distribution patterns and phylogeny of marine ... · diversity of small eukaryotes in the south...

Distribution Patterns and Phylogeny of Marine Stramenopiles in theNorth Pacific Ocean

Yun-Chi Lin,a,b,c Tracy Campbell,b Chih-Ching Chung,d,e Gwo-Ching Gong,d,e Kuo-Ping Chiang,a,d,e and Alexandra Z. Wordenb,c

Institute of Environmental Biology and Fishery Science, National Taiwan Ocean University, Keelung, Taiwan, Republic of Chinaa; Monterey Bay Aquarium ResearchInstitute, Moss Landing, California, USAb; Ocean Sciences Department, University of California Santa Cruz, Santa Cruz, California, USAc; Institute of Marine EnvironmentalChemistry and Ecology, National Taiwan Ocean University, Keelung, Taiwan, Republic of Chinad; and Center of Excellence for Marine Bioenvironment and Biotechnology,National Taiwan Ocean University, Keelung, Taiwan, Republic of Chinae

Marine stramenopiles (MASTs) are a diverse suite of eukaryotic microbes found in marine environments. Several MAST lineagesare thought to contain heterotrophic nanoflagellates. However, MASTs remain uncultured and data on distributions and trophicmodes are limited. We investigated MASTs in provinces on the west and east sides of the North Pacific Subtropical Gyre, specifi-cally the East China Sea (ECS) and the California Current system (CALC). For each province, DNA was sampled from threezones: coastal, mesotrophic transitional, and more oligotrophic euphotic waters. Along with diatoms, chrysophytes, and otherstramenopiles, sequences were recovered from nine MAST lineages in the six ECS and four CALC 18S rRNA gene clone libraries.All but one of these libraries were from surface samples. MAST clusters 1, 3, 7, 8, and 11 were identified in both provinces, withMAST cluster 3 (MAST-3) being found the most frequently. Additionally, MAST-2 was detected in the ECS and MAST-4, -9, and-12 were detected in the CALC. Phylogenetic analysis indicated that some subclades within these lineages differ along latitudinalgradients. MAST-1A, -1B, and -1C and MAST-4 size and abundance estimates obtained using fluorescence in situ hybridizationon 79 spring and summer ECS samples showed a negative correlation between size of MAST-1B and MAST-4 cells and tempera-ture. MAST-1A was rarely detected, but MAST-1B and -1C and MAST-4 were abundant in summer and MAST-1C and MAST-4were more so at the coast, with maximum abundances of 543 and 1,896 cells ml�1, respectively. MAST-4 and Synechococcusabundances were correlated, and experimental work showed that MAST-4 ingests Synechococcus. Together with previous stud-ies, this study helps refine hypotheses on distribution and trophic modes of MAST lineages.

Marine heterotrophic nanoflagellates (HNFs) are small uni-cellular eukaryotes typically ranging from 2 to 20 �m (13,

42, 46). They are thought to be important grazers of bacteria andpicophytoplankton (diameter, �2 to 3 �m) and to contribute tonutrient cycling (1). Results from environmental 18S rRNA geneclone library studies indicate that many HNFs are novel marinestramenopiles (MASTs) (25, 26). MASTs are composed of 12 in-dependent phylogenetic clusters (MAST-1 to MAST-12), none ofwhich appear to be represented by cultured isolates. Among theMAST groups, MAST-1, -3, -4, and -7 have been found in bothopen-ocean and coastal systems, on the basis of 18S rRNA geneclone libraries (24, 26, 31). The trophic roles of each MAST groupare not necessarily known. The MAST-3 cluster appears to containparasites, based on the fact that Solenicola setigera, a parasite ofdiatoms (Leptocylindrus mediterraneus), groups with the MAST-3clade (15, 16). MAST-1 and -4 contain HNFs that actively con-sume prey (27). Rates of MAST-1C and -4 consumption of fluo-rescently labeled bacteria (FLB) were estimated to be 3.6 and 1 to1.5 bacteria per predator per hour, respectively, in coastal waters.Few studies have quantified MAST populations in the natural en-vironment; combined counts of MAST-1 and MAST-4 togetherhave ranged from 2 to 658 cells ml�1 in the world oceans, account-ing for 5 to 35% of HNFs in the �5-�m size fraction (26).MAST-1, -4, -6, and -7 have been shown to be active predators onbacteria and/or picophytoplankton, based on microscopic obser-vations and isotope labeling (14, 27, 36).

Relatively few studies have investigated the abundance and 18SrRNA gene diversity of eukaryotes and, more specifically, MASTsin the North Pacific Ocean. The North Pacific is characterized by anumber of different provinces (21) that are influenced by the larg-

est of these, the North Pacific Subtropical Gyre (NPSG). Some ofthe most productive provinces are observed in regions heavilyinfluenced by boundary currents in the eastern and western NorthPacific Ocean. These regions can be highly complex. For example,the East China Sea (ECS) is the largest marginal sea in the westernNorth Pacific and receives input from coastal and oligotrophicsources. Coastal regions of the ECS are influenced by inflow fromthe Changjiang River (also known as the Yangtze River), the fifthlargest river in the world, and by anthropogenic activities thathave resulted in the loading of inorganic nutrients and dissolvedorganic matter. This is thought to have led to eutrophication andan increase in microbial abundance and biomass in the microbialfood web (45). The hydrographic characteristics of the ECS arealso influenced by the Kuroshio (a western boundary current toNPSG), Yellow Sea coastal water, and Taiwan Warm Current. Incontrast, the California Current (CC) system (CALC), a regionwhich is less enclosed by landmasses than the ECS, is hydrographi-cally dominated by the NPSG eastern boundary current, the Cal-ifornia Current. This dynamic region experiences strong seasonalvariation in upwelling, is susceptible to influences from El Niño

Received 20 September 2011 Accepted 9 February 2012

Published ahead of print 17 February 2012

Address correspondence to Kuo-Ping Chiang, [email protected], orAlexandra Z. Worden, [email protected].

