Ondrusek 1996, Peeken 1997), andmicroscopic methods (Sherr et al. 2005, Lassiteret al. 2006). Molecular approaches have also beenintroduced for phytoplankton: in analogy withmicrobial ecology, much greater diversity has beendetected at the 18S rRNA level, especially amongthe picoeukaryotes (Biegala et al. 2003, Massanaet al. 2004), than was detectable by pigment analysisor even by LM. Functional genes involved in carbonand nitrogen assimilation provide another windowinto the diversity of phytoplankton, one that mayprovide direct linkages to the important functionsin carbon and nitrogen biogeochemistry that phyto-plankton perform. Diversity, gene abundance, andgene expression studies based on the rbcL gene,which encodes the LSU of the CO2-fixing enzymeRUBISCO, have produced important insights intocommunity composition and environmental patternsin the Gulf of Mexico (Pichard et al. 1997a,b, Paul

1Received 27 August 2008. Accepted 12 June 2009.2Author for correspondence: e-mail [email protected] address: Indian Institute of Science Education and

Research-Kolkata (IISER-K), Mohanpur Campus, P.O. BCKV CampusMain Office, Mohanpur-741252, West Bengal, India.

J. Phycol. 45, 1335–1347 (2009)� 2009 Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2009.00766.x

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et al. 2000, Wawrik et al. 2003). Only recently havegene sequences for the essential nitrogen-uptakeprocesses been published (Hildebrand and Dahlin2000, Allen et al. 2005). Like rbcL, the genes encod-ing nitrate reductase and nitrate transporters mayenable us to link diatom diversity and biogeographyto primary production and nutrient utilization.Genes involved in nitrate assimilation may be partic-ularly useful for understanding the basis of diatomsuccess in upwelling and spring bloom environ-ments where nitrate availability drives primary pro-duction.

The question of how diatoms succeed and ‘‘win’’in bloom and upwelling events is related to funda-mental questions in marine phytoplankton ecology,such as community diversity, abundance, and vari-ability among species in response to environmentalfactors. Distribution patterns and ecological successare related to the evolutionary development of thegroup (Litchman et al. 2007) but must be expressedby real-time physiological and biochemical features.For diatoms, some of those features are related toefficient utilization of nitrate. In diatoms and otherphotosynthetic eukaryotes, NO3

) is reduced to nitriteby the assimilatory nitrate-reductase enzyme beforebeing incorporated into biomass. Most of what isknown about the regulation of NO3

) assimilation isderived from enzyme and whole-cell studies. Nitrate-reductase enzyme activity in diatoms is quantitativelyrelated to NO3

)-assimilation rates and qualitativelyrelated to NO3

) concentration (Berges and Harrison1995). The growth of diatoms and the uptake ofNO3

) from the medium are directly related to theactivity of the nitrate-reductase enzyme and theamount of enzyme present in the cells (Berges et al.1995). NR activity is tightly linked to the diel cycleand is regulated at the transcriptional level inresponse to photosynthetic carbon assimilation dur-ing the preceding light period (Vergara et al. 1998).Enzyme activity depends on temperature and light aswell and on the nitrogen source for growth (Lomasand Glibert 1999, Berges et al. 2002).

While enzyme activity is directly linked to the bio-geochemical function of interest, in this case, car-bon or nitrogen assimilation, it is not possible withthe present technology to determine enzyme activityassociated with individual components of a naturalassemblage. Size fractionation can provide someinsights, but it is not possible to determine the con-tributions of different kinds of phytoplankton,much less to distinguish among the activities of dif-ferent kinds of diatoms. Greater resolution can beobtained at the level of gene sequences. As the reg-ulation and activity of key genes in individual phyto-plankton cells lead to regulation of enzyme activity,and are therefore related to the success of the spe-cies during bloom and upwelling events, genesequences provide high-resolution, high-sensitivitytools for the investigation into the dynamics of indi-vidual taxa.

The two important metabolic genes that are ofgreatest interest to us are the chromosomallyencoded eukaryotic assimilatory nitrate reductase(NR) and the plastid-encoded rbcL gene. These twogenes were selected for this study because they rep-resent key steps in carbon and nitrogen transforma-tions and can provide direct information on theactivities and environmental response of the phyto-plankton to changes in physical and chemical char-acteristics of their environment.

The molecular basis of NO3) assimilation is best

understood in green algae (reviewed by Fernandezand Galvan 2007). Dawson et al. (1996) determinedthat NR of Chlorella and other algae is not inducedin the presence of (ammonium) NH4

+, but synthesisoccurs upon NO3

) addition, and enzyme activityincreases with increasing NO3

) as a result ofincreased gene transcription (Solomonson and Bar-ber 1990). NR gene sequences from members ofBacillariophyceae are 50%–60% identical to plantand green algae sequences at the amino-acid level(Allen et al. 2005), but there is little information onthe regulation of NR in diatoms (Parker and Armb-rust 2005). To characterize natural phytoplanktonassemblages in terms of diversity and environmentalregulation of individual phylotypes, sequence infor-mation for nitrogen- and carbon-assimilating genesis necessary. By expanding the NR sequence data-base, we will not only learn about the diversity ofthis gene but will also enable the development oftools to investigate its regulation in the environ-ment.

Photosynthetic fixation of carbon in the marineenvironment is mainly catalyzed by the enzymeRUBISCO (Tabita 1999). RUBISCO reductivelyincorporates CO2 into organic carbon via the Cal-vin-Benson-Basham cycle. Several forms of RUBI-SCO are distinguished on the basis of subunitassembly and biochemical characteristics (Watsonand Tabita 1996, Watson et al. 1999). rbcL genesequences are highly conserved, such that it is possi-ble to design probes that are specific for major phy-toplankton clades. Thus, rbcL-specific PCR has beenused to investigate the diversity of marine plankton(Wawrik et al. 2003, Wawrik and Paul 2004) and todocument spatial and temporal patterns in rbcLgene expression (Paul et al. 1999, Wawrik et al.2002, John et al. 2007). In this study, we havefocused on the form 1D because it occurs in dia-toms, a key constituent of bloom and upwellingevents, and is also present in the other major mar-ine eukaryotic phytoplankton (e.g., coccolithophor-ids, prymnesiophytes, pelagophytes, silicoflagellates,etc.) (Paul et al. 2000, Wawrik et al. 2003).

