Growth rates and pigment patterns of haptophytes isolated from estuarine waters

Journal of Sea Research 62 (2009) 286–294

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Journal of Sea Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s e a r e s

Growth rates and pigment patterns of haptophytes isolated from estuarine waters Sergio Seoane a,⁎, Manuel Zapata b, Emma Orive a a b

Department of Plant Biology and Ecology, University of the Basque Country, P.O. 644, 48080 Bilbao, Spain Instituto de Investigaciones Marinas, CSIC, c/ Eduardo Cabello 6, 36208 Vigo, Spain

a r t i c l e

i n f o

Article history: Received 14 October 2008 Received in revised form 24 June 2009 Accepted 16 July 2009 Available online 29 August 2009 Keywords: Carotenoids Chlorophylls Estuaries Growth Rates Haptophytes

a b s t r a c t Eleven haptophyte species were isolated over a two year period (2001–2002) from the Nervion River estuary (Biscay Bay) and batch cultured under non-axenic, isothermal (18 °C), isohaline (30 psu) conditions at three different light levels (60, 110 and 350 µmol photons m− 2 s− 1). The variation pattern of growth rate with light intensity was different among species. In all cases growth rates were maxima at 350 µmol photons m− 2 s− 1 and ranged from 0.93 divisions d− 1 in Chrysochromulina throndsenii, the slower growing species at the highest light studied, to 2.23 divisions d− 1 in Pleurochrysis roscoffensis, the faster one at the three light intensities tested. Differences in growth rate were less acute between cultures growing at 60 and 110 µmol photons m− 2 s− 1 but large species-specific differences were observed between 110 and 350 µmol photons m− 2 s− 1. Based on the occurrence of chl c-type pigments and fucoxanthin and its derivatives, the isolated species belonged to six of the eight pigment types known in Haptophyta. Most haptophytes isolated could be unequivocally distinguished from other algal groups based on pigment composition. Chlorophyll c- and fucoxanthin-type pigment ratios normalized to chl a showed a decrease with the increase in light intensity and growth rate. The observed decrease in pigment ratios were more marked for the fucoxanthin pool than for the chlorophyll c-pool. The intensity of the changes were highly species-specific reflecting both differences in the pigment composition of the different components of the light harvesting protein complexes as well as the balance between them and the reaction centre complexes. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The relative importance of haptophyte algae in the marine environment has been underestimated due to the difficulty to identify most of their species with the light microscope, the usual technique for counting phytoplankton cells. Together with other small cells, haptophyte algae have been included generally in the group of small flagellates. The use of HPLC to analyze the pigment composition of the phytoplankton assemblages has revealed, based on the presence of 19′hexanoyloxyfucoxanthin (Hex-fuco), that these algae are of paramount importance in both coastal waters (Gieskes and Kraay, 1986; Carreto et al., 2003; Rodríguez et al., 2003; Llewellyn et al., 2005; Seoane et al., 2005) and open-ocean (Gieskes and Kraay, 1989; Moon-van der Staay et al., 2000; Gibb et al., 2001; Obayashi et al., 2001; Barlow et al., 2002; Qian et al., 2003). The use of improved HPLC methods (revised by Jeffrey et al., 1999; Garrido and Zapata, 2006) and the occurrence of novel pigments isolated from haptophyte species (Garrido and Zapata, 1998; Egeland et al., 2000; Garrido et al., 2000; Zapata et al., 2001) have allowed a deep insight into pigment diversity within Haptophyta. From the 4 pigment types proposed by Jeffrey and Wright (1994) we have passed to distinguish 8 pigment types based on the distribution of 9 chl c-type pigments and 5 ⁎ Corresponding author. Tel.: +34 946012694; fax: +34 946013500. E-mail address: [email protected] (S. Seoane). 1385-1101/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.seares.2009.07.008