Supplemental material for this article may be found at http://aem.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.06952-11

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Southern Oscillation events, and undergoes mesoscale variationsin the form of fine filaments and eddies which occur between thehydrographic current boundaries of the California and Davidsoncurrents (reviewed in reference 9). Taken together, this region isknown as the California Current system (9) and is within theCALC province, as defined by Longhurst (21). With respect tothe diversity of small eukaryotes, two studies have reported on thediversity of small eukaryotes in the South China Sea (5, 6). In theCALC, picoeukaryote 18S rRNA gene diversity has been investi-gated with a strong emphasis on photosynthetic taxa (10, 47).

Here, we investigate MAST groups in two North Pacific prov-inces. We quantified MAST-1A, -1B, -1C, and -4 cells with specificfluorescent in situ hybridization (FISH) probes and explored theirrelationship with environmental parameters and other microbesin data from two ECS cruises. Prey ingestion was tested in preyaddition experiments. The diversity of MAST populations wasinvestigated in euphotic zone waters of both the ECS and CALCprovinces. Comparisons of 18S rRNA gene sequences with thosefrom previous studies, along with enumeration, were used to ex-plore the ecology and distribution of these uncultured eukaryotes.

MATERIALS AND METHODSStudy sites and general sampling. Three cruise transects were sampled,one in the eastern North Pacific Ocean (CALC) and two in the westernNorth Pacific Ocean (ECS) (Fig. 1). Contextual data were collected at 25stations in the CALC along California Cooperative Fisheries Investigation(CalCOFI) Line 67 between 1 and 10 October 2007 on the R/V WesternFlyer. Three of these stations, representing different water masses, werealso sampled for DNA and other biological measurements (Fig. 1B). TheECS cruises were performed on the R/V Ocean Researcher I, and sampleswere taken along a grid over the continental shelf in the ECS during latespring (29 April to 13 May 2009) and summer (29 June to 15 July 2009),with data collected at a total of 32 and 47 stations, respectively (Fig. 1C andD). Samples from all cruises were collected with either 20-L Go-Flo orNiskin bottles. The vertical profile of the hydrographic conditions, in-cluding temperature, salinity, density, and dissolved oxygen, was recordedby instruments mounted on the rosette, including a conductivity, temper-ature, and depth (CTD) sensor using a CTD collector (SeaBird SBE9/11plus).

Nucleic acid and microscopy sampling. At CALC sites, water wasfiltered through a 20-�m-mesh-size nylon mesh net and then onto3-�m-, 0.8-�m-, and 0.1-�m-pore-size 293-mm filters in series as de-scribed previously (11). Microbial biomass from three ECS sites (station

FIG 1 Study regions and sampling sites in the Central California and eastern Pacific Ocean provinces. (A) Location of the study regions within the North PacificOcean; (B) CALC sites sampled in October 2007; main stations are represented by white dots. (C and D) ECS sites sampled from April to May 2009 (C) and Juneto July 2009 (D). The color background represents sea surface salinity according to the gradient bars provided. Note the different scales between the top andbottom panels.

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[St] 12, St 19, and St 23) was collected for DNA extraction by filteringbetween 1.5 and 9 liters of seawater through a 20-�m-mesh-size nylonmesh net and subsequently prefiltering it through a 5-�m-pore-size filterand onto a 0.8-�m pore-size 47-mm filter (Whatman). Filters with mi-crobial biomass from the ECS or the CALC were stored at �75°C and�80°C, respectively. FISH samples were collected without a prefiltrationstep from surface water (2 or 3 m) at all ECS stations, preserved with 37%formaldehyde (final concentration, 3.7%), and incubated at 4°C for 1 to24 h. Subsequently, 100-ml (for coastal stations) or 200-ml (other sta-tions) subsamples were filtered onto a 0.8-�m-pore-size polycarbonatemembrane (47 mm) and then stored at �80°C.

Nutrient and Chl a measurements. CALC nutrient and chlorophyll a(Chl a) data and the data collection methodology have been reportedpreviously (32). In the ECS, macronutrients were measured according toprevious studies (30, 33) and modified according to Gong et al. (17). Oneto 2 liters of seawater was filtered onto 25-mm-diameter GF/F membranes(Whatman) for determining ECS Chl a concentrations (35). For bothsites, Chl a concentrations were measured with a fluorometer (TurnerDesigns) after extraction.

Flow cytometric analyses for picoplanktonic abundance. Two-mil-liliter ECS samples were fixed using paraformaldehyde (final concentra-tion, 0.2%) for 15 min in the dark, frozen in liquid nitrogen, and stored at�75°C for later analysis. Enumeration of Synechococcus and photosyn-thetic picoeukaryotes was performed on a FACSAria flow cytometer (Bec-ton Dickinson). Heterotrophic bacteria were enumerated by staining a1-ml subsample with SYBR green (Molecular Probes, Inc.) at a 1:10,000dilution and incubated in the dark for 15 min. Synechococcus was deter-mined by its characteristic orange fluorescence, while photosynthetic pi-coeukaryotes were counted using red fluorescence and scatter properties(23). Calibration beads (1-�m yellow-green fluorescence beads) wereadded to each sample as an internal reference. All flow cytometric datawere acquired for 2 min, and the flow rate ranged from 0.020 to 0.031 mlmin�1.