In the future, DNA sequence information forthese two critical functional genes will allow us toinvestigate species- or group-specific differences inregulation and activity on the basis of phylogeneticdifferences among members of the natural assem-blage. Genetic information makes it possible to

1336 PUNYASLOKE BHADURY AND BESS B. WARD

investigate community ecosystem function withmuch greater phylogenetic resolution than is possi-ble with enzyme assays. Thus, it is important toincrease our knowledge of the diversity of NR andrbcL gene sequences for the phytoplankton groupsthat are important in the marine environment. Tothat end, new partial NR and rbcL sequences fromcultured phytoplankton strains encompassing abroad range of classes are reported here.

The main objective of this study was 2-fold: (i) toexplore the functional gene diversity of marinephytoplankton and (ii) to use that information toinvestigate the community composition and biogeo-graphic patterns of natural phytoplankton popula-tions from two environments where diatoms aremajor contributors to primary production. MontereyBay (MB), an upwelling environment on the Pacificcoast of North America, and the Western EnglishChannel (EC) in the North Atlantic, an environ-ment that exhibits a classical spring bloom, wereinvestigated. Diatoms are important primary produc-ers in both environments, but they were expected toharbor different phytoplankton assemblages due totheir distinct biogeochemical and oceanographiccharacteristics. In addition, the same analyses wereperformed on three samples collected during a sim-ulated upwelling experiment using MB water, anexperiment designed to capture the transition fromnonupwelling to upwelling communities. Thus, geo-graphic, oceanographic regime, and seasonal signalsshould all be represented in the suite of samplesanalyzed here.

MATERIALS AND METHODS

Phytoplankton strains. Phytoplankton strains were obtainedfrom the Provasoli-Guillard National Center for Culture ofMarine Phytoplankton (CCMP, West Boothbay Harbor, ME,USA) and the Plymouth Culture Collection of Algae (PCC,Plymouth, UK): Chaetoceros muellerii CCMP 1316 (Bacillariophy-ceae), Cyclotella cryptica CCMP 332 (Bacillariophyceae), Nitzschiacf. pusilla CCMP 560 (Bacillariophyceae), Rhodomonas salinaCCMP 1419 (Cryptophyceae), Porphyridium purpureum CCMP1947 (Rhodophyceae), Isochrysis galbana PCC 565 (Prymnesio-phyceae), Gymnodinium chlorophorum (Dinophyceae), Pleurochr-ysis scherffelii CCMP 649 (Prymnesiophyceae), Heterosigmaakashiwo CCMP 452 (Raphidophyceae), and Chlorarachnionreptans CCMP 238 (Chlorarachniophyceae). Batch cultures andcell harvesting were carried out following the protocol of Allenet al. (2005). Following harvesting, cells were transferred byscraping with a sterile razor blade into sterile 2.0 mL micro-centrifuge tubes and stored at )80�C until further analysis.

DNA was extracted from cultures using a Puregene DNAisolation kit (Gentra Inc., Minneapolis, MN, USA) followingthe manufacturer’s instructions. Extracted DNA was stored at)20�C until further analysis.

Environmental samples. Water samples from Monterey Baywere collected from 20 m and 17 m depths at a midbay station(36�44.76¢ N, 122�01.30¢ W) in August 1998 (MBAug) and July1999 (MBJul), respectively. Seawater (4 L) was concentrated in47 mm diameter 0.2 lm pore-size Supor filters (Gelman,Covina, CA, USA) following the procedures previously de-scribed (Allen et al. 2005). The filters were archived at )80�Cuntil DNA was extracted. Samples (4 L) from the L4 station

(50�15 N, 4�13 W) in EC from the months of April (ECApr)and July (ECJul) 2004 were collected on 0.2 lm Sterivex filtercapsules (Millipore, Cork, Ireland) using a peristaltic pump.Filters were stored at )80�C until further analysis.

In August 2006, simulated bloom experiments were carriedout in three polypropylene 200 L barrels (designated as B1, B2,B3) (Fisher Scientific, Pittsburgh, PA, USA) filled with seawatercollected from Monterey Bay (36�50.86¢ N, 121�57.88¢ W) at adepth of 70 m and inoculated with 2 L of surface water(1–2 m). The progress of the bloom was monitored for 8 d,and samples (1 L each) were collected from the barrels eachday on 0.2 lm Sterivex filter capsules using a peristaltic pump.Filters were flash frozen in liquid nitrogen, transported on dryice, and stored at )80�C. Filters collected on day 4 of the barrelexperiment are included here; they correspond to the earlyexponential portion of the bloom that occurred in the barrels.DNA was extracted from Supor and Sterivex filters using thePuregene DNA Isolation Kit (Gentra Inc.).

PCR amplification of the NR and rbcL gene fragments. NR genefragments from both culture and environmental DNA wereamplified using PhusionTM DNA Polymerase (New EnglandBioLabs, Ipswich, MA, USA) using a nested PCR approach. Theouter primers consisted of NRPt907F and NRPt2325R, yieldingan amplification product of �1,418 bp; the inner reactionprimers (NRPt1000F, NRPt1389R) yield a product length of389 bp (Allen et al. 2005). Outer reactions contained 0.5 lL ofDNA template (3–5 lg Æ lL)1), 5 lL of 5X Phusion HF buffer,1 lL of dNTPs (0.4 mM final concentration), 0.25 lL of eachprimer (0.01 lM final concentration), 0.25 lL of Phusion DNApolymerase, and water to make a final volume of 25 lL. Innerreactions contained 10 lL of 5X Phusion HF buffer, 2 lL ofdNTPs (0.4 mM final concentration), 0.5 lL of primer each(0.5 lM final concentration), 0.5 lL of Phusion DNA poly-merase, 0.5 lL of the outer reaction as a template, and water tomake a final volume of 50 lL. Thermal cycle parameters aredetailed in Allen et al. (2005).

PCR primers specific for the type 1D rbcL gene (Wawrik et al.2002) were used to amplify a 554 bp fragment from culture orenvironmental DNA (3–5 lg Æ lL)1) by adding 10 lL of 5XPhusion HF buffer, 2 lL of dNTPs (0.4 mM final concentra-tion), 0.5 lL of each primers (0.5 lM final concentration),0.5 lL of Phusion DNA polymerase, and water to make a finalvolume of 50 lL. Thermal cycle parameters were as follows:3 min at 98�C, 37 cycles of 15 s at 98�C, 25 s at 52�C and 30 s at72�C, and a final extension for 5 min at 72�C. BSA (0.5 lL of10 mg Æ mL)1) was added to each PCR reaction for amplifica-tion of NR and rbcL genes from environmental DNA samples.Environmental amplicons were gel purified using the QiagenGel Purification kit (Qiagen, Valencia, CA, USA) following themanufacturer’s instructions.