fucoxanthin derivatives (Zapata et al., 2004). However, haptophytes belonging to pigment types 1 to 5 lack Hex-fuco and consequently this pigment cannot be used as the unique marker to estimate haptophyte abundance. On the contrary, Hex-fuco is present in dinoflagellates having haptophyte-like chloroplasts obtained by tertiary endosymbiosis (Tengs et al., 2000) and their presence in natural samples will lead to an overestimation of haptophytes abundance when using Hexfuco as a proxy of haptophyte relative importance. In addition to the relative importance of the haptophytes in mixed phytoplankton assemblages, several species of this group are known for their ability to form blooms in coastal as well as in open marine waters (Dahl et al., 1998; Edvardsen and Paasche, 1998; Moestrup and Thomsen, 2003; Seoane et al., 2005). Availability of nutrients and less grazing pressure are basic conditions for the development of a bloom of flagellated species capable of migrate to surface waters to intercept enough light. Estuaries are very dynamic systems and favourable conditions can change before a particular species reaches high densities unless it can grow at high rates. In the Nervion River estuary (Biscay Bay), the relative importance of haptophytes is generally moderately low, except during blooms, which have been found regularly in the case of Isochrysis galbana (Seoane et al., 2005), or less frequently in Phaeocystis (Orive, 1989) and Chrysochromulina lanceolata (Seoane et al., 2005). It was the aim of this study to know: (i) the growth capability of selected haptophytes isolated from the Nervion River estuary under no predation or nutrient limitation, and with the

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light intensity, temperature and salinity typical of the outer and middle estuary in later spring and summer (periods of active phytoplankton growth in the estuary), and (ii) the diversity of pigment patterns of these species and the changes in pigment ratios associated with variation in growth light intensity. We find these two aspects relevant to know to what extent these haptophytes are potentially blooming species and what kind of pigments should be used to identify them in natural samples when using a chemotaxonomic approach based on pigment types to characterize plankton assemblages.

2. Materials and methods 2.1. Algal cultures The eleven haptophyte species were isolated by pippeting or serial dilution from samples taken at four sites located in the outer edge (Abra of Bilbao) of the Nervion River estuary, a 17 km long and 3.8 km maximum width estuary draining to the Bay of Biscay (Fig. 1 and Table 1). Unialgal, non-axenic cultures were grown at a temperature of 18 ± 2 °C and a salinity of 30 psu. Filter-sterilized (0.22 µm Millipore) natural water from the own estuary enriched with f/2 medium (Guillard and Ryther, 1962) and provided with 10− 8 M Na2SeO3 was used as culture medium. Light was supplied by cool white fluorescent lamps and measured by a Li-Cor model UWQ4667 sensor. Stock cultures were grown in borosilicate tubes containing 10 mL culture medium, at 30 psu, 18±1 °C and a light intensity of 60 µmol photons m− 2 s− 1 under a light: dark cycle (12 h: 12 h). Cultures were acclimated to the three light intensities by following growth curves until the end of the exponential phase, which extended for at least 5 days. Flagellated cells of Phaeocystis globosa and flagellated non-calcifying cells of Emiliania huxleyi were used in the growth experiments.

287

Table 1 Haptophyte species isolated from the Nervion River Estuary. Taxa

Date

Station

COCOLITHOPHYCEAE Chrysochromulina acantha C. cf. cymbium C. pringsheimii C. simplex C. throndsenii Emiliania. huxleyi Isochrysis galbana Imantonia rotunda Phaeocystis. globosa Pleurochrysis roscoffensis

June 2002 September 2003 August 2003 June 2003 April 2002 June 2002 April 2002 April 2002 April 2003 July 2002

2 3 1 1 1 1 2 1 1 1

PAVLOVOPHYCEAE Pavlova gyrans

August 2003

0

2.2. Light experiments and data processing Triplicate samples were grown under light intensities of 60, 110 and 350 µmol photons m− 2 s− 1 in borosilicate tubes containing 10 mL culture medium. Growth rates were measured directly in the culture tubes by reading the in vivo chlorophyll a fluorescence (Graneli and Moreira, 1990) with a Turner Designs 10-100R fluorometer. Fluorescence data were taken daily at the same time. Plots of the ln of fluorescence against incubation time (days) were used to calculate the maximum growth rate (µmax), which was expressed on a ln basis as divisions d− 1 (µmax/ln 2). The growth rate at different light intensities was analyzed by two-way ANOVA using the SPSS 13.0 statistical package for Windows. 2.3. Pigment analysis Before being filtered, cultures were examined by light microscopy to check for cell health and absence of contaminant algae. Volumes of

Fig. 1. Location of sampling sites in the Nervion River estuary.