Microscopy. MAST-1 and -4 cells were labeled with specific FISHprobes. Different probe sequences (published previously) were used forenumerating three distinct clades within the MAST-1 cluster: NS1A (5=-ATTACCTCGATCCGCAAA-3=), NS1B (5=-AACGCAAGTCTCCCCGCG-3=), and NS1C (5=-GTGTTCCCTAACCCCGAC-3=) (26). The NS4probe (5=-TACTTCGGTCTGCAAACC-3=) was used to enumerate theMAST-4 cluster (25). Five ECS coastal stations (St 19 to 23) were testedusing a negative-control probe based on the antisense sequence of NS4.For all hybridizations, a portion of each filter was incubated at 46°C for 3 hwith hybridization buffer (30% formamide, 900 nM NaCl, 20 mM Tris-HCl, and 0.01% SDS) with oligonucleotide probes (final concentration, 5ng �l�1) containing a Cy3 moiety at the 5= end. After hybridization, thefilters were transferred into a wash buffer (110 mM NaCl, 20 mM Tris-HCl, 5 mM EDTA, and 0.01% SDS) and incubated at 48°C for 20 min(26). Finally, the filter was overlaid with a mixture of 1 �g ml�1 di-amidino-2-phenylindole (DAPI; final concentration) and antifading re-agent (Citifluor Ltd., London, United Kingdom). Probe-positive cellswere identified by their Cy3 fluorescence under green light excitation. Byswitching UV, blue, and green light excitation, MAST-1 and -4 cells couldbe differentiated from Chl a-containing eukaryotes. Enumeration of totalHNFs was carried out with a different section of the same filter accordingto the method of Porter and Feig (37). To determine the size of MASTcells, we measured the length and width and then converted these mea-surements into equivalent spherical diameter (ESD). The biovolume-car-bon conversion factor for MAST-1 and -4 was estimated to be 183 fgcarbon �m�3 based on results presented elsewhere (3). Three ocular linesof each FISH slide were inspected under epifluorescence at �1,000, andthe average area of each line scanned was 1.32 � 0.04 mm2. Pearson’scorrelation was performed with SPSS software (SPSS Inc., Chicago, IL) toevaluate MAST abundances and relationships with environmental andbiological factors.

Prey observation of MAST-4 by tracer addition. To explore MAST-4prey ingestion, a grazing experiment was conducted utilizing fluores-cently labeled Synechococcus (FLS) at St 24 during the ECS summer cruise.FLS was prepared on the basis of methods described by Sherr and Sherr(43) and stored at �20°C until use at sea. Synechococcus (sp. strainWH7803) was grown at 25°C in f/2 medium (18). FLS was added to 1-literpolycarbonate bottles containing natural seawater and incubated for 40min in an on-deck incubator with running seawater. Two hundred-mil-liliter and 400-ml FISH samples were collected at time zero (T0) and after40 min (T40), respectively. The food vacuole content of 50 MAST-4 cells atT0 and 100 MAST-4 cells at T40 was inspected by epifluorescence micros-copy. FLS could not be distinguished from natural Synechococcus cells;thus, counts represent both cell types.

DNA extraction and clone libraries. DNA was collected from surfacewaters at three ECS stations (St 12, St 19, and St 23) and three CALCstations (St H3, St 67-70, and St 67-155) and from the deep chlorophyllmaximum (DCM) at St 67-155, using the filtration methods detailedabove. DNA was extracted from CALC samples using a sucrose-basedprocedure as described previously (11). ECS samples were treated as fol-lows: cells on the membrane were disrupted using a lysis buffer (0.1 MEDTA [pH 8], 1 mM Tris HCl [pH 8], 0.25% SDS, and 0.1 mg ml�1

proteinase K) with gentle shaking at 55°C overnight. Subsequently, poly-saccharides were removed using cetyltrimethylammonium bromide for15 min at 65°C. Genomic DNA was purified by phenol-chloroform ex-traction and finally dissolved in 50 �l Tris-EDTA (pH 7.5) (7, 8). Theconcentration and purity of DNA were measured using a spectrophotom-eter (Nanodrop).

The 18S rRNA gene-specific PCR primers used on ECS (28) and CALC(29) samples amplified the same nearly full-length gene product. Forwardand reverse primer sequences for ECS samples (5=-AACCTGGTTGATCCTGCCAGTA-3= and 5=-GATCCTTCTGCAGGTTCACCTAC-3=) weresimilar to those used on CALC samples (5=-ACCTGGTTGATCCTGCCAG-3= and 5=-TGATCCTTCYGCAGGTTCAC-3=). PCR conditions forECS samples were as follows: 95°C for 2 min, followed by 30 cycles ofdenaturing at 95°C for 45 s, annealing at 55°C for 1 min, and extension at68°C for 2 min, with a final extension step at 68°C for 10 min with Advan-tage II DNA polymerase (Clontech). About 50 positive colonies were ran-domly picked from each library. ECS clones were sequenced using vector-targeted primers T7 and SP6, each rendering ca. 1,000-bp reads, whichwere then assembled. PCR conditions for CALC samples, cloning, andsequencing were performed as described in reference 11. Between 1,200and 2,000 clones were sequenced at each site using vector-targeted prim-ers M13F and M13R, as well as primers internal to the PCR product, 502Fand 1174R (47). Sequencing was performed on a Prism 3100 or 3730xlgenetic analyzer (Applied Biosystems).

Sequence and phylogenetic analyses. Stramenopile sequences wereidentified and selected for further analysis. Sequences were subjected toNCBI BLAST analysis to screen for stramenopile-derived sequences, andBLAST analysis was used to compare the sequences against those in theSILVA database, which contains 18S rRNA gene sequences representingall known stramenopile groups. Sequences with �97% nucleotide iden-tity to their closest relatives on NCBI BLASTn analysis were furtherchecked for chimeras by breaking each into several short fragments andassessing the apparent origin of each fragment. Sequences were alignedusing the SINA aligner tool and the SILVA SEED database (http://www.arb-silva.de/aligner/) (39). The alignment was manually adjusted accord-ing to secondary structure in the ARB software environment (22). Prior tophylogenetic analysis, gaps and regions where the alignment was ambig-uous based on inspection by eye were removed. Trees were inferred usingneighbor-joining (NJ) and maximum-likelihood (ML) methods using100 bootstraps in the PHYLIP program, version 3.69 (12). The nucleotidesubstitution model for ML was selected with the jModelTest tool by theBayesian information criteria (38).

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Nucleotide sequence accession numbers. Sequences from this studywere deposited in GenBank under accession numbers JQ781881 toJQ782099.