Clone library and DNA sequencing. Purified NR and rbcLamplicons from the environmental DNA samples were clonedwith the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA,USA) following the manufacturer’s instructions. Colonies werescreened for inserts of the expected size with standard T7 andM13R primers. Plasmid DNA containing the inserts was cyclesequenced using the Big Dye Terminator v3.1 Kit (AppliedBiosystems, Foster City, CA, USA). Cycle sequencing reactionswere cleaned using the isopropanol precipitation method.Sequencing was carried out in both directions using the T7 andM13R primers in an ABI Prism 310 Genetic Analyzer. NR andrbcL amplicons from pure cultures were directly sequencedusing the same sets of PCR primers. The sequences were thencompared with known NR and rbcL sequences from GenBank,European Molecular Biology Laboratory (EMBL), DNA DataBank of Japan (DDBJ), and Protein Data Bank (PDB) using theBLAST query engine (Altschul et al. 1997).

Sequence assembly and analysis. Sequences were evaluatedand assembly was performed with CodonCode Aligner, and

MARINE PHYTOPLANKTON MOLECULAR DIVERSITY 1337

subsequently, DNA sequences were translated into amino-acidsequences using Transeq (http://www.ebi.ac.uk/emboss/transeq). Amino-acid sequences from the 389 bp NR and554 bp rbcL fragments were aligned using ClustalW (http://www.ebi.ac.uk/clustalw). Additionally, published NR and rbcLsequences from the GenBank and EMBL databases were alsoincluded in alignments and subsequent phylogenetic analysis.Phylogenetic trees were constructed using the neighbor-joiningmethod in PAUP (version 4.0b10). Trees were bootstrappedwith 1,000 replicates to produce consensus trees retainingsignificant branching >50%. Pure culture and environmentalsequences reported in this paper have been submitted toGenBank, and their accession numbers are GQ921309–GQ921318 and GU203085–GU203455, respectively.

Diversity and rarefaction analysis. Operational taxonomicunits (OTUs) and Shannon-Wiener index were calculatedusing the Web version of FastGroupII (http://biome.sdsu.edu/fastgroup/fg_tools.htm). OTUs were defined with a sequenceidentity cutoff that corresponded to obvious clusters in thephylogenetic analysis: 4% distance for rbcL and 5% distance forNR. Rarefaction analysis was used to compare the diversity ofNR and rbcL clone libraries. Expected number of OTUs perclone sequenced was calculated using the Analytical Rarefac-tion program (http://www.uga.edu/strata/software). The totalnumber of OTUs present in each clone library was calculatedusing the Chao Estimator (http://www2.biology.ualberta.ca/jbrzusto/rarefact.php#ChaoEstimator).

Nonmetric multidimensional scaling (MDS). Multivariate statis-tical approaches such as the principal component analysis(PCA) and nonmetric MDS are used to compare microbialassemblages from diverse environments (Casamayor et al.2002, Lawley et al. 2004). Nonmetric MDS is favored overPCA because PCA assumes that data are normally distributed,which is not necessarily the case for OTUs in microbialassemblages (Clarke and Warwick 2001). Relative abundancesfor rbcL and NR OTUs were arranged in a matrix, and similaritymatrices were created in PRIMER v5.1 by selecting normalizedEuclidean distance and transforming each value by the squareroot (Clarke and Warwick 2001). Transformations downweighted the effect of abundant OTUs on similarities. Non-metric MDS (Clarke and Gorley 2001), an ordination tech-nique that is robust in representing multidimensional data(indicated by acceptable stress values), was then applied to thesimilarity matrices.

RESULTS

rbcL and NR sequences from cultured marine phyto-plankton species. Based on amino-acid sequences,the rbcL sequence from P. scherffelii was 100% identi-cal to that of its congener, P. haptonemafera, and the

C. muellerii rbcL sequence showed 98% identity withthe cultured C. gracilis rbcL sequence (Table 1).

Amino-acid sequences of the new NR gene frag-ments from cultured strains were 54%–91% identi-cal to their closest match in BLAST queries(Table 1), indicating that some of these were quitedistant from any previously sequenced species. Outof eight cultured taxa, two had introns in the frag-ment of their assimilatory nitrate-reductase genessequenced here. These were C. cryptica (Bacillario-phyceae) and P. purpureum (Rhodophyceae).

Diversity and phylogeny of rbcL from environmentalsamples. Environmental parameters for all the sitesare provided in Table 2. The environmental datapresent only a snapshot but clearly identify thecharacteristic differences between the regimes sam-pled. In the EC samples, spring (ECApr) was associ-ated with higher nitrate and lower temperaturecompared to summer (ECJul). For MB samples, theupwelling sample (MBJul) had higher nitrate andlower temperature compared to the nonupwellingsample (MBAug). The barrels started out with highnitrate (20 lM, which was still present as the bloombegan around day 4) and were incubated at surfaceseawater temperature.

All the major chromophytic algal groups thatcontain the Type 1D RUBISCO (Bacillariophyceae,Bolidophyceae, Pelagophyceae, Prymnesiophyceae,and Raphidophyceae) as well as alveolate sequences(represented by Dinophyceae) were detected inthe libraries in varying proportions (Fig. 1A).

Table 2. Environmental variables for sites from whichclone libraries were created.

SiteNitrate

(NO3)) (lM)

Salinity(ppm)

Temperature(�C)

Chl(lg Æ L)1)

ECApr 1.81 35.3 9.8 3.8ECJul 0.00 35.2 13.2 1.8MBAug 1.98 33.23 14.26 0.6MBJul 13.60 33.80 11.42 1.18Barrels day 4 20.0 NA 17.0 0.5–1.0

EC, Western English Channel; MB, Monterey Bay. NA, notavailable.

Table 1. Identity level of assimilatory NR and rbcL sequences from cultured phytoplankton strains and their closest rela-tives from the sequence databases.

