Algal Pigment Patterns and Phytoplankton Assemblages in Different Water

8/12/2019 Algal Pigment Patterns and Phytoplankton Assemblages in Different Water

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Algal pigment patterns and phytoplankton assemblages in different watermasses of the R o de la Plata maritime front Jose I. Carreto , Nora Montoya, Rut Akselman, Mario O. Carignan,Ricardo I. Silva, Daniel A. Cucchi ColleoniInstituto Nacional de Investigacio n y Desarrollo Pesquero, Paseo V, Ocampo No. 1, B7602HSA-Mar del Plata, Argentina

a r t i c l e i n f o

Article history:Received 1 November 2006Received in revised form24 February 2007Accepted 28 February 2007Available online 19 March 2008

Keywords:PhytoplanktonPigmentsCHEMTAXPhoto-adaptationSouth AmericaArgentina

a b s t r a c t

The composition of phytoplankton assemblages were studied in three sections across the continentalshelf between the R o de la Plata and the oceanic waters of the Subtropical Convergence, during latespring. Algal communities were examined using microscopy and HPLC-derived pigment concentrations.The CHEMTAX program was used to estimate the chlorophyll a (chl a) biomass of different algal classes.Trends in pigment ratios due to phytoplankton photo-adaptation and photo-acclimation were alsoexamined. In order to accommodate the natural diversity of phytoplankton assemblages the originaldata have been split to represent ve ecosystems. In addition, the pigment data for the Brazil Currentecosystem has been split by sample depth.

High chl a concentrations were recorded in the outer estuary region (up to 15.5 mgm 3 ) and in theshelf-break front associated with Subantarctic waters (24 mg l 1 ). In contrast, chl a concentrations wererelatively low over the continental shelf and in the oceanic region dominated by the Brazil Current,where the lowest values (0.10.2 mg l 1 ) were found. Both pigment patterns and microscopy-derivedinformation showed ve different phytoplankton assemblages spatially segregated by the prevailingenvironmental conditions. In the inner estuary assemblage green algae (5456% of total chl a) werealways the dominant group and most of the chl a, arises from chlorophyceans (4049%). In a decreasingorder, diatoms cyanobacteria and cryptophytes were also relevant. In the outer estuary assemblagediatoms and dinoagellates were the dominant groups but cryptophytes and euglenophytes werepresent as sub-dominant groups. In the coastal and shelf region, the algal assemblage showed an almosttotal dominance (59.387.6%) of diatoms. The usual diatom-pigment pattern (chl c 1 , chl c 2 ) group(diatom I), was the more abundant and widely distributed, but in some stations, diatoms containing chlc 2 and c hl c 3 (diatom II) were present as dominant group. A more complex phytoplankton communitydominated by coccoid and small agellates (25 mm) predominantly comprised by chlorophyceans(up to 50%) and haptophytes (up to 62%) was found near the shelf-break front. This is the rst time thathigh chl b concentrations associated to a bloom of a picoplanktonic ( o 3 mm) coccal chlorophyceanwas reported for this area. The Brazil Current assemblage showed the dominance (55.471.9%) of thepicoplanktonic cyanobacteria Synechococcus spp. (32.345%) and Prochlorococcus spp. (41.410.4%).Haptophytes were also present as sub-dominant group being particular abundant at the deepchl a maxima. A sharp transition in photo-collectors/(chl a+Dv chl a) and photo-protectors/(chl a+Dv chl a) ratios at depth near the base of the euphotic zone was observed in the water column of this ecosystem. These results are discussed in relation to the complex environmental features of the

region. & 2008 Elsevier Ltd. All rights reserved.

1. Introduction

The physical oceanography and the sediment transportprocesses of the R o de la Plata estuary and plume are wellunderstood and documented ( Framinan and Brown, 1996 ;

Guerrero et al., 1997 ; Framin an et al., 1999 ; Campos et al., 1999 ;Piola et al., 2000, 2005 ; Mianzan et al., 2001 ; Simionato et al.,2004 ). Recently, Acha et al. (2008) summarize present knowledge,concluding that the estuary is a highly variable environment,strongly stratied most of the time but that can be mixed during afew hours by strong wind events that occur in an unpredictablemanner, generating stratication/partially mixed pulses all alongthe year ( Acha et al., 2008 ). The shallow, high turbidity tidal riveris separated from the mixohaline area by a turbidity front, closely

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Continental Shelf Research

0278-4343/$- see front matter & 2008 Elsevier Ltd. All rights reserved.doi: 10.1016/j.csr.2007.02.012

Corresponding author. Tel.: +54 223 4862586; fax: +54 223 4861830.E-mail address: [email protected] (J.I. Carreto).

Continental Shelf Research 28 (2008) 1589 1606
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related to the salinity front ( Framin an and Brown, 1996 ).Within the nutrient rich turbid river, phytoplankton growth islightlimited ( Carreto et al., 2003 ; Acha et al., 2004, 2008 ; Huretet al., 2005 ). In the outer estuary zone the phytoplanktonproduction appears to be regulated by light penetration and theassimilable nitrogen ow, while silicate and phosphate are inexcess ( Carreto et al., 1986 ; Nagy et al., 1997 ). In this area, the

concentrations of inorganic nutrients decrease rapidly with thedecrease of turbidity along the salinity gradient ( Carreto et al.,2007 ; Huret et al., 2005 ).

Simionato et al. (2004) , showed that in the absence of windsthe normal path of the estuary ow should be a buoyant plume tothe NNE direction along the Uruguayan coastline. When the meanwind blows from directions between SSE and NNW this pattern isintensied. During winter, the inuence of the plume has evenbeen noticed along the Brazilian coast as far as 28 1 S (Camposet al., 1999 ), while in summer it is constrained to south of 32 1 S (Piola et al., 2000 ). However, during summer its inuencemay be detected sporadically in a southward direction along thecoastal region of Argentina ( Carreto et al., 2007, and referencestherein ). Even though a seasonal (summer and winter)

circulation pattern has been reported ( Guerrero et al., 1997 )recent model studies suggest that variability in the estuarinecirculation is highly sensitive to the atmospheric forcing andthat the dynamic condition of summer and winter are likelyto occur during any season ( Simionato et al., 2004, 2006 ). Overthe continental shelf, a sharp contrast in water mass character-istics exists below the low salinity plume. The relatively coldnutrient-rich Subantarctic Shelf water dominates south of 33 1 S,while warm, salty Subtropical Shelf water extend north of thatlatitude. These water masses are separated by relatively narrowfrontal zone in subsurface referred to as Subtropical Shelf Front(Piola et al., 2000 ). Occasionally, the inuence of the R o de laPlata waters may extend to the shelf-break front where theseestuarine waters contact the nutrient-rich waters of Malvinas ow(Negri et al.,1992 ), and the warm, nutrient-poor subtropical BrazilCurrent water or it may be found between both currents(Lusquin os and Valde s, 1971 ; Provost et al., 1995 ). These waterscan be carried offshore by the return ux of the Malvinas Current(Provost et al., 1995 ) in an area referred to as the SubtropicalConvergence.

However, relatively few phytoplankton studies have carriedout in this region ( Carreto et al., 2007 and references therein) andthe recognition of the importance of delicate agellates andminute coccoid forms has been recognized only recently, aftermore appropriate techniques for studying the picoplankton wereapplied ( Silva and Negri, 2000 ; Carreto et al., 2003 ). Using theseapproach, diatoms, cryptophytes, prasinophytes, dinoagellates,haptophytes and cyanobacteria were found forming differentassemblages spatially segregated by the prevailing hydrographicconditions ( Carreto et al., 2003 ).

Pigment chemo-taxonomic approach based on chromato-graphic pigment analysis has been useful for distinguishing themain algal classes, especially of the small-sized phytoplankton.The signature role of the pigments has been summarized invarious papers, although the interpretation of HPLC eld data isnot a straightforward task ( Jeffrey and Wright, 1997 ). TheCHEMTAX program has proven to be a solid method to calculatethe abundance of phytoplankton groups, if correct information onthe phytoplankton groups present in the sample is loaded in theprogram. Screening the samples in the microscope prior to dataanalysis, minimize the possibility of overlooking phytoplanktongroups with no or overlapping diagnostic pigments ( Wright et al.,1996 ; Wright and van den Enden, 2000 ; Mackey et al., 1998 ;

Rodrguez et al., 2002 ; Carreto et al., 2003 ; Schlu ter andMhlenberg, 2003 ; Wright and Jeffrey, in press ). However,

choosing starting pigments/chlorophyll a (chl a) ratios for theCHEMTAX remains the biggest problem due to insufcientknowledge of these ratios in the eld ( Schlu ter et al., 2000 ;Wright and Jeffrey, in press ).