RESULTS AND DISCUSSIONCharacteristics of study sites. MAST diversity was investigated atoceanic provinces on either side of the NPSG. The ECS lies overthe Asian continental shelf, while the CALC region is off CentralCalifornia but extends northwards to Alaska and south to BajaCalifornia. The latter includes the eastern boundary current of theNPSG and a highly productive inshore upwelling region. In eachof these provinces, three sites were used to represent differenthabitats for MAST populations. Specifically, we sampled inshoreat highly productive sites ECS St 19 and MBARI Time Series St H3,with the latter being a midbay site in Monterey Bay. We also sam-pled at transitional stations under the influence of intersectingwater masses, ECS St 23 and CalCOFI Line 67 St 70 (St 67-70).Finally, we sampled a more oligotrophic site in each region: in theECS, St 12 was on the shelf break and influenced by the Kuroshio,and in the CALC, the station (St 67-155) was located at the NPSG-California Current boundary.

The major stations in the two provinces had distinct character-istics and were subject to different seasonal influences. Surfacewater temperatures in the ECS ranged from 15.8 to 25.3°C inspring and 23.3 to 29.6°C in summer 2009, while salinity rangedfrom 27.9 to 34.7 and 23.8 to 34.1 in spring and summer, respec-tively (Fig. 1; see Fig. S1 in the supplemental material). Hydro-graphic features could be a summarized as follows. In spring, theChina Coastal Current, low in both temperature and salinity(temperature, �20°C; salinity, �31), flowed along the coast of themainland and warm, higher-salinity Kuroshio (�20°C, �34 psu)water entered onto the shelf from the southeastern shelf break.Cold, saline Yellow Sea waters (�20°C) intruded into ECS fromthe north. St 23 was located midshelf and was heavily influencedby Yellow Sea waters (Fig. 1C). Hydrographic features changeddramatically by the time of the summer cruise, when two watermasses dominated, a warm stream of the Taiwan Warm Current(�26°C), which flowed northward from Taiwan Strait, and waterdiluted by Changjiang River inputs (�31), resulting in mixed sea-water and freshwater discharge (Fig. 1D) induced by the south-western monsoon. Coastal St 19 was situated in this area and pre-sumably subject to the fluctuations in surface (freshwater) runoff,

especially during summer. Even St 23 was influenced by the lower-temperature, lower-salinity Changjiang River waters during thesummer cruise (Table 1; Fig. 1D).

Overall, sites along Line 67 had salinity within a tighter rangethan the ECS stations (Fig. 1). The warmest sites encounteredover the 3 cruises were during the ECS summer cruise, and con-versely, the St H3 and 67-70 surface waters were cooler than thoseat any of the ECS sites (see Fig. S1 in the supplemental material).Along Line 67, water temperatures were relatively low inshore andbecame warmer toward the open ocean (see Fig. S1B in the sup-plemental material). They were also more saline in Monterey Bay,freshened slightly over the transition into the California Current(CC), and were more saline again beyond the core of the CC (Fig.1B). Conditions observed in the CALC during October 2007 wereconsistent with a relaxation in seasonal upwelling (9), includingrelatively warm temperatures (although they were still cooler thanECS sites) at coastal and midbay stations. Surface NO3 concentra-tions were higher at the midbay station (St H3; 8.86 �M) than anyother CALC or ECS stations for which clone libraries were con-structed (Table 1). However, this was considerably lower than atsome ECS sites where MAST cells were enumerated during cruises(e.g., ECS St 30, 25.3 �M; see Table S1 in the supplemental mate-rial). Surface NO3 concentrations at St 67-155 (0.01 �M) weresimilar to values observed at Station ALOHA in the NPSG (20),and St 12 also had relatively low NO3 concentrations. AlthoughPO4 concentrations were considerably higher at all CALC stationsthan those in the ECS, NH4 concentrations were lower. Chl aconcentrations were highest at inshore stations (St 19 and H3) anddecreased moving off shore (Table 1).

Distribution of MAST-1 and MAST-4 cells in ECS. Maximumabundances of MAST-1A, -1B, and -1C were 14 cells ml�1, 114cells ml�1, and 543 cells ml�1, respectively (Table 2). No cells weredetected with the negative (antisense) probe when applied to en-vironmental samples. Very few MAST-1A cells were detected atthe ECS stations, and none were detected in the plume area (n �18), with the exception of a single station (St 29) in spring (seeTable S1 in the supplemental material). In contrast, MAST-1Ccells were observed frequently and at greater abundances (Fig. 2Cand D; Table 2; see Table S1 in the supplemental material).MAST-1C contributed 1% of the total HNFs detected in both ECScruises. The maximum abundance of MAST-4 occurred in the

TABLE 1 Environmental data corresponding to 18S rRNA gene clone library samples

Date(day-mo-yr) Station

Depth(m)

Longitudea

(°E)Latitudea

(°N)Temp(°C) Salinity

Chl a concn(mg m�3)

Concn (�M)No. of MASTs/no.of stramenopilescNH4

b NO2 NO3b PO4

10-Oct-07 H3 5 �122.02 36.74 12.28 33.47 4.19 NMd 0.31 8.86 1.12 47/759-Oct-07 67–70 10 �123.49 36.13 15.57 33.12 2.72 NM 0.05 0.51 0.61 33/347-Oct-07 67–155 5 �129.43 33.29 19.02 33.19 0.10 0.02 0.07 0.01 0.66 33/346-Oct-07 67–155 86 �129.43 33.29 13.39 33.13 0.94 0.02 0.13 0.40 0.58 54/567-May-09 19 2 123.15 31.63 16.79 29.61 3.32 1.0 0.27 7.9 0.07 2/37-May-09 23 2 126.23 30.46 16.51 33.04 0.64 1.0 0.02 1.0 0.03 0/02-May-09 12 2 125.13 27.55 22.97 34.53 0.28 1.8 0.00 0.0 0.01 3/32-Jul-09 19 2 123.14 31.63 24.52 30.38 5.13 0.9 0.36 3.8 0.28 1/35-Jul-09 23 2 126.23 30.46 25.71 31.30 0.56 0.3 0.03 0.0 0.02 12/139-Jul-09 12 2 125.12 27.49 28.51 33.90 0.14 0.7 0.00 0.0 0.03 1/1a Specified in decimal degrees.b The numbers of significant digits were 1 decimal place in the ECS and 2 decimal places in the CALC.c The number of MAST clones relative to the number of stramenopile clones.d NM, not measured.