Phytoplankton strain Gene sequenced Amplicon size (bp) Identity (%) Closest match

Cyclotella cryptica CCMP 332 NR 519 80 Uncultured eukaryote clone (AAV67020)Nitzschia cf. pusilla CCMP 560 NR 390 91 Uncultured eukaryote clone (ABK32641)Rhodomonas salina CCMP 1419 NR 390 62 Phaseolus vulgarisPorphyridium purpureum CCMP 1947 NR 531 54 Nicotiana benthamianaIsochrysis galbana PCC 565 NR 408 60 Uncultured eukaryote clone (ABK32715)Chlorarachnion reptans CCMP 238 NR 390 61 Phaseolus vulgarisGymnodinium chlorophorum NR 393 60 Uncultured eukaryote clone (ABK32715)Heterosigma akashiwo CCMP 452 NR 387 63 Physcomitrella patensPleurochrysis scherffelii CCMP 649 rbcL 554 100 Pleurochrysis haptonemaferaChaetoceros muellerii CCMP 1316 rbcL 554 98 Chaetoceros gracilis


Bacillariophyceae was one of the most abundantgroups in the rbcL libraries, followed by Prymnesio-phyceae for both sites. The 296 rbcL-deducedamino-acid sequences from environmental samples,along with additional sequences from GenBank andEMBL databases, are shown in the neighbor-joiningtree (Fig. S1 in the supplementary material).

In total, 135 clones were sequenced from the ECrbcL libraries (ECApr and ECJul). The clones includedrepresentatives of several important bloom-formingspecies previously detected in the MB and EC environ-

ments by chemotaxonomic or microscopic methods(Table 3). For several of the previously identified spe-cies (Eucampia, Leptocylindricus, Ceratulina, and Rhizo-solenia), no rbcL sequences are available, so it was notpossible to detect them in the clone libraries;although they may be present among our sequences,they cannot be identified. All of the others, for whichrbcL sequences are available, were detected in at leastone of the clone libraries. Several different OTUs withclosest identity to Thalassiosira oceanica were detectedin both EC libraries and in MBAug. Pseudo-nitzschia

B

A

Fig. 1. Percentage representation of phytoplankton groups detected from the rbcL (A) and NR clone (B) libraries from Western Eng-lish Channel (EC) and Monterey Bay (MB) samples. (For rbcL libraries, ECApr = 63 clones, ECJul = 72 clones, MBAug = 50 clones,MBJul = 30 clones, B1 = 24 clones, B2 = 32 clones, and B3 = 25 clones. For NR libraries, ECApr = 21 clones, ECJul = 31 clones,MBAug = 33 clones, MBJul = 36 clones, B1 = 20 clones, B2 = 32 clones, and B3 = 34 clones.)


delicatissima was found in one EC library and both theMB libraries, but not in the barrels. Navicula sp. com-monly found in the L4 database was detected only inthe ECApr library.

Diatom-like rbcL sequences (Bacillariophyceae)comprised a large fraction of both ECApr (22 of 63sequences) and ECJul (52 of 72 sequences)libraries. Sequences from the ECJul library showedidentity with diatom species including Thalassiosirapacifica, T. aestivalis, T. oceanica, T. mediterranea,Skeletonema menzellii, Placoneis cf. constans, Placoneis cf.paraelginensis, Stellarima microtrias, P. delicatissima,Navicula sp., and Cyclotella bodanica. Fourteensequences from ECJul showed 97%–99% identitywith the diatoms T. oceanica and T. mediterranea atthe amino-acid level. Diatom-like sequences fromthe ECApr rbcL library showed similarity with Odon-tella sinensis, Minidiscus trioculatus, Helicotheca tamesis,Fragilaria striatula, T. oceanica, T. mediterranea, andPseudogomphonema cf. kamschaticum.

In the ECApr library, 23 out of 63 clones showedidentity with cultured haptophytes at the amino-acidlevel (89%–100%). Seven out of 72 clones in theECJul library were prymnesiophyte-like (91%–100%identity with Chrysochromulina spinifera, Platychrysis sp.TKB8934, and E. huxleyi). Five sequences fromECApr and two from ECJul were 100% identical atthe amino-acid level with E. huxleyi (YP277313)(Fig. S1). The sequences from the ECApr libraryshowed varying identities with other prymnesio-

phytes, such as P. pouchetii (13 clones at 95%–99%identity) and Chrysochromulina hirta.

Several other phylotypes were present in EC rbcLlibraries from both April and July. Both librariescontained sequences (two clones from ECApr andseven clones from ECJul) with 89%–95% identity toBolidomonas mediterranea and B. pacifica var. eleuthera(Bolidophyceae, Heterokonta). Also present in thelibraries were 20 clones (15 sequences from ECAprand five sequences from ECJul) with 90%–99%identity to dinophytes such as Peridinium quinquecorne,Galeidinium rugatum, Dinophysis tripos, and D. fortii. Inaddition, both EC libraries had two clones showing87%–98% identity with the dictyophyte Pseudochatto-nella verruculosa.

The most divergent rbcL sequences from the EClibraries based on BLAST query were two sequencesfrom the ECApr library showing only 86% identitywith Aulacoseira sp. (AAT74854) (Bacillariophyceae)and, in the ECJul library, one sequence with only87% identity to the stramenopile P. verruculosa(BAF80665) rbcL sequence (Dicytophyceae).

Eighty rbcL clones were sequenced in total fromthe MBJul and MBAug libraries. Six chromophyticalgal groups (Bacillariophyceae, Bolidophyceae,Cryptophyceae, Dictyophyceae, Pelagophyceae, andPrymnesiophyceae) as well as the alveolates(Dinophyceae) were represented in the MontereyBay rbcL clone libraries (MBAug and MBJul)(Figs. 1A and S1). The amino-acid identities to

Table 3. List of known bloom-forming species from EC and MB environments and their detection (�) in the rbcL clonelibraries based on BLAST queries.

ECApr2004

ECJul2004

MBAug1998

MBJul1999

Barrelsamples 2006

Total number ofinserts sequenced

DiatomsThalassiosira oceanica � � � – – 18Odontella sinensis � – – – – 3Pseudo-nitzschia delicatissima – � � � – 7Pseudo-nitzschia dolorosa – – � ) – 3Pseudo-nitzschia calliantha – – – ) � 4Chaetoceros sp. – � – ) � 3Eucampia sp. – – – ) – –Skeletonema sp. � � – ) � 3Leptocylindricus sp.a – – – ) – –Ceratulina pelagicaa – – – ) – –Navicula sp. – � – ) – 3Nitzschia delicatissum – – – ) � 1Rhizosolenia sp. – – – ) – –

DinoflagellatesDinophysis fortii ⁄ tripos � – � � � 16Peridinium quinquecorne – � � ) � 20

FlagellatesEmiliania huxleyi � � � ) – 9Phaeocystis pouchetii � – � � � 33Chrysochromulina sp. � � � � � 23Heterosigma akashiwo – – – ) � 18

PicoeukaryotesPelagomonas calceolata – – � � � 7

EC, Western English Channel; MB, Monterey Bay.aIndicates rbcL sequences not available for these strains.


previously published sequences ranged between85% and 100%.