Recently, concurrent analyses of microscope enumeration of phytoplankton species, HPLC-derived pigment concentrations andthe CHEMTAX software were used for the rst time in this region

to evaluate the surface distribution and abundance of the differentphytoplankton groups ( Carreto et al., 2003 ). Results showed thatthe inclusion in the pigment matrix of the most abundantmembers of the chlorophyll c (chl c ) pigment family (chl c 1 , chlc 2 , chl c 3 and chl c 2 monogalactosyldiacylglyceride esters)improved CHEMTAX interpretation of eld data ( Carreto et al.,2003 ; Rodr guez et al., 2002 ). Using this novel approach vedifferent surface phytoplankton assemblages, spatially segregatedby the prevailing environmental conditions, were distinguishedacross the studied section. All of them showed a complexcommunity structure, formed by a background of small-sizedcells such as cyanobacteria, cryptophytes, haptophytes andprasinophyceans on which, in the more eutrophic waters, diatom,cryptophytes or some haptophyte blooms were overlapped

(Carreto et al., 2003 ). In the surface phytoplankton communitystructure of the nutrient-poor Subtropical Brazil Current Watersthe picoplanktonic cyanobacteria Synechococcus , appeared to bethe most important group ( Carreto et al., 2003 ) although thevariability in the vertical distribution has not yet been reported.Recently, Barlow et al. (2002) showed that divinyl chl a (DV chl a),an exclusive biomarker of Prochloroccocus , was prominent atdepth in the tropical and subtropical region of the Atlantic Oceanfrom 40 1 N to 35 1 S. The highest DV chl a concentrations wereobserved in the equatorial region at the chlorophyll maximalocated at 7080 m ( Barlow et al., 2002 ).

In this study, the structure of phytoplankton communities wasstudied during November 2001 in tree sections across thecontinental shelf between the R o de la Plata and the oceanicwaters of the Subtropical Convergence ( Fig. 1), using microscopy,HPLC-pigment patterns and the chemical taxonomy softwareCHEMTAX. In addition, we describe the relationship betweenphytoplankton communities and the environmental features of the region.

2. Material and methods

2.1. Sample collection

Sampling procedures were performed from 19 November to 1December 2001, on board the R.V. Dr. E.L. Holmberg (Fig. 1).Vertical proles for temperature and salinity were obtained with aCTDSBE911. CTD data were calibrated and reduced to 1-m bins.Light penetration was measured with a PUV 500 (BiosphericalInstruments Inc.) underwater radiometer. Surface water samples(0 m depth) were collected with a clean plastic bucket, anddiscrete sampling of the water column was carried out usingNiskin bottles attached to the CTD rosette.

2.2. Remote-sensing data of SST and ocean color

Sea surface temperature (SST) eld was derived from datacollected by the Advanced Very High Resolution Radiometer(AVHRR) installed on the NOAA 11 polar satellite and processedby the Argentine National Space Research Commission (CONAE)using a multi-channel algorithm. Chlorophyll images were pro-

vided by the SeaWiFS Project, NASA/Goddard Space Flight Centerand ORBIMAGE (http://seawifs.gsfs.nasa.gov/ ) processed to level 3.

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2.3. Microscopic analysis

Surface water sub-samples (0 m depth) for phytoplanktonidentication and cell counts were obtained from selectedstations and xed with neutral Lugols iodine. Sample volumesof 50 and 100 ml were settled. Phytoplankton species wereidentied and enumerated with an Olympus IX-70 inverted

microscope using bright-eld optics and 200, 400, 600 and 1000(oil) magnications. Algal cells were identied to species levelwhen possible, except for the small and fragile ones for whichonly the genus or algae group was recorded. In occasions, watersamples consisting about 50100 ml were ltered throughpolycarbonate membrane (0.2 mm pore size, 47mm diameter)and left in plastic Petri slides. A portion was cut from each lterand glued to aluminum stubs, which were coated with gold/palladium and examined by scanning electron microscopy (Jeol6460-LV).

2.4. Pigment analysis

Seawater samples of 0.53l were ltered through WhatmanGF/F glass ber lters. Filters were deep-frozen immediately andstored in liquid nitrogen to prevent pigment modication.Pigments were extracted with 100% methanol and sonicated(Vibra Cell, Sonic and Materials) 0 1 C. The extracts were lteredthrough GF/F lters to remove cell debris. Water was added to theextract immediately before injection to obtain an 80% methanoldilution ( Jeffrey and Wright, 1997 ). Sample solution aliquots wereautomatically injected into an HPLC system Shimadzu LC 10 AC.For pigment elution we use the method of Zapata et al. (2000) .A C8 column (symmetry 150 4.6mm 2 , 3.5 mm particle size, 100 Apore size) was used protected with a C8 (Symmetry) guardcolumn. The mixing chamber and column were thermostated at25 1 C with a CTO-10 AC (Shimadzu) column oven. Mobile phaseswere: (A) methanol:acetonitrile:aqueous pyridine solution

(0.25M, pH adjusted to 5.0 with acetic acid) (50:25:25, v/v/v),and (B) methanol:acetonitrile:acetone (20:60:20, v/v/v). A linear

gradient from 0% to 40% B was pumped for 22 min, followed by anincrease to 95% at minute 28 and isocratic hold at 95% B forfurther 10 min. Peak detection was carried out using a diode arraydetector (SPD-M10Avp). Chlorophylls were also detected byuorescence: Excitation at 440 and emission at 650 nm (spec-tral-uorometer FR-10Axl). Pigments were identied by theirretention time and absorption spectra obtained from the on-line

diode array detector (350750nm). High-purity standards wereprovided by VKI (The International Agency for 14 C Determination,Denmark) or isolated from cultures of Emiliania huxleyi (cloneCCMP370) and Alexandrium tamarense (clone MDQ1096). TheHPLC system was calibrated using genuine standards from VKI orthe extinction coefcients reported by Jeffrey and Wright (1997)for prepared standards. Novel pigments were quantied consider-ing the extinction coefcients for the most similar documentedchromophore (e.g. DV chl a was measured as chl a , and chl c 3 andchl c 2 -MGDG esters were measured as chl c 2 equivalents).

2.5. Data processing

The contribution of different phytoplankton groups to the totalchl a , at each sampled station were calculated using CHEMTAX, amatrix factorization program running under MATLAB ( Mackeyet al., 1996 ). All diagnostic pigments detected, as well as thoseambiguous markers indicative of only a few groups, were includedin the program matrix. Chl c 1 , chl c 2 , chl c 3 and chl c 2 -MGDG-esters were also included, as the HPLC method used is very usefulto separate these pigments ( Zapata et al., 2000 ). The original datahas been split into ve geographic groups. The distinction of vegroups accommodated the natural diversity of phytoplanktonassemblages between the different ecosystems previously distin-guished for this area ( Carreto et al., 2003 ). These categories weredelineated on the basis of: (1) identication of pigment assem-blages and their relation to the hydrography and (2) themicroscopic analysis of representative samples from each pigment

assemblage. In addition, using the photo-collector pigments(PCPs)/total chl a and the photo-protector pigments (PPPs)/total

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Fig. 1. AVHRR sea surface temperature ( 1 C) mapped image from 27 November 2001, showing location of occupied stations and distribution of surface salinity (PSU).



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chl a ratios as indicator of phytoplankton photo-adaptation, thepigment data for the more oceanic ecosystem has been split bysample depth. For each group, a matrix using initial pigment: chl aratios derived from Carreto et al. (2003 and references therein) ,supplemented by recent surveys ( Van Lenning et al., 2003 ; Latasaet al., 2004 ; Zapata et al., 2004 ) was constructed, since data onpigment ratios and their changes with light climate for the

dominant phytoplankton species of the studied area were notavailable. It should be emphasized that in some cases a pigmentgroup may be composed of several phytoplankton taxonomicclasses.