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Changjiang River plume during both cruises and extended to themiddle of the continental shelf during spring (Fig. 2E and F).MAST-4 abundance ranged from 12 to 712 cells ml�1 (average,187 � 29 cells ml�1, n � 32) and 8 to 1,896 cells ml�1 (average,264 � 60 cells ml�1, n � 47) in spring and summer, respectively.A significantly positive relationship between the abundance ofMAST-1B and MAST-4 was observed (r � 0.39, P � 0.001, n �79) (Table 3). Notably, four 18S rRNA gene sequences in ourlibraries (represented by 155D1Ae4Eh in Fig. 7A below) had amismatch with the MAST-4 probe. Likewise, the 3 probes target-ing the 3 different MAST-1 subclades first described (MAST-1A,-1B, -1C) probably do not account for cells within a fourth cladedefined here as an additional sublineage, MAST-1D (representedby clone 155S8Be86f; see below), due to mismatches. These mis-matches may have resulted in underestimates of overall MAST-1and MAST-4 abundance.

MAST-1B and -1C as well as MAST-4 had peak abundances inthe plume area during summer (Fig. 2). Previous studies havesuggested that discharge from the Changjiang River has an effecton the nanoflagellate community (45). Therefore, we comparedMAST abundances between what we defined herein, using salinitymeasurements, as coastal waters (�31) and more oceanic waters(�31) using correlations and t tests. MAST-1C abundance washigher in the coastal water (n � 18) than at the more oceanic sites(n � 61) (P � 0.002), and its abundance was negatively correlatedwith nitrite and phosphate in the oceanic samples (Table 3). Like-wise, MAST-4 abundance was significantly higher in coastal wa-ters than in the more oceanic waters (P � 0.02). Photosyntheticpicoeukaryotes and bacteria also had higher abundances in thecoastal water (P � 0.03 and P � 0.001, respectively). In addition,concentrations of NO2, NO3, and Chl a were significantly greaterin coastal water than more oceanic water (P � 0.05, P � 0.001, andP � 0.001, respectively). Thus, it appears that MAST-1C andMAST-4 thrive in the inner shelf of the ECS, and this may berelated to higher prey availability, higher productivity in general,or other environmental parameters associated with these sites.

MAST cell size, biomass, and prey. Cell sizes of MAST-1 and-4 from smallest to largest were MAST-4, MAST-1B, MAST-1C,and finally, MAST-1A (Table 2). MAST-1A cells in the ECS weresmaller (Table 2) than in data from high latitudes, where cell sizewas 7.7 � 0.3 �m (26), but our values are based on very few cells.The average cell size of MAST-1C was about 4.9 �m (Table 2),although at two coastal stations, some cells were �8 �m in diam-eter (2% of measured cells; Fig. 3). In general, MAST-1 cells werelarger than MAST-4 cells. Thus, although MAST-4 dominatednumerically, composing 10% (spring) and 6% (summer) of thetotal HNFs, total biomass contributions of MAST-1C (1.7 � 0.4

�g liter�1) were equivalent to those of MAST-4 (1.7 � 0.3 �gliter�1) in spring. In summer, the total biomass of MAST-1C (3.8� 1.3 �g liter�1) was greater than that of MAST-4 (2.4 � 0.6 �gliter�1). The cell sizes of MAST-1A, -1B, and -4 and HNFs werenegatively correlated with temperature (Fig. 3), whereas in a pre-vious study a significant (negative) relationship betweenMAST-1C and cell size was found (26).

A grazing experiment using tracer addition was carried out atECS St 24 during the summer cruise. FLS were added at a slightlyhigher concentration (final concentration, 14 � 104 cells ml�1)than the natural abundance at T0 (12 � 104 cells ml�1). At T0, 2%of MAST-4 cells contained Synechococcus in their vacuole. AfterT40, FLS were observed in 11% of MAST-4 cells; 6% contained 3FLS per cell, 3% contained 2 FLS per cell, and 2% contained 1 FLSper cell (see, e.g., Fig. 7B). A similar experiment using fluores-cently labeled heterotrophic bacteria (FLB) appears to have faileddue to the addition of too few FLB (less than 1% of the naturalbacterial abundance). By this experiment alone, it is unclearwhether the Synechococcus cells ingested during the grazing exper-iments were then digested and assimilated (19) or, alternatively,possibly later egested. However, there was also a statistically sig-nificant positive relationship between the abundance of MAST-4cells and Synechococcus (Table 3). With respect to other potentialpicophytoplankton prey, the abundance of photosyntheticpicoeukaryotes in the ECS was higher in coastal waters than inmore oceanic waters and higher in spring than in summer (P �0.05). Additionally, photosynthetic picoeukaryotes were occa-sionally observed within MAST-4 cells. Previously, MAST-4 hasbeen detected by PCR (followed by cloning and sequencing) in asample sorted by flow cytometry, based on chlorophyll-derivedred fluorescence; the red fluorescence was attributed to prey con-tained in the MAST-4 food vacuole (44). Taken together, theseresults indicate that picophytoplankton may serve as importantprey items for MAST-4.