In the MBAug library, diatom-like sequenceswere mainly represented by species of Pseudo-nitzs-chia, including P. pseudodelicatissima (96% identity),P. dolorosa (97%–100%), and P. cuspidata (96%).Sequences showing identity with P. delicatissima(94%–95%) and P. calliantha (96%) were alsodetected in the MBJul library and in the barrelsamples (discussed later). Other diatom-likesequences showing identities with S. microtrias, Chae-toceros neogracile, S. menzellii, Placoneis cf. paraelginen-sis, Thalassionema frauenfeldii, Nitzschia longissima,Fragilaria sp., C. bodanica, T. oceanica, Minidiscus trio-culatus, and Bacterosira sp. were also detected in thelibraries of MBAug and MBJul.

Prymnesiophyte-like sequences showing identitieswith P. pouchetii, Platychrysis sp. TKB8934, Chrysoch-romulina parva, C. alifera, C. hirta, and Chrysochromuli-na sp. TKB8936 were detected in both the MB rbcLlibraries. A single prymnesiophyte-like clone fromMBAug (MBAug42) also showed 100% identity withE. huxleyi (YP277313). From the MBJul library, oneof the clones had 100% identity with the cultureddinophytes D. tripos and D. fortii.

Pelagophyte-like clones were found in both MBlibraries (97%–100% amino-acid identity). CloneMBAug18 (and three additional clones) and MBJul3(and one additional clone) were 100% identical toPelagomonas calceolata (AAC02816).

rbcL in the simulated upwelling experiment. Form IDrbcL gene was amplified, and 81 clones weresequenced from the barrel libraries. Unlike the otherMB libraries, diatoms were not a major component.One sequence from the B1 library was 90% identicalto the cultured diatom S. microtrias (ABU62934), butmost of the diatom-like sequences were not closelyrelated to cultivated phylotypes. A single prymnesio-phyte-like sequence from the B2 library showed 100%identity with Imantonia rotunda (BAB20786). A dino-phyte-like clone from the B3 library showed 90%identity with G. rugatum (BAD98975) and Peridiniumbalticum (BAD98976).

Nineteen of the sequences from the barrelsbelonged to Raphidophyceae (Fig. S1). Eighteen ofthese sequences showed 100% identity at the amino-acid level with H. akashiwo (BAD15118), a raphido-phyte that causes red tide. One sequence from theB2 library was 92% identical to the H. akashiwo rbcLsequence. All the chromophytic algal groups,including the Raphidophyceae, were detected inthis study, but the raphidophytes were exclusive tothe barrel samples (Figs. 1A and S1). A single clonesequence showing 98% identity with the cryptophytePlagioselmis sp. was detected from the B1 rbcLlibrary.

Diversity and phylogeny of NR from environmentalsamples. The 207 new NR amino acid sequences,along with previously published NR sequences (Gen-Bank and EMBL) and the new culture sequences

generated in this study, are shown in Figure S2 (seethe supplementary materials). Sequences represent-ing Bacillariophyceae, Chlorarachniophyceae, Crypt-ophyceae, Dinophyceae, and Prymnesiophyceaewere detected in the environmental libraries. OneNR sequence from the MBAug library showed signif-icant identity with a fungal NR sequence and wasnot included in further analysis. The NR librariesfrom both sites were dominated by diatom-likesequences (Fig. 1B).

Fifty-two assimilatory NR clones were sequencedfrom the EC libraries, and all of the sequences werediatom like. Two diatom-like sequences from theECApr and ECJul libraries were 99% identical at theamino-acid level to an uncultured diatom-like cloneMBap19 (AAV67026) sequenced previously from theMB waters (Allen et al. 2005). Also present was aclone from the ECApr library that showed only 58%identity at the amino-acid level with an unculturedeukaryote clone Leo2121e (AAV67015) from theinner continental shelf waters off the coast of NewJersey (USA). A single sequence from the ECJullibrary had 58% identity with an embryophyte, Physc-omitrella patens (BAE06056), indicating that thesesequences were largely distinct from previouslyknown phytoplankton NR sequences.

Sixty-nine NR clones were sequenced from thelibraries of MB environmental samples (MBAug andMBJul) representing three chromophytic algal phyla(Figs. 1B and S2) plus Dinophyceae. In the MBAuglibrary, a clone sequence was 93% identical to a dia-tom-like NR sequence (ABK32683) detected from aseagrass community on the Florida coast. Eightclones from the MBJul library showed 100% identitywith an uncultured diatom-like sequence, MBap24(AAV67029), from Monterey Bay.

The most divergent NR sequences were onesequence from MBAug with 54% identity to Cucurbitamaxima NR gene fragment (AAA33114), a member ofEmbryophyta, and one sequence with 55% identity toan uncultured eukaryote clone (ABK32625)sequenced previously from a seagrass bed off thecoast of Florida. This finding indicated that some ofthe MB environmental sequences were quite diver-gent from the previously known NR sequences, as wasalso true for the EC samples. Five sequences fromMB and EC libraries showed identities <55% at theamino-acid level.

NR sequences from the simulated upwelling experi-ment. Eighty-six NR clones from the three barrellibraries (B1, B2, B3) clustered with Bacillariophyceae,Chlorarachniophyceae, Dinophyceae, and Prymnesio-phyceae (Figs. 1B and S2). Bacillariophyceae was byfar the biggest group in the barrel libraries. Twosequences from B2 showed 99% identity with the twodiatom-like sequences MBap14 and MBap20 fromMonterey Bay waters (AAV67025 and AAV67027,respectively) at the amino-acid level. Seven sequenceseach from B1 and B3 stations, respectively, showed90%–91% identities at the amino-acid level with the


diatom T. oceanica (AAV67000). Sequences were alsodetected in the barrel libraries showing significantidentity (96%–97%) with the pennate diatom Pseudo-nitzschia calliantha. Low identity ranging between 40%and 46% was found in four sequences from the barrellibraries.

Three prymnesiophyte-like NR sequences weredetected in the barrels, as were 13 dinoflagellate-likesequences (80%–84% identity with G. chlorophorum).Although very abundant in the rbcL libraries,sequences from H. akashiwo were not detected inthe NR library. This may represent specificity of theNR primers and suggests the need for further pri-mer development based on the broader database ofNR sequences now available.