3. Results and discussion

3.1. Environmental conditions

The satellite-derived SST data on 29 November ( Fig. 1) showedthat the Brazil Current separated from the shelf-break at 36 1 Swhere it encountered a narrow lament of cooler waters owingalong the upper shelf-break between 39 1 S and the latitude of

Brazil Current separation. This transition zone of intermediatetemperatures seems to be lled with patches and laments of Subantarctic and Subtropical waters. On the shelf the occurrenceof well-dened thermal fronts was also evident: (1) a thermalfront between the R o de la Plata outow and coastal waters, (2) athermal front separating Coastal from Subantarctic waters and (3)a thermal front between the shelf waters and the SubtropicalBrazil Current waters. In coincidence with SST data, surfacesalinity distribution ( Fig. 1) indicated that the R o de la Plataoutow forms a low-salinity tongue which extends northwardalong the Uruguayan coast, with salinity between 28 and 30 psu(Sts. 26, 25). In the head of the estuary (Sts. 20, 19, 18), and in themore shallow outer estuarine stations (Sts. 17, 16) ( Fig. 1), thevertical distribution of temperature ( Fig. 2a) and salinity ( Fig. 2b)

showed no stratication. Under calm winds freshwater overlyingsea water denes a salt wedge with a typical scale of 100km(Guerrero et al., 1997 ). However, during the cruise the prevailingstrong onshore winds (mainly SE and ESE) eroded the quasi-permanent halocline by wind-induced vertical mixing. Theinuence of the R o de la Plata extends offshore up to the surfacesalinity front ( Mianzan et al., 2001 ) capping the coastal waters(3033.5psu) produced by the mixing of the estuarine and theSubantarctic Shelf waters ( Carreto et al., 2003 ).

In the central section, the vertical light distribution showed(Fig. 2c) that in the head of the estuary the incident photo-synthetically active radiation (PAR) was reduced to less than 10%in the rst centimetres depth and that the ratio of mixing depth toeuphotic depth ( Z m : Z eu ) was about 3. In contrast, the highesttransmission values were observed at the oceanic region of theoligotrophic Brazil Current waters, where the euphotic zonereached up to 80 m depth. In this region the Z m : Z eu ratios wereabout 0.2. Intermediate values were observed in the coastal andshelf regions.

3.2. Chlorophyll a distribution

The surface chl a distribution based on the SeaWiFS imagery(Fig. 3A) and the obtained from sampled in situ data ( Fig. 3B)showed the same distributional pattern. In both cases data revealsa band along the edge of the shelf extended northward up to thelatitude of the Brazil Current separation, where high surfacechlorophyll concentrations (34 mg l 1 ) were found ( Fig. 3A). This

chlorophyll band has been regularly observed from satellites onthe shelf-break off Argentina during late spring and summer

(Podesta, 1997 ; Brandini et al., 2000 ; Carreto et al., 2003 ; Romeroet al., 2006 ). The chl a concentrations in the water column werealso high, especially at St. 10 where a chl a maximum (5.81 mg l 1 )near the base of the euphotic zone was found ( Fig. 4b).

In contrast, at the edge of the shelf area dominated by thewarm, nutrient-poor waters of the Brazil Current the lowestsurface chl a concentration (0.10.2 mg l 1 ) were found ( Fig. 4a).Another characteristic of this area is the presence of a deep chl amaximum situated near the base of the euphotic layer at St. 24.Another noteworthy area was the R o de la Plata estuary, where anextremely high Sea WiFS chlorophyll signal was found ( Fig. 3A).However, in situ surface chl a measured was very much lower(o 1.4 mg l 1 ) than that obtained by the Sea WiFS ( 20.0 mg l 1 )and did not vary signicantly with depth ( Fig. 4b). As has beenpreviously reported, the application of the classical algorithms(OReilly et al., 2000 ) over-estimate the chl a concentration in theRo de la Plata estuary, mainly at stations near the turbidity front(Armstrong et al., 2004 ; Carreto et al., 2003 ; Huret et al., 2005 ). Inthe outer estuary area in situ surface chl a was much higher (up to15.5 mg l 1 ) (Fig. 4b). However, due to constrains imposed by thelight limitation, the total chl a , integrated over the euphotic layerof the outer estuary area were much lower (up to 46.3 mg m 2 ,St. 17) to that recorded at the shelf-break front (up to 81.2 mg m 2 ,St. 10). The surface chl a concentrations over the entire shelf werelower than that observed in the mentioned frontal areas. However,

in some shelf stations high chl a concentrations near the base of the euphotic zone was found ( Fig. 4).

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Fig. 2. Temperature, salinity and solar radiation along the central section.(a) Temperature distribution ( 1 C). (b) Salinity distribution (PSU). (c) Photosyntheticactive radiation (PAR) penetration distribution as percentage of the incidentradiation.



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3.3. Phytoplankton communities, photo-adaptation and photo-acclimation

Based on previous results ( Carreto et al., 2003, 2007 andreferences therein), the obtained pigment patterns ( Table 1 andFig. 5) and the species composition of the surface phytoplanktoncommunities ( Table 2 ) ve phytoplankton communities (seeFig. 3B) closely related with the hydrography of the area weredistinguished: (a) inner estuary, (b) outer estuary, (c) coastal,(d) Subantarctic Shelf-break front, and (e) Brazil Current. Thechromatograms obtained ( Fig. 5) from six selected stationsshowed the characteristic pigment patterns that prevailed in theconsidered phytoplankton assemblages.

In addition to water masses and nutrient conditions, factorscontrolling the integrated irradiance exposure (transparency of thewater and vertical mixing) should be considered to breaking thedata set up ( Mackey et al., 1998 ; Wright and van den Enden, 2000 ).Trends in pigment ratios due to phytoplankton photo-adaptationand phytoplankton photo-acclimation (used here to describereversible light-induced alterations in the physiological character-istics of a population) were examined using the water columnvariability in the PCP and in the PPPs/chl a ratios ( Fig. 6). As in most

eld studies in estuarine and coastal zones ( Fig. 6a), the PCP/chl aratio observed in the water column showed small variability with

irradiance, probably because the phytoplankton in the mixed layernever get the chance to acclimate their pigmentation to a constantirradiance over a generation time and because they experienced asimilar average light exposure. However, the synthesis of thephoto-protective carotenoids diadinoxanthin (Diadino) and diatox-anthin (Diato) is a more rapid process (hours) and the variability inthe PPP/chl a ratio with the measured sample depth or irradianceappears to be evident ( Fig. 6a). On the other hand, in stratiedwaters photo-acclimation of phytoplankton populations is usuallyreected by a change in pigment ratios. Dramatic changes in thePCP/Total chl a (chl a+Dv chl a) ratio was observed in the watercolumn where subtropical waters dominated (Sts. 8 and 22),ranging from 0.4 in the surface layer to 1.1 near the base of theeuphotic layer ( Fig. 6b). Inversely, the PPP/Tchl a ratio decreasedfrom 0.6 in the surface to 0.1 at the base of the euphotic layer(Fig. 6b). The sharp transition of these ratios at deep near the baseof the euphotic zone may be due in part to photo-acclimationprocesses, but mainly to changing differently photo-adaptedpopulations ( Goericke and Repeta, 1993 ; Moore et al., 1995 ). Theseresults indicate that the pigment data of this oceanic ecosystemshould be split by sample depth. Since the small number of samples adversely affected the accuracy of the CHEMTAX calcula-

tion only two pigment matrix (surface to 5% PAR and lower than 5%PAR) were considered in our calculation.

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Fig. 3. Surface chlorophyll a distribution ( mg l 1 ). (A) SeaWiFS level 3 standardmapped image for chlorophyll, 1724 November composite. (B) Data derived fromHPLC measurements of pigment extracts from surface samples. The ve identiedphytoplankton communities are indicated as follows: (a) inner estuary; (b) outerestuary; (c) coastal; (d) Subantarctic Shelf-break front and (e) Brazil Current.