Stramenopile 18S rRNA gene sequences and phylogenetics.We obtained a total of 222 stramenopile sequences, 23 from theECS (6 in spring, 17 in summer) and 199 from the CALC (Table1). MAST-1, -3, -4, and -7 18S rRNA gene sequences have beenfound in a number marine environments in previous work (seethe review in reference 24); many of the available sequences arepartial length. Therefore, we cloned, sequenced, and analyzednearly full-length 18S rRNA gene sequences. Two phylogeneticapproaches were used to integrate these new sequences with datafrom past studies. In the first approach, only full-length sequenceswere analyzed, allowing us to analyze more nucleotide positionsand differentiate lineages more clearly on the basis of strongerbootstrap support (Fig. 4). The second set of phylogenetic analyses

TABLE 2 Abundance and size of MAST-1 and -4 cells in the ECS

Parameter

MAST-1A MAST-1B MAST-1C MAST-4

Spring Summer Spring Summer Spring Summer Spring Summer

Mean diam (�m) 5.4 (0.1)a 4.5 (0.2) 4.0 (0.1) 3.7 (0.1) 4.9 (0.2) 4.9 (0.1) 2.2 (0.0) 2.2 (0.0)Presenceb 12/32 (38) 7/47 (15) 18/32 (56) 23/47 (49) 27/32 (84) 40/47 (85) 32/32 (100) 47/47 (100)Avg no. of cells ml�1 2 (0)a 0 (0) 11 (3) 7 (3) 16 (3) 40 (15) 187 (29) 264 (60)Maximum no. of cells ml�1 14 6 72 114 47 543 712 1,896a Data in parentheses represent standard errors.b The numerator reflects the number of samples in which the group was detected, and the denominator represents the total number of samples interrogated. The number inparentheses reflects the percentage of samples in which the group was detected.



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used fewer nucleotide positions in order to analyze a broader suiteof sequences, including published partial-length sequences fromlow to high latitudes as well as the deep sea (Fig. 5 to 8). Thissecond set of analyses did not always retain the levels of bootstrapsupport seen in the analysis that used more phylogenetic informa-

tion (Fig. 4). For each clone library generated, a single represen-tative sequence for 98% identity groups was included in the align-ment (Fig. 4 to 8). This resulted in 87 selected representatives, 17and 70 from the ECS and CALC provinces, respectively.

MAST-3 sequences were the most frequently detected among

FIG 2 Abundance during spring and summer of MAST-1B (A and B, respectively), MAST-1C (C and D, respectively), and MAST-4 (E and F, respectively) inthe ECS, as determined by FISH (units are cells ml�1). Note the differences in the color scales.

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MAST sequences (13 in the ECS provinces and 77 in the CALCprovinces) and were extremely diverse (Table 1 and Fig. 4 and 6).Our analysis supported the inference that MAST-3 organismscomprise an independent cluster with high bootstrap values. Thedata also indicated that MAST-3 contains two distinct, supportedsubclades (Fig. 4). One MAST-3 subclade contained sequencesfrom more oligotrophic (St 12), DCM (St 67-155), and mesotro-phic transitional (St 67-70) stations as well as sequences from theparasite Solenicola setigera (Fig. 4). The other MAST-3 subcladecontained sequences from the ECS transitional station (St 23) andall CALC samples, of which 37 had 98% identity to each other(represented by clone H3S1Be4Kp from St H3; Fig. 4). Overall,MAST-3 18S rRNA gene sequence diversity was relatively high, asreported previously (24). Most MAST-3 sequences appeared to befrom between latitudes 20° and 45°, and few have been retrievedfrom colder deep waters (�200 m) at high latitudes (�60°) (Fig.6). Organisms that have a parasitic lifestyle, as reported for S.setigera, have been hypothesized to be characterized by extremelyhigh diversity (41). Solenicola, which may alternatively be an epi-biont, is broadly distributed, with proposed differences betweencoastal and oligotrophic communities (2, 15). Although the extentto which other MAST-3 organisms are parasites is not known andsome are free-living HNFs (25), the presence of parasites in thislineage might contribute to its relatively high diversity.

We did not recover MAST-4 clones in the ECS but did findthem at all CALC sites. Besides the biases inherent to PCR-clonelibrary studies, the number of CALC clones sequenced was greaterthan the number of ECS clones sequenced and the size fractionssequenced differed in the two provinces, making comparison ofnumbers between provinces invalid. A relatively large number ofMAST-4 sequences were retrieved from the CALC oceanic site;DCM clone 155D8Be8Za represented 24 sequences from thatsample, and the clone 155S8Aeagi represented 15 sequences fromthe surface at St 67-155. At the mesotrophic transitional station,clone 70S1Be7jE represented 12 sequences. MAST-4 cells werealso detected by FISH at all ECS stations (Table 2). In the IndianOcean, MAST-4 sequences were more abundant in coastal sitesthan at open-ocean sites, although they also appeared to be moreabundant in the DCM than at the surface, on the basis of quanti-tative PCR data (40). MAST-4 organisms have not been detectedin polar regions (�60°), by either 18S rRNA gene clone libraries(Fig. 7) or FISH (26). The distribution of MAST-4 organisms mayrelate in part to prey availability or other factors associated withtemperature. Overall, MAST-4 18S rRNA gene sequences ap-peared to have relatively less genetic distance and fewer subcladesthan MAST-3 or MAST-1 (Fig. 4).

In the CALC province, representatives of the 3 previously de-scribed MAST-1 subclades, MAST-1A, -1B, and -1C (26), wereretrieved along with one from the ECS (from MAST-1C; Fig. 4 and5). The MAST-1A clone 155D3Ae6ho was obtained from theDCM at the most oligotrophic station sampled. MAST-1A se-quences have primarily been acquired from higher latitudes, andmany of them composed a large supported subgroup (ECS andCALC sequences did not belong to this subgroup; Fig. 5). The factthat there are several phylogenetically distinct groups from highand low latitudes that are targeted by the same FISH probe (Fig. 5)could potentially explain the observed differences in the sizes ofthe ECS cells measured here and those measured in samples fromhigher latitudes (26).