Introns were found in 50 NR sequences (60% ofthe clones sequenced) from the barrel libraries.Only one sequence from the EC NR libraries con-tained an intron. The intronic sequences were gen-erally similar to those observed in nitrate-reductasesequences in diatom-like genes from planktonic andepiphytic samples associated with seagrass communi-ties (Adhitya et al. 2007) and were consistent withthe canonical splice site pattern (Breathnach et al.1978).

Diversity index and rank abundance of OTUs. TheShannon-Wiener diversity index (H) was calculatedfor the clone libraries (Table 4). The overall rbcLand NR diversity for pooled libraries (296 and 207sequences, respectively) was >3 (Table 4). For rbcLlibraries, H was highest in the MBAug library fol-lowed by ECJul library. For NR, H was higher in thebarrel libraries, and highest in the B2 library. Rank-abundance analysis representing the frequency dis-tribution of OTUs was also performed for thelibraries. In NR libraries, 65% of the sequences

belonged to nine common OTUs (‡7 sequences perOTU), while singletons represented 64% of NROTUs. The highest number of NR singletons wasfound in the barrel libraries, with the maximum inthe B2 library. In the rbcL libraries, 55% of theOTUs were singletons, while 59% of the sequencesbelonged to 11 common OTUs (‡7 sequences perOTUs). The highest number of rbcL singletons wasin the MBAug library, followed by the ECJul library.

Rarefaction analysis. Richness estimates were nor-malized to the library with the least number ofclones sequenced. The OTU richness of the rbcLlibraries increased in the following order: B3, B1,B2, MBJul, ECApr, ECJul, and MBAug (Fig. S3A inthe supplementary material). The sequencing extentwas generally undersaturated, which was expectedbecause an exhaustive sequencing strategy was notundertaken. For NR libraries, richness increased inthe order ECApr, ECJul, MBAug, B3, MBJul, B1,and B2 (Fig. S3B). The sequencing extent appearedto approach saturation in several NR libraries, asindicated by the curvature of the rarefaction curves.Nonparametric ChaoI estimates clearly indicatedthat the expected number of OTUs (species rich-ness) for rbcL is highest in the MBAug library,followed by the B3 library (Table 4). For NR, theexpected species richness based on ChaoI was high-est in B2, followed by ECJul and B1 (Table 4).

Nonmetric MDS. On the basis of the two-dimen-sional MDS plot, the three barrel rbcL libraries weremost similar to MBJul library (Fig. 2A). Thus, thesimulated upwelling community, even at this earlystage of the bloom, resembled the MBJul upwellingsample more than the nonupwelling assemblagefrom MBAug. In contrast, the MDS analysis for NRshowed the three barrels to be most different(Fig. 2B). The two EC NR libraries were very similarto each other, and the MB libraries were both dis-similar from the barrel libraries (Fig. 2B).

DISCUSSION

The primary objective of this study was to investi-gate the diversity of eukaryotic functional genes,especially in the Bacillariophyceae, and to investi-gate the composition of phytoplankton communitiesfrom the MB and EC environments. NR showedgreater variation at the species level than did rbcL,suggesting that it will be more powerful as a markerfor detection of phytoplankton taxa from marineenvironments. The higher resolution in NR wasobserved in the gene sequence fragments analyzedhere and in sequences already available in pub-lished databases (species-level variation for NR,15%–17%; rbcL, 2%–3%).

Diversity and community composition of phytoplanktonassemblages. Clone libraries for both NR and rbcLcontained sequences that were closely related tothose of cultivated algae and sequences that were notclosely related to any in the database. It is clear that

Table 4. OTU distributions and diversity and richnessestimates for rbcL and NR clone libraries from the threesites.

Site N OTUs H ChaoI estimator (SD)

Total rbcL 296 91 3.90ECApr 63 19 2.50 60.5 (13.4)ECJul 72 28 2.80 47.1 (5.3)MBAug 50 29 3.04 125 (40.4)MBJul 30 14 2.38 47.5 (13.6)B1 24 13 2.33 44 (23.3)B2 32 11 1.95 33.6 (11.0)B3 25 12 1.89 73.5 (37.7)Total NR 207 61 3.3ECApr 21 5 1.11 23.6 (7.9)ECJul 31 7 1.36 68 (27)MBAug 33 11 1.75 26 (7.9)MBJul 36 11 1.97 47 (23.3)B1 20 12 2.31 62 (32.6)B2 32 23 2.99 184 (64.6)B3 34 12 1.91 23.1 (6.5)

EC, Western English Channel; MB, Monterey Bay; N, num-ber of clone sequences; OTU, operational taxonomic unit; H,Shannon-Weiner diversity index.


many of the NR sequences retrieved from the envi-ronment belonged to phytoplankton, the sequencesof which are not yet in the database. It is not known,however, how many of these are currently in cultureand how many may represent unknown phylotypes.Thus, a concerted effort to sequence the culture col-lection would be very useful in gauging the extent ofunknown environmental diversity.

Ten cultures were sequenced for this study(Table 1), and the new NR sequences will be usefulin identifying unknown environmental NRsequences from other eukaryotic algal groups. Ofparticular interest among the new cultured NRsequences is that of H. akashiwo. This cultivated algawas detected a number of times in the rbcL librariesof the simulated upwelling experiment (see Discus-sion later) and may provide vital information aboutthe progression of the bloom and interactionamong members of the assemblage.

It is interesting that we retrieved rbcL sequenceswith high identities to cultured members of alveo-lates (Dinophyceae) from all the libraries (Fig. 1A).Dinophytes possess the type II form of RUBISCO(Morse et al. 1995), but the type 1D chromophyticrbcL primers used here yielded clones with highidentities to published Dinophyceae-like sequencesfrom both environments. Three distinct clusters inthe rbcL tree are affiliated with sequences from culti-vated dinophytes. The split lineages may arise fromendosymbionts or captured plastids, as some dino-flagellates are heterotrophic but known to utilize

ingested plastids for photosynthesis (McFadden andGilson 1995, Saldarriaga et al. 2001, Chesnick et al.2008). The plastid-borne rbcL gene might thenreflect a past evolutionary event, a current endosym-biont, or a prey item rather than the host organism.

The lack of a comprehensive sequence databasefrom cultivated algae limits the comparisons we canmake between our clone library results and thecomposition of algal assemblages expected on thebasis of previous microscopy data. Several speciesknown to occur in blooms from the EC and MBenvironments were detected in the rbcL libraries(Table 3), but the NR database was so slim at thetime of this work that no similar comparisons couldbe made for NR.