Fig. 4. Vertical chlorophyll a concentration ( mg l 1 ) from HPLC measurements of pigment extracts along the sections. (a) Northern section. (b) Central section. (c)Southern section.



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Changes in pigment ratios (PCP/Total chl a, and PPP/Tchl a)with depth showed a more complex scenario at the shelf-breakfront. Although in most of the studied stations, the PCP/total chl aratio increased moderately with depth, variability betweenstations are evident, changing from about 1.0 7 0.2 in the surfacelayer to 1.2 7 0.2 at the base of the euphotic zone ( Fig. 6c). Incontrast, the high PCP/total chl a ratio observed (1.5 7 0.1) at thesurface of station 10 do not changed with sample depth or watercolumn irradiance. Nevertheless, as can be expected by the rapidsynthesis of photoprotective carotenoids, the PPP/total chl a ratiodecreased signicantly with decreasing irradiance in all stations,ranging from about 0.4 7 0.15 at the surface layer to 0.2 7 0.15 nearthe base of the euphotic zone ( Fig. 6c). The studied stations aresituated at the northern Subantarctic Shelf-break front, near thehigh SST gradient region associated to the Brazil/MalvinasConuence where the Malvinas Current veers offshore ( Podesta,1997 ; Saraceno et al., 2005 ). Recent numerical simulationssuggest that bottom friction associated to the presence of a strongslope current, such as the Malvinas Current, creates along-shelf pressure gradients and lead to robust upwelling at the shelf break(Romero et al., 2006 ). The heterogeneity of the frontal area andthe instability related with the alternation of upwelling andvertical stratication periods appear to be relevant processesexplaining the strong contrast observed in photo-acclimationstage of these phytoplankton populations. Another contributing

factor is the high complexity in the pigment patterns andphytoplankton composition observed in this region. Therefore,

the pigment data for this ecosystem has not been split by sampledepth and only one pigment matrix was considered in ourCHEMTAX calculation.

3.4. Pigment pattern and abundance of algal groups

3.4.1. Inner estuary communityPigments associated with green algae (Chlorophyceae and

Prasinophyceae) were relevant in the inner estuary. Although theamount of chl b was not high in this ecosystem, lutein the majorcarotenoid of chlorophyceans , was found in high proportion inthis assemblage ( Figs. 5a and 7). Prasinoxanthin (Pras), anunequivocal marker of some prasinophyceans (Mamiellales,Peudoscoureldiales and Prasinococcales, Latasa et al., 2004 ),was only detected in trace amounts. Consequently, more of the chlb arises from chlorophyceans or prasinophyceans lacking Pras. Themicroscopic examination of these samples showed that a notidentied palmeloid chlorophycean from freshwater origin wasthe most abundant green algae, indicating that most of the chl bdetected arises from chlorophyceans.

Fucoxanthin (Fuco) and zeaxanthin (Zea) were also found inhigh proportion in the inner estuary ( Fig. 5a). Microscopic analysisindicated that several diatoms species present in low abundance(Table 2 ) were co-dominant in this phytoplankton assemblage,explaining the observed Fuco concentrations. Accordingly, it isinteresting to note that in our chromatograms, the chl c pigmentscharacteristics of diatoms (chl c 1 and chl c 2 ) and other Chromo-phyte algae are present at trace levels ( Fig. 5a). We estimated thatthese polar compounds were strongly adsorbed in the activesurface of the particulate material retained by the lters in a formnot accessible to methanol. Zeaxanthin, a pigment present inrather high proportion in cyanobacteria, was found in all samples,whereas canthaxanthin was measured only in one station (St. 20)where the colonial cyanobacteria Microcystis aeruginosa wasdetected ( Table 2 ).

In terms of chl a calculated by CHEMTAX, green algae (5658%of total chl a) were always the dominant group. Most of the chl aarises from chlorophyceans (4355%) and the contribution of prasinophyceans was small (313%). As expected from pigmentresults, diatoms were the subdominant group (1442%) beingrelatively abundant in the deeper samples of the border of theturbidity front. In a decreasing order, cyanobacteria (919%) andcryptophytes (811%) were also relevant. The contribution of other groups such as dinoagellates (08%) and haptophytes(03%) was small ( Figs. 9 and 10 ). Similar results were reported byGomez et al. (2004) in their study of phytoplankton samples takenin the turbidity front during the same spring cruise.

3.4.2. Outer estuary communityFucoxanthin and peridinin (Perid) were the most abundant

carotenoids in the overall area of the outer estuary ( Fig. 7), beingparticularly high (3.11 and 4.56 mg l 1 , respectively) at the surfacemaximum chl a concentration (15.5 mg l 1 , St. 17) observed in thisarea ( Fig. 5b). Chl c 1 , chl c 2 and Mg 3,8-divinyl pheoporphyrin(MgDVP) were the major chl c pigments whereas chl c 3 waspresent only in minor amounts. In particular, chl c 2 and chl c 1attained their highest concentrations of the study area ( Figs. 7and 8 ). Microscopic analysis ( Table 2 ) conrmed that severaldiatom species ( Thalassiosira spp., Thalassionema nitzschioides ,Rhizosolenia setigera and Pseudo-nitzschia spp.) were very abun-dant in this phytoplankton assemblage explaining the observedFuco dominance. In contrast, the unequivocal marker for dino-agellates, Perid, attained their highest concentration at

the surface ( Fig. 8) and was related with a bloom of thedinoagellate Prorocentrum minimum (up to 397,600cells l 1 ).

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Table 1Peak identication and abbreviation pigment name used in the text

Peakno.

Pigment Abbreviation

1 Peridininol Peridol2 Chlorophyll c 3 (PCP) Chl c 3 ,3 Monovinyl chlorophyll c 3 MV chl c 34 Chlorophyllide a Chlide5 Mg 3,8-divinyl pheoporphyrin Mg DVP6 Chlorophyll c 2 (PCP) Chl c 27 Chlorophyll c 1 (PCP) Chl c 18 Methyl-chlorophyllide a Me chlide9 Peridinin (PCP) Perid

10 19 0-Butanoyloxyfucoxanthin (PCP) But-fuco11 Fucoxanthin (PCP) Fuco12 9 0 -cis-neoxanthin Neo13 Prasinoxanthin (PCP) Pras14 4-Keto-19 0-hexanoyloxyfucoxanthin 4 k-Hexa-fuco15 Violaxanthin (PPP) Viola16 19 0-Hexanoyloxyfucoxanthin (PCP) Hexa-fuco17 Diadinoxanthin (PPP) Diadino18 Antheraxanthin Anth19 Dinoxanthin Dino20 Alloxanthin (PCP) Allo

21 Diatoxanthin (PPP) Diato22 Zeaxanthin (PPP) Zea23 Lutein (PPP) Lut24 Gyroxanthin diester Gyr25 Chlorophyll b (PCP) Chl b26 Chlorophyll c 2 monogalactosyldiacylglyceride [18:4/

14:0] ester from E. huxleyiChl c 2 MGDG[18:4/14:0]

27 Divinyl chlorophyll a DV chl a28 Chlorophyll a allomer Chl a allom29 Chlorophyll a Chl a30 Chlorophyll a epimer Chl a epi31 Chlorophyll c 2 monogalactosyldiacylglyceride [14:0/

14:0] ester from C. polylepisChl c 2 MGDG[14:0/14:0]

32 Phaeophytin a Phaeo33 b,e-Carotene ( a -carotene) a-Car34 b,b -Carotene ( b -carotene) b -Car

PPP: photo-protector pigments, PCP: photo-collector pigments.



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Other dinoagellate species were also present but in lowerconcentrations ( Table 2 ).

In coincidence with the distribution of dinoagellates, therelative concentration of the unequivocal Cryptophyceae markeralloxanthin (Allo) was high in the surface samples of the outerestuarine area, but sharply dropped with depth ( Figs. 7 and 8 ).Microscopy ( Table 2 ) showed that free-living cryptophytes(Hemiselmis sp. and Plagioselmis sp.) were abundant in outerestuary waters, explaining the observed distribution of Allo. It isinteresting to note that some cryptomonads may be found in

endosymbiotic association with other organisms ( Hackett et al.,2004 ), as for example some toxic dinoagellate species of the

genus Dinophysis (D. caudata and D. acuminata ) that also werepresent at relatively high abundance in our samples ( Table 2 ).Although red-tide concentrations of the autotrophic ciliateMyrionecta rubra has been reported ( Montoya et al., 2006 andreferences therein ), in our samples was present only in lownumbers.