MAST-1B sequences were retrieved from the DCM only at themost oligotrophic CALC station (represented by 155D3Ae6hF;Fig. 4 and Fig. 5). An additional sublineage, MAST-1D, was ap-parent. MAST-1B contained DCM clones and appeared to have abroader distribution from low to high latitudes, while MAST-1Dwas detected only in tropical and subtropical/temperate locations(latitudes � 45°) (Fig. 5). MAST-1C contained two distinctgroups of sequences (Fig. 4), one with those from the transitionaland oligotrophic CALC stations (70S3Be9KQ and 155D3Be4vg)and the other with sequences from coastal St H3 and St 19 (Fig. 4).The former group contained (within it) two additional supportedclades which separated sequences from low and high latitudes

TABLE 3 Significant Pearson correlations observed between MAST abundances and other factorsa

Data subset

Pearson correlation

MAST-1A MAST-1B MAST-1C MAST-4

All stations (n � 79) Salinity, 0.29** MAST-4, 0.39*** MAST-1B, 0.39***; Syn, 0.56***�31 psu (n � 61) Salinity, 0.31* Temp, �0.28* NO2, �0.33**; PO4, �0.27* Syn, 0.55***�31 psu (n � 18) HNF, 0.57*; PNF, 0.58*; MAST-4,

0.66**; Syn, 0.72**HNF, 0.70**; PNF, 0.54*; MAST-1B,

0.66**; Syn, 0.77***a Only factors that had a significant correlation are shown. Dissolved oxygen, NH4, NO2, NO3, PO4, Chl a, heterotrophic nanoflagellates (HNF), pigmented nanoflagellates (PNF),Synechococcus (Syn), photosynthetic picoeukaryotes, and heterotrophic bacteria were included in the analysis. r values are significant at P � 0.05 (*), P � 0.01 (**), or P � 0.001(***).

FIG 3 Relationship between water temperature and cell diameter of each ofthe MAST groups enumerated as well as HNFs as a whole. Significant relation-ships were determined for HNFs (r � �0.70, P � 0.001, n � 79), MAST-1B(r � �0.39, P � 0.05, n � 41), and MAST-4 (r � �0.25, P � 0.05, n � 79) butnot MAST-1C (r � �0.21, P � 0.91, n � 67). For MAST-1A, the relationshipwas also (negatively) significant, but the analysis involved very few cells. MASTand HNF cell sizes were estimated using different methods, the former by FISH(effectively, the cytoplasm) and the latter by DAPI staining (effectively, thenucleus).



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(Fig. 5). Those from lower latitudes (including CALC sequences)seemed to be from more oceanic settings, such as the Sargasso Sea(Q2H11N10 [31]), although nutrient data were not available fromall previous studies (Fig. 5). Sequences composing the group con-

taining clones 897St19-43 and 14H3Te6QW from our most eu-trophic sites have also been recovered from sediments, anoxic, andpelagic environments (Fig. 5). Although the ECS has been re-ported to be a low-oxygen area (4), hypoxia was not observed at St

FIG 4 Stramenopile phylogeny by ML methods of ECS (open squares) and CALC (solid squares) sequences. Nearly full-length 18S rRNA gene sequences fromcultured or described organisms (gray) or environmental sequences (black) were used, resulting in 1,399 analyzed positions. ML and NJ bootstrap values areshown at nodes retaining �70% support (100 replicates total). Solid circles, significant support by both methods; gray circles, supported only by ML; open circles,only NJ support. Actual bootstrap values (ML/NJ) are shown for MAST clusters. The TrN�I�G substitution model (I � 0.206 and � � 0.438) was used, and fourdinoflagellate sequences served as an outgroup. ECS clone names contain the cruise (897, spring; 905, summer) and station information, while in CALC clonenames, the first number after the station identifier, i.e., 1, 8, and 3, corresponds to the sequenced size fractions 0.1 to �0.8 �m, 0.8 to �3 �m, and 3 to �20 �m,respectively; S and D indicate surface and DCM samples, respectively. Numbers in parentheses beside sequence names from our study indicate the number ofclones represented by that sequence (all having �98% identity).

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19 in spring during this study. We hypothesize that this MAST-1Csubclade can acclimate to dramatic variations in environmentalconditions, including estuarine or anoxic environments.

In the ECS, only a single clone each was obtained fromMAST-2 (clone 897St19-5) and MAST-7 (clone 905St19-25), and

both were from the coastal station. In the CALC, although noMAST-2 sequences were recovered, MAST-7 sequences werefound at all stations. The basal part of the MAST-7 tree was rela-tively diverse, and only the upper half of the tree contained se-quences from between latitudes 20° and 60° (Fig. 8). Sequences

FIG 4 continued



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from the CALC province were spread throughout the tree andprimarily seemed to come from the most oligotrophic station, St67-155 (Fig. 8). The MAST-2 sequence from St 19 was phyloge-netically close to a sequence (MB04.36; 99% identity over 713 bp)

FIG 5 Phylogenetic analysis of MAST-1 using NJ distance methods with par-tial-length sequences and containing representative ECS (open squares) andCALC (solid squares) sequences. Bootstrap values estimated from ML and NJmethods (100 replicates) are shown for nodes with �70% support, as de-scribed in the legend to Fig. 4. A total of 499 positions were analyzed, aftermasking and gap removal. Latitude and depth at the site of sequence origin areprovided in color bars. Samples from latitudes of �20° are from the CariacoBasin, Caribbean (A95, AA, AB, BC, and CA), Indian Ocean (IND58, IND60,IND70, and IND72), and Pacific Ocean (OLI); those from latitudes from 20° to45° are from the Atlantic Ocean (AMT15_33, ENI, N5, N10, SSRP, and nu-merical sequence identifiers), Indian Ocean (IND1, IND2, IND31, andIND33), Mediterranean (BL and ME), and Pacific Ocean (TH and this study);those from latitudes from between 45° and 60° are from the Antarctic (ANTand DH), Atlantic Ocean (NA, AMT15_1, and OR), English Channel (RA),Framvaren Fjord (FV and SIF), and Helgoland (HA); and those from latitudesof �60° are from the Arctic Ocean (MD and NW), adjacent seas of the ArcticOcean (NOR), Franklin Bay (CS), and Norwegian Sea (AD and CD). All cloneswere derived from the water column, except those from sediments (BAQ,TAGIRI, and DSGM). FIG 6 Phylogenetic reconstruction of MAST-3 by NJ distance methods with

partial-length sequences, including 7 representative sequences from ECS(open squares) and 27 representative CALC sequences (solid squares). A totalof 471 positions were analyzed, after masking and gap removal; bootstrapvalues were estimated using ML and NJ methods with 100 replicates and areshown for nodes with �70% support. Clonal libraries represented, latitudinalranges, and depths are as described in the legend to Fig. 5.