The fact that some of the database sequenceswith greatest similarity to the environmentalsequences were from plants may indicate that themajority of relevant phytoplankton either have notbeen cultured, or if in culture, have not yet beensequenced, rather than that the environmentalsequences represent marine organisms that are clo-sely related to terrestrial plants. Even some of thenear-complete new NR gene sequences from cul-tured phytoplankton strains showed significant iden-tities (amino-acid level) with moss and plant-like NRsequences from GenBank and EMBL (e.g., P. pur-pureum, C. reptans, and H. akashiwo in Table 1), indi-cating the wide diversity that remains to bediscovered in marine phytoplankton communities.

Low stress values indicated that the two-dimen-sional MDS plots (Fig. 2, A and B) were good repre-sentations of the relationships between the clonelibraries from EC and MB. Libraries with the high-est H-values also had higher numbers of singletonOTUs and tended to be most distinct in the MDSanalysis as compared to the other libraries. Thus, allmodes of quantitative comparison detected similarpatterns in OTU composition among libraries.

Although MBJul and ECApr both had highnitrate and low temperature, and MBAug and ECJulboth had low nitrate and higher temperature, thesepairs of libraries did not cluster together in MDS.This finding suggests that other oceanographic orregional constraints were more important in deter-mining community composition. Differences indiversity and relationships derived from rbcL and NRmay be related to the different levels of interspeciesdiversity of the two genes.

Biogeography of eukaryotic phytoplankton assem-blages. The environmental clone libraries were notsequenced very deeply, but biogeographic patternscan be suggested based on the available sequences.Several rbcL clone sequences appear to be ‘‘cosmo-politan’’ since these were detected in both EC andMB libraries. For example, sequences with 100%identity with E. huxleyi, a ubiquitous marine species,were detected in both environments. E. huxleyi hasbeen implicated in major blooms in the EC duringJuly and August (Garcia-Soto et al. 1995, Schroeder

A

B

Fig. 2. Two-dimensional nonmetric multidimensional scalingplots of operational taxonomic units for rbcL (A) and NR (B)clone libraries generated from the Western English Channel(EC), Monterey Bay (MB), and barrel samples. 0.01 and 0.04 indi-cate acceptable stress values for the plots.


et al. 2003, Llewellyn et al. 2005). RbcL sequencetypes representing the diatom genus Pseudo-nitzschiaand the prymnesiophytes, such as Phaeocystis orChrysochromulina, were also detected in both sites,suggesting ‘‘cosmopolitan’’ distribution. The pres-ence of Phaeocystis in the upwelling (MB, seawatersamples from 2 years and all three barrels) andbloom (EC) environments is consistent with its glo-bal importance as a major bloom-forming species(Irigoien et al. 2004). Different Pseudo-nitzschia spe-cies occurred in all the libraries—members of thisgenus appear to be both cosmopolitan and success-ful in exploiting a variety of environments (de Baaret al. 2005, Hasle and Lundholm. 2005, Ryan et al.2005). Dinoflagellate-like sequences were alsodetected in both environments. Llewellyn et al.(2005) detected peridinin, a pigment of dinoflagel-letes, throughout the year, including a sharpincrease in July ⁄ August in the waters of EC. Thepresence of this group confirms their importance asa bloom constituent. A series of samples collectedfrom Plymouth to Roscoff (1967–1983 time series)revealed dinoflagellates to be one of the majorgroups in those waters (Robinson and Hunt 1986).While the presence of an OTU in all libraries cansuggest cosmopolitanism, absence cannot be takenas indicating a restricted distribution at this level ofsequencing.

Many phylotypes were present in libraries fromboth environments, but community compositionalso varied both between and within environments.The community composition of the two rbcLlibraries from the EC differed markedly. Membersof Bacillariophyceae and Prymnesiophyceae wererepresented in almost equal proportions (�36%) inthe ECApr library, whereas in the ECJul library,72% of the sequences were diatom like (Fig. 1A).The absence of nitrate in the water column of sam-ple ECJul (Table 2) implies total nitrate drawdownby the phytoplankton community (mainly diatoms).Diatom blooms in the EC in May and July (South-ward et al. 2005) are usually dominated by phyto-plankton, such as T. sp., and the molecular datafrom our study are consistent with the presence ofthis abundant and dominant phytoplankton inECApr and ECJul libraries. Among the Prymnesio-phytes, the harmful bloom-forming alga P. pouchetiihas been recorded previously from the surfacewaters of EC (Southward et al. 2005, Wilson et al.2006), and its rbcL sequence was found only in theECApr library. Prymnesiophytes were more impor-tant in the April library, but some types, such asE. huxleyi and species of Chrysochromulina, were pres-ent in both seasons.

Both EC libraries contained clones with varyingdegrees of identity with the Bolidophyceae. This is agroup that is not easily detected and identified byconventional methods. Bolidophyceae have beendetected in the Mediterranean Sea and equatorialPacific (Guillou et al. 1999) but are not routinely

enumerated in the long-running L4 data set, so verylittle is known about the presence of this picoplank-ton group from the English Channel.

Major groups such as diatoms and dinoflagellateswere present in both barrel and water samples fromMB, but the two MB libraries were different bothfrom each other and from the three barrel librariesin important ways. There was no overlap in the typi-cal bloom-type diatom species that were detected inthe rbcL libraries from water samples versus barrels(Table 3). In addition, rbcL sequences identical tothe red-tide alga H. akashiwo were numerous in thebarrel libraries, but no H. akashiwo–like clonesequences were detected from the MBAug andMBJul rbcL libraries, suggesting the episodic dynam-ics of this microalga in MB waters (O’Halloran et al.2006). This observation suggests that the diatomassemblages that occur with H. akashiwo blooms aredifferent from the diatom assemblages that occur intheir absence, representing major ecological shiftsaccompanying such a bloom.

Sequences showing significant identity (96%–97%) with the pennate diatom P. calliantha weredetected in the barrel and MBAug libraries. Diatomsof this genus have been reported to dominate thewaters of Monterey Bay (Nicholson et al. 2006). TherbcL sequences for P. australis, the important speciesfor domoic acid production in this environment(Buck et al. 1992), were not available for compari-son, so we do not know whether it was included inthe libraries. Diatoms of the genera Skeletonema andChaetoceros are important in the California upwellingsystem, based on microscopic analysis (Lassiter et al.2006), and were detected in this study based on themolecular approach.

Both MB and barrel rbcL libraries containedsequences showing identities with the pelagophyteP. calceolata. The presence of this picoplankton inMB and barrel libraries, but not in the EC, suggeststhat upwelling environments may harbor uniquephytoplankton taxa. This picoplankton has beendetected in other environments, such as the Gulf ofMexico, and is thought to be an important constitu-ent of the phytoplankton assemblages (Paul et al.2000).