Another pigment pattern can be dened in association with thedetected bloom (St. 14) of the silicoagellate Dictyocha bula (upto 14,700 cells l 1 ; Table 2 ). The pigment data showed that inaddition to Fuco and chl c 3 , 190-butanoyloxyfucoxanthin (But-

fuco) was the dominant acyl-fucoxanthin ( Fig. 7) with minorcontributions of 19 0-hexanoyloxyfucoxanthin (Hexa-fuco). This

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Fig. 5. HPLC chromatogram of pigment extracts from surface samples (0 m depth) of six selected representative stations. (a) Inner estuary (station 18). (b) Outer estuary

(station 17). (c) Coastal (station 13). (d) Subantarctic Shelf-break front (station 10). (e) Subantarctic Shelf-break front (station 5). (f) Brazil Current waters (station 21).Detection was by absorbance at 436 nm; peak identication as in Table 1.



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pigment prole is similar to that reported for the silicoagellateD. speculum (Daugbjerg and Henriksen, 2001 ), but also with thatdescribed for some pelagophytes ( Jeffrey and Wright, 1997 ).

Pigments associated with green microalgae (Chlorophyceae,Prasinophyceae and Euglenophyceae) were not relevant in theouter estuary phytoplankton community. In this assemblage,the amount of chl b with respect to chl a was low and lutein (Lut),the major carotenoid of chlorophyceans, was also found in lowproportion ( Fig. 5b). Prasinoxanthin, an unequivocal marker of some prasinophyceans ( Latasa et al., 2004 ), was only detected intrace amounts. However, the high amount of Diadino (3.4 mg l 1 ),the major carotenoid of marine euglenoids, and the Diadino/Fucoratio ( Fig. 5b) are indicative that part of the chl b arises fromeuglenophyceans. The microscopic examination showed a patchydistribution of a non-identied microplanktonic euglenophyte, inconcentration of up to 30,271 cellsl 1 at the chl a maximum(St. 17). The nanoplanktonic euglenophyte Eutreptiella sp. was alsodetected in this estuarine area (up to 10.7 10 4 cellsl 1 ) but thediagnostic pigment siphonein, which is present in some eugleno-phyte species, was not detected. On the other hand, Pyramimonassp., a prasinophycean lacking Pras, was also detected as numeri-cally abundant (13.6 10 4 cellsl 1 ). These results suggest that chl

b could be shared between euglenophyceans and prasinophyceanslacking prasinoxanthin.

Zeaxanthin, a pigment present in rather high proportion incyanobacteria, was found in small amounts in all samples.However, in the Uruguayan coast associated with the river plume,Zea was the most abundant carotenoid. Minor contributions byhaptophytes were also detected in stations 14, 15 and 16. Thepresence of a non-identied species of Prymnesiaceae ( Imantonia /Chrysochromulina ) (Table 2 ) was in coincidence with the presenceof both chl c 2 -MGDG (18:4/14:0) and chl c 2 -MGDG (14:0/14:0) intrace amounts. These haptophyte markers appear to be associatedwith the presence of chl c 3 , Fuco, Hexa-fuco and 4-keto-19 0-hexanoyloxyfucoxanthin (4 k-Hexa-fuco), as in the Pigment-type7 described by Zapata et al. (2004) .

As expected from pigment results, the relative contribution of the various groups to chl a calculated by CHEMTAX ( Figs. 9and 10 ), showed that with exception of some surface samples(St. 26), diatoms and dinoagellates were the dominant groups inthe phytoplankton community of the outer estuary. The highestspecic diatom-chl a concentration was attained at station 17(7.24 mg l 1 , 47% of total chl a). Also the highest absolute(4.81 mg l 1 ) and relative contribution (31% of total chl a) of dinoagellates to the total chl a calculated by CHEMTAX wasattained at the surface of St. 17, and was related with a bloom of

the dinoagellate Prorocentrum minimum (up to 397,600 cellsl1

).However, in some surface samples cyanobacteria was the most

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Table 2Main identied taxa of surface microplankton, nanoplankton and picoplankton rank abundance values in the ve geographic areas considered

Shelf-break front Brazil Coastal Outer estuary Inner estuary

MicroplanktonBacillariophyceae

Pseudo-nitzschia spp. 0960 a 1604800 0200Rhizosolenia setigera Brightwell 240680 48012,800 203460Thalassionema nitzschioides (Grunow) Grunow ex Hustedt 1202520 1202520 0200Thalassiosira spp. 0200 040 8401000 4004720 0400Total diatoms 0200 401840 216015,120 392011,200 01600

DinophyceaeCeratium furca (Ehrenberg) Clapare `de et Lachmann 0120 3602120Ceratium tripos (O.F. Mu ller) Nitzsch 040 40400 02160Dinophysis acuminata Clapare`de et Lachmann 601760 4802500Dinophysis caudata Saville-Kent 040 802340Prorocentrum minimum (Pavillard) Schiller 0440 40160 080 1120397,600 01800Prorocentrum scutellum Schro der 020 0180 280540 805400Total photosynthetic dinoagellates 0980 280360 13605480 6400398,490 01800

DictyochophyceaeDictyocha bula 576014730Dictyocha speculum 402360

Euglenophyceae 4030,270

Nano and picoplanktonCryptophyceae

Hemiselmis or Plagioselmis 00.687 021.8

PrymnesiophyceaeImantonia or Chrysocromulina 027.7 01.37 013.8Gephyrocapsa or Emiliania 013.0 00.687Other Coccolithophore species 09.09 076.3

PrasinophyceaePyramimonas spp. 013.6ChlorophytaCoccal cells 05910

EuglenophyceaeEutreptiella spp. 010.7BacillariophytaCentric cells ( o 5 mm) 066.5ChrysophytaOllicola spp. 01.38

Microplankton abundance in cells per liter; nano- and picoplankton abundance in cells per liter 10 4 .a 219,260cells l 1 at 20 m depth.



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important group (St. 26: 39.0% of total chl a). In decreasing order,green algae (euglenophyceans+prasinophyceans lacking Pras,

30.71%), cryptophytes (19.23.4%) and silicoagellates(14.23.8) were also relevant. The contribution of other groups

such as haptophytes (100%) and prasinophyceans containing Pras(10.20%) was small.

3.4.3. Coastal communityIn contrast to the estuarine waters, the pigment markers for

green algae (chl b, Lut), dinoagellates (Perid) and cryptophytes

(Allo) were in relatively low concentrations in the coastalecosystem ( Fig. 7). Fuco was largely the most abundant carotenoidin the whole area, being particularly high at the chl a maximumobserved at station 12 (3.81 mg l 1 ) near the base of the euphoticzone ( Fig. 8). In a decreasing order, chl c 2 , chl c 3 and chl c 1 werethe major chl c pigments of this assemblage. In the surface, chl c 3was detected in relatively low amounts but a sharp increase in thechl c 3 / chl a ratio was observed at the chl a subsurface maximum,where the highest chl c 3 concentration (St. 12, 0.35 mg l 1 ) wasfound. However, Hexa-fuco was present at very low concentra-tions ( Fig. 8), and some other haptophyte markers such as chlc 2 -MGDG (18:4/14:0) and chl c 2 -MGDG (14:0/14:0) were onlydetected at trace levels. Moreover, in this station the verticaldistribution of phytoplankton showed that diatoms prevailed at

all depths of the water column whereas haptophytes were absent.However, associated with the subsurface chl c 3 maximum, a sharpchange in the diatom species composition was observed. Incoincidence with the chl c 3 maximum, a mono-specic bloom of the diatom Pseudo-nitzschia sp. was found ( Fig. 11 ), explaining theobserved pigment pattern ( Zapata et al., 2000 . These resultsallowed us to distinguish between two pigment-types diatompopulations (Diatom I and Diatom II) within the coastalphytoplankton community.