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from the Hong Kong coast in the subtropical western PacificOcean. These two sequences have a mismatch to the NS2 probe(26), which may explain why MAST-2 sequences were rarely ob-served by FISH (less than 10 cells detected in a total of 79 samples).There are still few published MAST-2 sequences, making it diffi-cult to further inspect the relationship between the phylogeny anddistribution.

MAST-8 and -11 sequences were retrieved only from St 67-70and the DCM at St 67-155 (Fig. 4). All of these had low identitiesto deposited sequences (91 to 96%), except MAST-11 clone905St23-34. Phylogenetic analysis supported MAST-8 being anindependent stramenopile lineage. Most MAST-8 clones in theanalysis were from our study because only one other nearly full-length sequence (that for clone OR000415.113) was available (24).Finally, the position of MAST-11 and most other MAST groupswas not supported in the 18S rRNA gene tree (Fig. 4).

Other heterotrophic stramenopile sequences included quitedivergent sequences from the most nutrient-rich stations sampledin both provinces, which grouped with the Bicosoecida andOomycetes (Fig. 4). Labyrinthulida-like sequences were also

detected, and one was similar to clone BL010625.31 from thecoastal Mediterranean Sea. In addition, a colorless chrysophyte(Spumella) sequence was retrieved from the ECS transitional sta-tion (St 23) in summer. Chrysophytes were also detected in theCALC but belonged to uncultured taxa for which the trophicmode is unknown. A single pelagophyte sequence was identified(St 67-155). Twenty-two diatom sequences were found at themore eutrophic midbay station (St H3), and most belonged to aclade largely composed of Thalassiosira sequences. Only one wasfound at St 67-70, and none was found at St 67-155 or any of theECS stations. Most diatom sequences were detected in the largestCALC size fraction (3 to 20 �m), and a similar size fraction wasnot sequenced in the ECS (where water was prefiltered through a5-�m-pore-size filter).

In conclusion, our results indicate that many of the previousobservations on the distribution and phylogeny of MAST lineages(6, 24, 25, 31, 26, 48) hold true in the North Pacific Ocean. Withinthe stramenopiles, many trophic modes exist, and this is presum-ably the case across MASTs as well. Their ecology, physiology, anddiversity are still little understood, in part due to their uncultured

FIG 7 (A) Phylogenetic reconstruction of MAST-4 by NJ distance methods using partial-length 18S rRNA gene sequences. Nine representative CALC sequences(solid squares) were included, and 489 nucleotide positions were analyzed, after masking and gap removal. Bootstrap values were estimated using ML and NJmethods with 100 replicates and are shown for nodes with �70% support. Vertical red lines indicate probe mismatches with environmental sequences. (B)MAST-4 cells and ingested Synechococcus. The images are the result of an overlay of images from blue and green light excitation; red represents the MAST-4 cell,and yellow represents Synechococcus cells. Bar, 10 �m.



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status. The addition of sequences as well as abundance and sizedata from previously unexplored oceanographic provinces ishelping to refine hypotheses on the distribution and activities ofthese organisms. We found that the cell size of MAST-1B andMAST-4 varied with temperature, indicating that differentecotypes may exist within these lineages. The analysis of full-length 18S rRNA genes also indicated that MAST-1 and MAST-7contain distinct subclades, and groups within those were associ-ated with specific latitudinal zones. Thus, perhaps not surpris-ingly, temperature or other environmental parameters associatedwith these zones appear to play a role in the 18S rRNA gene diver-sity and distribution of these eukaryotes.

Finally, MAST-4 organisms have been shown to consume het-erotrophic bacteria. Here we observed their consumption of pico-phytoplankton, specifically, Synechococcus. Combined with thestatistically significant relationship observed between the naturalabundance of MAST-4 and Synechococcus, this may indicate thatthe latter serves as a food resource for MAST-4. Herbivory haspreviously been reported for MAST-1, MAST-6, and MAST-7, onthe basis of label incorporation in nucleic acids from incubationexperiments with cyanobacterial prey cells (Prochlorococcus orSynechococcus) in NPSG surface waters (14). MAST-6 organismshave been reported to ingest more algae than heterotrophic bac-teria in brackish waters of the Baltic Sea, on the basis of visual

assessment of food vacuole contents (36). The factors that shapeprey selection by these MAST lineages and now MAST-4 influencetheir ecosystem roles, particularly with respect to control of pri-mary producer communities.

ACKNOWLEDGMENTS

We thank the captains and crews of the R/V Ocean Researcher I and theR/V Western Flyer as well as M. P. Simmons, E. Demir, R. M. Welsh, andA. Engman for CALC sample collection. We are grateful to H. Wilcox forprocessing CALC DNA samples, G. Weinstock, E. Sodergren, andWUSTL Genome Center staff for CALC clone library sequencing underGBMF1668 (see below), as well as D. McRose and A. Monier for initialscreening of CALC libraries. We also thank S. B. Johnson for assistancewith jModelTest and are deeply grateful to R. Massana for training Y.-C.L.in FISH methods. Finally, we appreciate the constructive comments andsuggestions from two anonymous reviewers.

Y.-C.L. was supported by a fellowship (NSC98-2917-I-019-101) forvisiting an external lab from the National Science Council, Taiwan. Wethank M. Silver for cosponsoring Y.-C.L. with A.Z.W. Major funding wasprovided through the National Science Council, Taiwan (NSC98-2611-M-019-021-MY3), to K.-P.C. and the Gordon and Betty Moore Founda-tion (GBMF1668) and David and Lucille Packard Foundation to A.Z.W.

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