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10 mL of exponentially growing unialgal cultures were vacuum filtered onto 25 mm Whatman GF/F glass fibre filters, which were maintained frozen until extraction and analysis. Frozen filters were extracted under low light with 1 mL 90% acetone, grinded with a glass stick and ultrasonicated for 5 min. The resulting slurry was removed with a syringe filter and the filtrate was maintained refrigerated until analysis. Just before injection, 0.5 mL of extracts was diluted with 0.2 mL of distilled water to avoid peak distortion (Zapata and Garrido, 1991). Pigment separation was performed using the HPLC method of Zapata et al. (2000), with a reformulated mobile phase A using a Waters (Waters Corporation, Milford, MA) Alliance HPLC System consisting of a 2695 separations module, a Waters 996 diode-array detector (1.2 nm optical resolution) interfaced with a Waters 474 scanning fluorescence detector by means of a Sat/In analog interface. The column was a C8 monomeric Waters Symmetry (150 × 4.6 mm, 3.5 μm particle-size, 100 Å pore-size). Eluent A was methanol: acetonitrile: 0.025 M aqueous pyridine (50:25:25 v/v/v); pyridine concentration was reduced one-tenth respect to the initial formulation. Eluent B was methanol: acetonitrile: acetone (20:60:20 v/v/v). Elution gradient was as follow: (time: %B) t0: 0%, t22: 40%, t28: 95%, t37: 95%, t40: 0%. Flow rate 1.0 mL·min− 1 and column temperature was 25 °C. Solvents were HPLC grade (Romil-SpS™), pyridine was reagent grade (Merck, Darmstadt, Germany). Pigments were identified by their retention times and absorbance spectra. Retention times were compared with those of pure standards, which were obtained from microalgal cultures as reported by Zapata et al. (2000). HPLC calibration by external standard was performed using chlorophyll and carotenoids standards isolated from microalgal cultures (Zapata et al., 2000). The molar extinction coefficients obtained from Jeffrey (1997) were used for pigment quantification. For chl c-like pigments whose molar extinction coefficients are not available (i.e. chl c2-like from Pavlova gyrans and MV chl c3) the molar extinction coefficient of chl c2 was used. The chls c2-MGDG was quantified by using the molar extinction coefficient of chl c2 chromophore. Following Jeffrey (1997) for fucoxanthin related compounds (i.e. acyloxy and 4-keto derivates), the molar extinction coefficient for fuco was used. 2.4. Abbreviations ββ-car: β,β-carotene; β,ε-car: β,ε-carotene; 4 k-Hex-fuco: 4-keto19′-hexanoyloxyfucoxanthin; But-fuco: 19′-butanoyloxyfucoxanthin; chl a: chlorophyll a; chl c1: chlorophyll c1; np-chl c2-Cp: chl c2 monogalactosyldiacylglyceride ester from Chrysochromulina polylepis; np-chl c2-Eh: chl c2 monogalactosyldiacylglyceride ester from Emiliania huxleyi; chl c2: chlorophyll c2; np-chl c2-Ch: chl c2 monogalactosyldiacylglyceride ester from Chrysochromulina hirta; chl c2Pg: chlorophyll c2-like from Pavlova gyrans; chl c3: chlorophyll c3; Diadino: diadinoxanthin; Diato: diatoxanthin; Fuco: fucoxanthin; Hex-fuco: 19′-hexanoyloxyfucoxanthin; LHCs: light harvesting complexes; MgDVP: Mg-2,4-divinyl phaeoporphyin a5 monomethyl ester; MV chl c3: monovinyl chl c3; Unk min 28: gyroxanthin-like pigment eluting at min 28; Unk min 29: gyroxanthin-like pigment eluting at minute 29; Unk 440 nm: unknown carotenoid eluting at minute 29; Viola: Violaxanthin; Zea: Zeaxanthin. 3. Results 3.1. Effect of light intensity on growth rates Growth rates increased significantly with increasing light intensity (ANOVA, F20, 66 = 18.957, P < 0.001) for all species, showing always values greater than 0.5 divisions d− 1. At 60 μmol photons m− 2 s− 1, the minimum growth rate (0.54 divisions d− 1) was exhibited by Pavlova gyrans and the maximum (1.20 divisions d− 1) by Pleurochrysis roscoffensis. At 350 μmol photons m− 2 s− 1, the highest growth