Some of the NR clone sequences from MBshowed 100% identity with uncultured eukaryotesequences reported earlier by Allen et al. (2005),which indicate that these organisms are present atdifferent times of the year and especially duringthe upwelling period. Sequences detected by Adh-itya et al. (2007) from Tampa Bay in Florida weresimilar to some clones found in the MB and bar-rel samples, suggesting that some organisms con-taining assimilatory nitrate reductase are commonto these two sites. Both Monterey Bay and TampaBay are both coastal environments; hence, it ispossible that the organisms common to these twosites may be also ubiquitous in other geographicregions.


Methodological constraints and implications. WhilePCR bias ensures that the clone library is not quan-titatively representative of the natural assemblage,the Type 1D rbcL primers used here do not exhibita pro-diatom bias (Wawrik et al. 2003). Therefore,the frequent occurrence in both EC and MBlibraries of Bacillariophyceae sequences probablyindicates the prevalence of this group in both envi-ronments.

Unlike the rbcL primers, the NR primers usedhere were developed to target diatoms, althoughthe original primer development was based onhigher plants and green algae (Allen et al. 2005).Thus, it might not be surprising if the clonelibraries were biased toward diatoms. AssimilatoryNR libraries from the English Channel, MontereyBay, and barrel samples were indeed dominated bydiatom-like sequences, including several clustersthat apparently represent diatoms but were not clo-sely related to anything previously sequenced. Forexample, 23 NR sequences from the English Chan-nel formed a cluster showing very little similaritywith other NR sequences from other sites (Fig. S2).Phylogenetic analysis of the diatom-like sequencesfrom previous studies by Allen et al. (2005) andAdhitya et al. (2007) from Monterey Bay, coastalNew Jersey, and Tampa Bay did not reveal signifi-cant identities with EC NR libraries, suggesting thatthese sequences may be characteristic of EC envi-ronments. The rbcL clone libraries from the EC arealso overwhelmingly dominated by diatom-likesequences. Therefore, the dominance of diatoms inthe DNA extract may have intensified the selectivityof the NR primers toward diatoms. More extensivesampling in time would be needed to make robustbiogeographic conclusions.

Although dominated by diatom-like sequences,some NR sequences from the Monterey Bay and barrelsamples were closely related to other phytoplanktongroups, such as Cryptophyceae, Chlorarachniophy-ceae, Prymnesiophyceae, and Dinophyceae. Thus, theprimers were able to detect sequences from other phy-toplankton groups, despite a bias toward diatoms.The detection of NR sequences from these samplesrepresenting other phytoplankton groups is consis-tent with results from the rbcL libraries, showing thatother groups, including Prymnesiophyceae and Dino-phyceae, were significant components of the phyto-plankton assemblages in MB and barrel samples.

Although limited in size, the clone libraries of NRand rbcL sequences show that diatoms are importantcomponents of the natural assemblage in both Mon-terey Bay and the Western English Channel. Theresults are consistent with previous observationsfrom both these environments that diatoms domi-nate primary production in upwelling and coastalenvironments (Kudela and Dugdale 2000).Although the same type 1D rbcL primers were usedin several studies (this and previous studies as high-lighted below), the results indicate that these envi-

ronments harbor different groups of phytoplankton.A diverse range of chromophytic rbcL sequences(diatoms, haptophytes, and pelagophytes) weredetected in the Gulf of Mexico (Paul et al. 2000,Wawrik et al. 2003). Wawrik et al. (2003) observedhigh diversity of prymnesiophytes within the coastalplume of the Gulf of Mexico and were also able todetect groups such as the Rhodophyta, Xanthophy-ceae, and Eustigmatophyceae within the Gulf ofMexico coastal environment. They did not detectRaphidophyceae and Dinophyceae (nonchromophy-tic group), which occurred in our study. Paul et al.(2000) detected sequences showing low identity withH. akashiwo, but not the 100% identity found for asignificant number of clone sequences from the bar-rel libraries.

The environmental and pure culture sequencesgenerated in this study should be useful for design-ing more robust PCR primers for detecting phyto-plankton communities and unraveling the stages ofbloom formation. The diversity present in the envi-ronmental libraries is compelling evidence for theneed to expand the database of cultivated species.Finally, the sequences generated from this study willform the basis for next-generation functional genemicroarrays that will be used to understand themechanism of bloom formation and the associatedphytoplankton groups, including diatoms.

This work was supported by the U. S. National Science Foun-dation grants to B. B. W. We thank Antoinetta Quigg for sup-plying algal biomass for the cultivated sequencedeterminations, and Nicholas Bouskill for statistics advice. Wegratefully acknowledge the marine operations group at MossLanding Marine Laboratory who made possible the collectionof both MB and barrel samples, and the L4 sampling team atPlymouth Marine Laboratory for collecting the EC water sam-ples.

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Supplementary Material

The following supplementary material is avail-able for this article:

Figure S1. Consensus tree obtained from aneighbor-joining analysis of deduced amino-acidalignments of rbcL sequences from English Chan-nel (ECApr and ECJul), Monterey Bay (MBAugand MBJul), and simulated upwelling experiment(B1, B2, and B3). The scale bar indicates substi-tutions per site. Bootstrap values for nodes >50%are shown in the tree.

Figure S2. Consensus tree obtained from aneighbor-joining analysis of deduced amino-acidalignments of NR sequences from English Chan-nel (ECApr and ECJul), Monterey Bay (MBAugand MBJul), and simulated upwelling experi-ments (B1, B2, and B3). The scale bar indicatessubstitution per site. Bootstrap values for nodes>50% are shown in the tree.

Figure S3. Rarefaction curves for rbcL (A) andNR (B) clone libraries from Western EnglishChannel (EC), Monterey Bay (MB), and simu-lated upwelling experiments.

This material is available as part of the onlinearticle.

Please note: Wiley-Blackwell are not responsi-ble for the content or functionality of any supple-mentary materials supplied by the authors. Anyqueries (other than missing material) should bedirected to the corresponding author for thearticle.


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Figure 4A: Rarefaction curves for rbcL clone libraries from WEC, MB and simulated upwelling experiments.

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Figure4B:Rarefaction curves for NR clone libraries from WEC, MB and simulated upwelling experiments.

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MOLECULAR DIVERSITY OF MARINE PHYTOPLANKTON … · Punyasloke Bhadury2,3 and Bess B. Ward Department of Geosciences, Guyot Hall, Princeton University, Princeton, New Jersey 08544,