The algal group composition obtained by CHEMTAX for thecoastal area showed an almost total dominance (59.393.5% of total chl a) of diatoms. With the exception of some samples(St. 12: 20 and 30 m, and St. 8: 30 m) the usual diatom-pigmentpattern group (chl c 1 , chl c 2 and Fuco, Diatom I), was the moreabundant and widely distributed in the coastal area. However,the CHEMTAX calculation showed total dominance (87.3%) of thepigment pattern group Diatom II (chl c 2 and chl c 3 ) within thePseudo-nitzschia spp. bloom. In a decreasing order, dinoagellates(160%), haptophytes (121.6%), cryptophytes (160%) and cya-nobacteria (110.5%) were also relevant. The contribution of greenalgae (prasinophyceans and chlorophyceans) was small.

3.4.4. Shelf-break front communityPigments associated with green algae were relevant in this

phytoplankton assemblage, particularly at the chl a maximumdetected in the central stations (Sts. 6 and 10) of this frontal area.Chromatograms showed ( Fig. 5d and e) high absolute and relativeconcentrations of chl b (chl b:chl a ratio up to 0.65) and lutein(Lut:chl a ratio up to 0.25), associated with minor amounts of neoxanthin (Neo), violaxanthin (Viola) and Zea. Prasinoxanthin,an unequivocal marker of some prasinophyceans was alsodetected in trace or minor amounts, being relevant at the deepeuphotic zone. The Lut/chl b ratio has found to be higher inchlorophytes (0.301.77) than in prasinophytes lacking Pras(00.18) ( Schlu ter et al., 2000 ; Henriksen et al., 2002 ; Latasaet al., 2004 ). The observed Lut/chl b ratios averaged 0.37 7 0.2indicating that mainly chlorophyceans contributed to the mea-sured chl b. In coincidence with pigment analysis, electronic andlight microscopic examination of these samples revealed a bloom(up to 59.1 10 6 cellsl 1 ) of a picoplanktonic ( o 3 mm) coccal cellsof a not identied chlorophycean. Therefore, most of the chl bappears to be related to picoplanktonic chlorophyceans. The high

abundance of chl b-containing organisms in the shelf-break areaduring this study is in contrast with a previous investigation

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Fig. 6. Changes in photo-collector pigments/chlorophyll a (empty symbols) andphoto-protector pigments/chlorophyll a ratios (full symbols) with PAR irradianceas percentage of the incident radiation, in some selected representative stations.(a) Coastal. (b) Brazil Current waters. (c) Subantarctic Shelf-break front. Drawnlines show general tendencies in photo-adaptive processes. No tting was done.



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Fig. 7. Surface distribution of principal accessory biomarker pigments concentration ( mg l 1 ) grouped in separate panels: (a) fucoxanthin, peridinin, chlorophyll b and19 0-hexanoyloxyfucoxanthin and (b) chlorophyll c 1 and c 3 , prasinoxanthin, alloxanthin, lutein, 19 0-butanoyloxyfucoxanthin, divinyl chlorophyll a and zeaxanthin.



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(Carreto et al., 2003 ) reporting insignicant concentrations of chlb during late spring 1999 in this area.

At the maximum chl a detected in the central stations of theshelf-break front, the concentration of chl c 3 , Fuco, Hexa-fuco andBut-fuco were high ( Fig. 5d and e) indicating that haptophytesand/or pelagophyceans could be also abundant. However, somespecies of Parmales with the same pigment signature, wereobserved in a sample from the Ross Sea ( Wright and van denEnden, 2000 ), indicating that sources other than haptophytes andpelagophytes could be related with the presence of the mentionedpigment pattern. In our samples, the additional useful haptophyte

marker pigments, chl c 2 -MGDG (18:0/14:0), chl c 2 -MGDG (14:0/14:0), monovinyl chl c 3 and 4 k-Hexa-fuco ( Zapata et al., 2004 ),

were also detected in minor or trace amounts. Using thesepigment markers, at least two haptophyte pigment types weredistinguished in the shelf-break area. Chl c 2 -MGDG (18:0/14:0)was the dominant non-polar chl c 2 at the sub-surface chl amaximum detected in the central St. 10, whereas at the borders of the front (Sts. 5, 7 and 9), in correspondence with the lowest chl avalues, both non-polar chl c 2 [chl c 2 -MGDG (18:0/14:0) and chlc 2 -MGDG (14:0/14:0)] were found in similar amounts. Thepresence of a non-identied prymnesiophycean species (possiblyChrysochromulina ) in concentrations of up to 27.7 10 4 cellsl 1

(Table 2 ), can explain the presence of both non-polar chl c 2

(Pigment type 7 of Zapata et al., 2004 ) and the relative amountsof Hexa-fuco detected at the borders of the front. In contrast, in

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Fig. 8. Vertical distribution of principal accessory biomarker pigments concentration ( mg l 1 ) along the central section. (a) Fucoxanthin, peridinin, chlorophyll b and 19 0-

hexanoyloxyfucoxanthin grouped in separate panels. (b) Chlorophyll c 1 and c 3 , prasinoxanthin, alloxanthin, lutein, 190

-butanoyloxyfucoxanthin, divinyl chlorophyll a andzeaxanthin.



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some stations (Sts. 6 and 7) at the shelf-break front thehaptophyte populations were mainly comprised by coccolitho-phorids ( E. huxleyi and/or Gephyrocapsa oceanica ), a familycharacterized by a single pigment type (Type 6) containing chlc 2 -MGDG (18:0/14:0) as unique non-polar chl c 2 (Zapata et al.,

2004 ). Similarly to that observed previously in this area ( Carretoet al., 2003 ), the Hexa-fuco biomass estimated by microscopic

coccolithophorid cell counts (assuming a cell content of 0.1 mgHexa-fuco per 10 6 cells, Stolte et al., 2000 ; Galmozzi et al., 2001 )was three orders of magnitude lower than that determined byHPLC. Moreover, no other haptophytes were microscopicallyobserved in samples containing the highest acyl-fucoxanthins

concentration (St. 10,1.31 mg l1

). The dominance of naked or scalycoccolithophorid cells ( Paasche, 2001 ) and the presence of other

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Fig. 9. Surface distribution of percentage of different phytoplankton groups contributing to the total chlorophyll a concentration, estimated by interpretation of pigmentHPLC data using CHEMTAX program, grouped in separate panels.



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haptophytes too fragile to be adequately preserved and thereforeundetected by microscopy, could explain such inconsistencybetween E. huxleyi/G. oceanica cell biomass and Hexa-fucoconcentrations. For instance, recent molecular studies in photicoceanic regions have revealed a large diversity of photosyntheticpicoeukaryotic heterokonts, including haptophyceans ( Moon-vander Staay et al., 2000 ). Our chromatograms also showed ( Fig. 5d) asignicant But-fuco/Hexa-fuco ratio, indicating that Pigmenttype 8 of haptophytes was probably present ( Zapata et al., 2004 ).However, the presence of pelagophyceans and/or other But-fuco-containing picoeukaryotes, such as the small and too fragileagellate Florenciella parvula (Dictyochophyceae; Eikrem et al.,2004 ), could not be discarded. Microscopy also showed that in onestation of this area, some non-identied, very small diatom cells( 5 mm) were present in high concentrations (up to66.5 10 4 cellsl 1 at St. 10). These results indicated that inaddition to haptophytes, the Fuco concentration was also relatedwith the abundance of these small diatoms. It is interesting tonote that signicant amounts of chlorophyllide a and lesseramounts of their methyl-ester, were found in our chromatograms(Fig. 5d) indicating that the diatom population probably was in asenescent stage. In addition to the major pigment markers, Pras,Allo, Zea and Perid were also detected in minor or trace amounts.

As expected from pigment results, the relative contribution of the various groups to the total chl a calculated by CHEMTAX,

showed that in contrast with the almost exclusive diatomdominance observed in the estuarial and coastal systems, the

shelf-break community was more complex, being principallycomprised by chlorophyceans (up to 50% of total chl a in St. 6) andhaptophytes (up to 62% in St. 5). Three categories of haptophytes(of pigment types 6, 7 and 8; Table 3 ) were considered in ourcalculations in view of the high complexity in the pigmentpatterns and phytoplankton composition observed in this region.Results showed that haptophyte pigment type 7 was abundant inall the studied stations, being dominant at the surface samples(75100% of total haptophytes). However, at the chl a sub-surfacemaximum a noticeable increase in the relative contribution of haptophyte pigment types 6 and 8 was found in some stations(Fig. 12 a and b).