rate (2.23 divisions d− 1) was also that of P. roscoffensis while the lower one (0.94 divisions d− 1) was observed in Chrysochromulina throndsenii. The growth rate arrange among the 11 species studied depended on the light intensities. Pavlova gyrans showed the slowest division rates at 60 and 110 μmol photons m− 2 s− 1 but it reached the fourth position at 350 μmol photons m− 2 s− 1. Similar trends were observed in C. cymbium, P. globosa and E. huxleyi. On the other hand, C. simplex and I. galbana ranked better at 60 and 110 μmol photons m− 2 s− 1 but they were comparatively less efficient at 350 μmol photons m− 2 s− 1. Pleurochrysis roscoffensis and Imantonia rotunda were the first and third faster species irrespective of differences in growth light (Fig. 2). Considering the growth rate pattern, four groups were defined. Two of them showed the same percentage of increase in growth rate between two consecutive light intensities, but whereas the first one, including Chrysochromulina simplex, Imantonia rotunda and Isochrysis galbana experienced an increase of 10–25%, the growth rate change of Chrysochromulina cf. cymbium and Phaeocystis globosa was of the order of 40–50%. The third group (Chrysochromulina pringsheimii, Emiliania huxleyi, P. gyrans and P. roscoffensis) showed a small increase in growth rates (2–15%) between 60 and 110 μmol photons m− 2 s− 1 and a higher increase (50–150%) between 110 and 350 μmol photons m− 2 s− 1. The last group included Chrysochromulina acantha and C. throndsenii which showed smaller growth rate changes between 110 and 350 μmol photons m− 2 s− 1 than between 60 and 110 μmol photons m− 2 s− 1 (Fig. 2). 3.2. Pigment composition and pigment types 3.2.1. Pigment composition Among chlorophylls, all species analyzed contained MgDVP and chl c2 (Table 2). Most species, except I. galbana, P. gyrans and P. roscoffensis, contained chl c3. The np-chl c2-Eh was present in almost all species, except P. gyrans and P. roscoffensis. Other chlorophylls detected were: np-chl c2-Cp in C. acantha, C. cf. cymbium, C. simplex and C. throndsenii; MV chl c3 in E. huxleyi and I. rotunda; and chl c1 in I. galbana, P. gyrans, P. roscoffensis and C. simplex (as traces). Two chlorophylls were found in only one species: np-chl c2-Ch in C. pringsheimii, and chl c2-Pg in P. gyrans. All species shared the carotenoids Fuco, Diadino and ββ-car (Table 2). The fucoxanthin derivatives But-fuco, 4 k-Hex-fuco and Hex-fuco appeared in Chrysochromulina spp., E. huxleyi, I. rotunda and P. globosa. Three unknown carotenoids were detected. Two of them shared the same absorption spectrum (λmax = 444.5 nm) eluting at minute 28 in E. huxleyi and I. galbana or at minute 29 in Chrysochromulina spp., Imantonia rotunda and P. globosa. The third unknown carotenoid (λmax at 440.9 nm) eluting at minute 29 was detected in I. galbana, P. gyrans and P. roscoffensis. 3.2.2. Pigment types Haptophyte species analyzed belong to six pigment types of the eight types defined in Haptophyta according to specific combination of chls c and fucoxanthin-type carotenoids (Zapata et al., 2004). Representative chromatograms are shown in Fig. 3. The coccolithophorid Pleurochrysis roscoffensis belongs to pigment type 1, the simplest pigment composition, with chl c1 and c2, Fuco, Diadino, Diato and an unknown vaucheriaxanthin (Vauch) ester-like pigment (λmax = 440.9 nm) eluting at min 29. The pavlovophycean Pavlova gyrans belongs to pigment type 2 having chl c2-Pg, together chl c1 and chl c2, and Fuco, Diadino, Diato and the unknown Viola-like pigment. Isochrysis galbana belongs to pigment type 3 with chl c1 and c2, and np-chl c2, Fuco, Diadino, Diato, the unknown carotenoids eluting at min 28 (λmax = 444.5 nm) and the unknown eluting at min 29 (λmax at 440.9 nm). Emiliania huxleyi is a member of pigment type 6 with chl c3, its monovinyl (MV) derivative (MV chl c3) (Garrido et al., 1995; Garrido and Zapata, 1998), chl c2 and np-chl c2-Eh; the fucoxanthin pool, included Fuco, 4 k-Hex-fuco, Hex-

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289

Fig. 2. Specific growth rates as a function of light intensity. Bars represent average values and error bars represent 1 SE (n = 3).