The CHEMTAX calculation showed that diatoms were relativelyabundant in the deeper samples of the borders of the front (St. 5:47.7% and St. 7: 44.5%). Cryptophytes, prasinophyceans andpelagophyceans were also present in surface samples of this areabut in relatively low proportions. Furthermore, the CHEMTAXcalculation showed a marked increase in the relative contributionof cryptophytes (up to 20.4%) and prasinophyceans (up to 36.6%)at the base of the euphotic layer. The cyanobacteria ( Synechococ-cus) was only detected in minor amounts with the exception of station 7 (21.5%).

3.4.5. Brazil Current community

Zeaxanthin was the principal carotenoid in the phytoplanktoncommunity of the oligotrophic Brazil Current waters ( Fig. 5f),

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Fig. 10. Vertical distribution of percentage of different phytoplankton groups contributing to the total chlorophyll a concentration along the central section, estimated byinterpretation of pigment HPLC data using CHEMTAX program grouped in separate panels.



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indicating that cyanobacteria were the most abundant cells in thephytoplankton assemblage. Zeaxanthin is found in both Synecho-coccus and Prochlorococcus , whereas divinyl chl a is exclusive toProchlorococcus . Although in our samples Synechococcus appearsto be dominant, the relative proportion of Prochlorococcus wasimportant, particularly at the surface of the more oceanic station(St. 21) where a high contribution of DV chl a ( 37%) to the total

chl a (chl a +DV chl a) was found ( Fig. 5f). Divinyl chl b (DV chl b)could not be separated from chl b with the method employed(Zapata et al., 2000 ), and our data refer to the sum of DV chl b andchl b (chl b1+2 ). The co-elution of these pigments could be adrawback if green algae without specic marker was also presentin the assemblage and its contribution to the total chl a shouldalso be evaluated. In this phytoplankton assemblage, Lut was notdetected and consequently the chl b of green algae arises onlyfrom prasinophyceans containing prasinoxanthin. On the otherhand, DV chl b is a less specic pigment marker for Prochlor-ococcus since some clones contain both DV chl b and chl b in asimilar proportion ( Moore et al., 1995 ; Moore and Chisholm,1999 ).

Although prochlorophytes were dominant at depth in thetropical and subtropical regions of the Atlantic Ocean ( Barlowet al., 2002 ), differences among ecotypes could be important indetermine the relative distribution of these cyanobacteria in thewater column and throughout the oceans ( Moore et al., 1995 ). Therecent divergence of the Prochlorococcus lineage into a distinctclade that is better adapted for growth at higher irradiance levels

could explain the ability of this genus to dominate at severaldifferent depths in the water column ( Moore and Chisholm, 1999 ;Ting et al., 2002 ). Our results showed that in the Brazil Currentwaters during spring, the concentration of DV chl a was high atthe surface ( Fig. 7), and uniformly distributed throughout the

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Fig. 11. Vertical distribution of chlorophyll c 3 concentration ( mg l 1 ), the concen-tration of the diatom Pseudo-nitzschia sp. (cellsml 1 ) and of the others diatomscounts (cells ml 1 ) in the Station 11 of the coastal area.

Table 3Pigment: chlorophyll a ratio in the haptophyte pigment-types considered for calculations after tting by CHEMTAX program

Haptophyte pigment type Fuco Hexa-fuco But-fuco Chl c 3 Chl c 2 Chl c 2 MGDG [18:4/14:0] Chl c 2 MGDG [14:0/14:0]

Type 6 0.10 1.06 0.021 0.24 0.23 0.087 Type 7 0.36 0.72 0.062 0.09 0.11 0.034 0.023Type 8 0.48 0.45 0.22 0.31 0.10 0.049

Fig. 12. Vertical distribution of chlorophyll a (mg l 1 ) in the different haptophytepigment types (6, 7 and 8), estimated by interpretation of pigment HPLC datausing CHEMTAX program, in some selected stations of the shelf-break area:(a) Station 5 and (b) Station 1.



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euphotic zone ( Fig. 8), suggesting the existence of at less one highlight adapted ecotype that is capable to growth at great irradiancelevels and that posses a low chl b1+2 : DV chl a ratio ( Fig. 13). At thesurface of station 21 where no traces of other green pigmentmarkers was found, the chl b1+2 :DV chl a ratio was 0.22 ( Fig. 5f).However, the range of chl b1+2 :DV chl a ratios measured in thedeep euphotic zone (70150 m) of station 22 ( Fig. 13 ) was similar(2.012.99) to that of members of the more deeply branchingclades ( Ting et al., 2002 ). As suggested by Goericke and Repeta(1993) , the sharp transition of chl b1+2 :chl a ratio observed atdepth may be due in part to photoacclimation processes, butmainly to changes in differently photoadapted Prochlorococcu spopulations ( Moore et al., 1995 ). However, in this assemblage,Pras concentrations were also important, indicating that chl b1+2should be shared between prasinophytes and Prochlorococcu s.Neverthless, picoplanktonic prasinophytes and Prochlorococcuscould not be detected in the microscope. The Pras-containingprasinophytes are represented by three phylogenetic groups:Mamiellales , Pseudoscoureldiales and Prasinococcales (Guillouet al., 2004 ). These groups can be classied based in thepresence/absence of uriolide, micromonal and other pigments(Latasa et al., 2004 ). We could not detect these pigments in our

samples and therefore some members of Pseudoscoureldialeswere probably present ( Latasa et al., 2004 ).

The relative proportions of chl c 3 , Fuco, Hexa-fuco and But-fucowere also relatively high ( Fig. 5f) indicating that haptophytes and/or pelagophytes were abundant. Chl c 2 -MGDG (18:4/14:0) was theonly non-polar chl c 2 detected at minor or trace levels. Althoughinterpretation is complicated by the fact that some additionalmarkers for distinguishing between the different haptophytetypes ( Zapata et al., 2004 ) were below the limits of detection,the observed proles can be associated with the Pigment-type 6.Microscopy showed that although E. huxleyi and/or G. oceanicawere present in this assemblage, some unidentied coccolito-phorid species were the dominant haptophyte (up to760 cellsml 1 ; Table 2 ). The ratio of But-fuco to total fucoxanthins(Fig. 5f) was signicantly higher than that observed for anystudied haptophyte species ( Zapata et al., 2004 ), indicating thatpelagophyceans were probably present.

Minor amounts of Perid, the unequivocal marker for dino-agellates, were also detected in the Brazil Current assemblage(Fig. 5f). Microscopy ( Table 2 ) conrmed that several dinoagellatespecies of the genera Prorocentrum, Ceratium and Torodinium including thermophilous species such as Ceratium lanceolatum andOxytoxum scolopax were present in low abundance in thisphytoplankton assemblage, in coincidence with the observeddistribution of Perid. Microscopy also showed that several diatomspecies were found in some samples at very low numericalabundance (up to 1.5 cells ml 1 ) indicating their minimal con-tribution to the observed Fuco concentration.

The algal group composition obtained by CHEMTAX for theBrazil Current assemblage showed the dominance of the pico-planktonic cyanobacteria Synechococcus (23.654.5% of total chl a)and Prochlorococcus (10.438% of total chl a). Haptophytes werealso present as sub-dominant group, being particularly abundant(up to 41.8%) in the deep chl a maximum situated near the base of the euphotic layer ( Fig. 14 ). Prasinophyceans and pelagophyceanswere present in surface samples but in relatively low proportions(up to 6.2% and 3.7% respectively). However, at the base of theeuphotic layer the CHEMTAX calculation showed a marked

increase in the relative contribution of prasinophyceans (up to16.0%) and pelagophyceans (up to 13.3%).

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Fig. 13. Vertical distribution of chlorophyll a, divinyl chlorophyll a and totalchlorophyll b (chlorophyll b1 +divinyl chlorophyll b2 ) concentrations ( mg l 1 ) instation 22 at the Brazil Current area.

Fig. 14. Vertical distribution of chlorophyll a contribution ( mg l 1 ) of the majorphytoplankton groups, estimated by interpretation of pigment HPLC data usingCHEMTAX program, in station 22 at the Brazil Current area.



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3.5. Phytoplankton communities and environmental conditions

Our results are consistent with the view that the size structureand taxonomic composition of phytoplankton communities areprincipally regulated by the linkage between nutrient availabilityand turbulent mixing ( Margalef, 1978 ). However, this linkage isexpressed differently in the different studied ecosystems ( Cullen

et al., 2002 ). Although large diatoms occupy the high-turbulence/high-nutrient ecosystems ( Margalef, 1978 ), our study showed thatwithin the tidal-mixed and nutrient-rich turbidity front, phyto-plankton growth is light-limited and freshwater chlorophytes,diatoms and cyanophytes were the dominant groups . Theseresults are consistent with previous records for this area ( Gomezet al., 2004 ). A high-nutrient/low-turbulent regimen, as can beexpected along the river plume ( Acha et al., 2004 ; Huret et al.,2005 ; Carreto et al., 2007 ), was associated with specially adapteddinoagellate species that can form red tides. However, phyto-plankton community in the outer estuary, as in most of theprevious reports for this area ( Negri et al., 1988 ; Carreto et al.,2003 ; Carreto et al., 2007 ; Gomez et al., 2004 ), was dominated bylarge diatoms. Nevertheless, bloom-forming dinoagellate species

were also abundant, co-dominating in some cases with the diatomora. Small-scale temporal variability in the turbulence/nutrientratio, produced by changes in the wind-induced vertical mixing,can modulate the relative abundance of both life forms in thephytoplankton assemblage of this ecosystem ( Acha et al.,2008 ).According to Guerrero et al. (1997) , a total disruption of watercolumn stratication and mixing of the salt wedge occurs afterseveral hours of strong onshore winds ( 4 11ms 1 ). During themore turbulent period, mucous secretion, chain formation andhigh swimming speeds may be environmental engineeringstrategies that dinoagellates and other agellates as thosepresent in this ecosystem can use to offset physical displace-ment through mixing, advection and turbulence ( Smayda, 2002 ).

Along the shelf region, seasonal changes in the turbulence/nutrient relationship drive the typical phytoplankton successioncharacteristics of temperate ecosystems ( Carreto et al., 1995,2004 ; Lutz et al., 2006 ). After the spring bloom, the slightlycoastal stratied low-nutrient waters ( Carreto et al., 2007 )showed low chl a concentration and the dominance of diatomsType I ( Thalassiosira sp.). Their abundance in the subsurface anddeep samples of the more coastal stations was probably related totheir transport in the buoyant plume from the estuary and, then,sedimentation. However, in the offshore stations, a more ad-vanced succession stage forms of diatoms (Type II) speciallyadapted to minimize sedimentation ( Pseudo-nitzschia sp. andRhizosolenia setigera ) were the dominant ora, suggesting theirlocal origin.

Typical phytoplankton succession can also provide a basis forunderstand large-scale horizontal distribution of phytoplanktoncommunities. It is well known that growth of phytoplankton inthe quasi permanent low-turbulence/low nutrient ecosystems islargely supported by regeneration of nutrients and that nano- andpicoplankton are the main components in these communities(Cullen et al., 2002 ). Along the offshore trajectory from theeutrophic estuarine waters ( Carreto et al., 2007 ) to oligotrophicsubtropical waters ( Brandini et al, 2000 ) our results showed anotably gradient in abundance, size structure and taxonomiccomposition of phytoplankton communities from high micro-plankton biomass to low picoplankton biomass. However, in spitethat specic growth rates of larger phytoplankton are relativelylow, some forms ( Ceratium spp.) can persist through specialstrategies, such as vertical migration ( Margalef, 1978 ; Smayda,2002 ).

At the central and southern sections the deepening of thestratied layer was interrupted at the shelf-break-front by a

moderate upwelling of subantarctic waters that forms a two-layerwater column with a well-developed, relatively shallow thermo-cline. Fast-growing more r-selected small agellates (haptophytesand chlorophytes) were abundant in this environment, explainingthe characteristic high surface chlorophyll concentration bandobserved from satellites along the edge of the shelf, during latespring ( Podesta, 1997 ; Carreto et al., 2003 ; Huret et al., 2005 ;

Romero et al., 2006 ). The occurrence of patches of water withspectral signatures identical to those of blooms of the coccolitho-phore E. huxleyi have been regularly observed on the shelf-breakoff Argentina during late spring and summer ( Brown and Podesta ,1997 ). In this area the most remarkable hydrographic featureassociated with previously reported E. huxleyi bloom was theexistence of a well-developed shallow pycnocline separatingsurface water from deep nutrient-rich Subantarctic waters(Gayoso and Podesta , 1996 ; Carreto et al., 2003 ). Recently, theubiquitous coccolithophorid species G. oceanica was also observedin high abundance (up to 3 10 5 cellsl 1 ) at the edge of the shelf-break ( Negri et al., 2003 ). On the other hand, this is the rst timethat high chl b concentrations associated to a bloom of apicoplanktonic ( o 3 mm) coccal chlorophycean cells was reported

for this region. Notably, picoplankton cells (o

2 mm) weredominant in the Antarctic Circumpolar Current and Chlorellalike organisms dominated the autotrophic pico- and nanoplank-ton ( Peeken, 1997 ; Detmer and Bathmann, 1997 ).

Preliminary studies showed that the high surface chlorophyllconcentration band observed along the shelf-break front duringOctober 2005, was produced by a typical temperate diatom springbloom. The predominance of small agellates in the shelf-breakarea during this study suggests that the observed scenariocorresponded to a more advanced phytoplankton succession stage(Margalef, 1978 ). These results are in coincident with recentstudies using satellite-derived data ( Signorini et al., 2006 ).

In the warm, highly stratied nutrient-poor oceanic waters of the Brazil Current, picoplanktonic cyanobacteria ( Synechococcusand Prochlorococcus ) were the dominant groups, being moreabundant in the deep chl a maximum situated near the base of theeuphotic layer. As was observed in other subtropical waters(Bouman et al., 2006 ), in this ecosystem, the strong straticationisolate deep illuminated waters from the surface, producingselection of genetic and physiological traits of marine microbialcommunities. In particular, the high light plasticity of the genusProchlorococcus has been explained by the discovery of geneticallyand physiologically distinct populations, commonly referred to ashigh light (HL)- and low light (LL)-adapted ecotypes ( Goericke andRepeta,1993 ; Moore et al.,1995 ; Bouman et al., 2006 ). Our resultsindicate the existence of at least one HL-adapted ecotype that iscapable to growth at great irradiance levels, posses a low chlb1+2 :DV chl a ratio and contains relatively high amounts of UVphoto-protective substances, mycosporine-like amino acids(Carreto et al., 2005 ). Recently, it has shown that the HL strainhas a photolyase gene, which serves to repair ultravioletdamage, and which is absent in the LL-adapted strain ( Rocapet al., 2003 ).

The observed variations in community structure along thestudied transects highlighted the adaptability of the phytoplank-ton to changing environmental conditions. In turn, the highvariability in the size structure and pigment composition of thecommunities strongly inuences the pattern of absorption inphytoplankton cells ( Yentsch and Phinney, 1989 ; Barlow et al.,2002 ; Bricaud, 2004 ; Sathyendranath et al., 2004 ). A morefrequent sampling strategy in space and time combined withanalysis of satellite images should be undertaken to improve ourunderstanding of phytoplankton dynamics in this region, and

therefore to improve the synoptic census of phytoplankton byocean-colour remote sensing.

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Acknowledgments

We are grateful to Dr. M.D. Mackey for providing the computerprogram CHEMTAX. We wish also to thank Dr. DomingoGagliardini for NOAA images processing and to SeaWiFS ProjectNASA Goddard Space Flight Centre and ORBIMAGE for chl aimages. This study was partially supported by Antorchas Founda-

tion (Grant 13900-13) and CONICET PIP 5009. This is contributionno. 1443 of INIDEP.

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Algal Pigment Patterns and Phytoplankton Assemblages in Different Water

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