fuco and low concentrations of But-fuco; in addition to Diadino and Diato, the unknown pigment (λmax = 444.5 nm) eluting at min 28 (Unk28/chl a = 0.037–0.050). The five Chrysochromulina species studied belong to pigment type 7, four of them contain np-chl c2-Cp, first detected in C. polylepis (see Zapata et al., 2001): C. acantha, C. cf. cymbium, C. simplex and C. throndsenii, while C. pringsheimii lacked np-chl c2-Cp but it contained np-chl c2-Eh as well as a non-polar chl c2, previously detected in C. hirta (np-chl c2-Ch, see Zapata et al., 2001),

eluting at min 27.8 (np-chl c2-Ch/chl a = 0.03). Imantonia rotunda belongs to type 8. The dominant pigment in the fucoxanthin pool was variable within Chrysochromulina species but it was unaffected by the growth light. Hex-fuco was the major carotenoid in C. acantha, C. pringsheimii and C. throndsenii, whereas Fuco was the major carotenoid in Chrysochromulina cf. cymbium and C. simplex. It was noteworthy the absence of Hex-fuco in Chrysochromulina cf. cymbium (at the 3 light

Table 2 Chlorophylls and carotenoids to chl a ratios (mol: mol) of the haptophytes studied growing at different light intensities. Light intensity COCCOLITHOPHYCEAE Chrysochromulina 60 acantha 110 350 C. cf cymbium 60 110 350 C. pringsheimii 60 110 350 C. simplex 60 110 350 C. throndsenii 60 110 350 Emiliania huxleyi 60 110 350 Imantonia rotunda 60 110 350 Isochrysis galbana 60 110 350 Phaeocystis globosa 60 110 350 Pleurochrysis 60 roscoffensis 110 350 PAVLOVOPHYCEAE Pavlova gyrans

60 110 350

chl c3

MV chl c3

MgDVP

chl c2

chl c1

np-chl c2-Eh

np-chl c2-Cp

But-fuco

Fuco

4 k-Hex-fuco

Hex-fuco

Dd

Dt

βε-car

ββ-car

0.225 0.223 0.214 0.264 0.216 0.179 0.256 0.190 0.192 0.182 0.163 0.179 0.233 0.137 0.200 0.229 0.182 0.130 0.249 0.227 0.171 – – – 0.319 0.286 0.230 – – –

– – – – – – – 0.038 0.041 – – – 0.026 0.018 0.025 – 0.019 0.042 0.010 0.010 0.020 – – – – – – – – –

0.012 0.019 0.013 0.010 0.013 0.018 0.004 – – 0.018 0.025 0.035 0.009 0.016 0.031 0.018 0.016 0.022 0.013 0.016 0.019 0.012 0.018 0.028 0.009 0.012 0.019 0.012 0.013 0.022

0.215 0.224 0.219 0.199 0.191 0.200 0.306 0.309 0.327 0.254 0.247 0.282 0.222 0.178 0.225 0.341 0.309 0.290 0.194 0.228 0.249 0.355 0.350 0.313 0.269 0.249 0.290 0.062 0.056 0.063

– – – – – – – – – 0.009 0.009 0.025 – – – – – – – – – 0.085 0.075 0.111 – – – 0.102 0.089 0.097

– – – – – – 0.137 0.150 0.154 – – – – – – 0.099 0.097 0.043 0.097 0.093 0.078 0.066 0.062 0.029 0.097 0.099 0.053 – – –

0.094 0.124 0.121 0.059 0.060 0.102 – – – 0.232 0.235 0.300 0.111 0.060 0.105 – – – – – – – – – – – – – – –

– – – – – – 0.023 0.024 0.014 – – – – 0.011 0.011 – – – 0.119 0.146 0.172 – – – 0.006 0.007 – – – –

0.291 0.101 0.038 1.052 0.937 0.412 0.218 0.130 0.089 1.153 1.184 0.707 0.101 0.065 0.049 0.316 0.218 0.044 0.147 0.162 0.128 0.779 0.801 0.277 1.404 1.239 0.464 0.469 0.416 0.235

0.128 0.075 0.030 0.103 0.093 0.035 0.184 0.155 0.072 0.020 0.035 0.031 0.064 0.031 0.016 0.125 0.088 0.029 0.037 0.033 0.011 – – – 0.058 0.092 0.039 – – –

0.602 0.417 0.561 – – – 0.666 0.639 0.708 – 0.007 0.029 0.535 0.577 0.559 0.534 0.523 0.445 0.891 0.850 0.620 – – – 0.025 0.068 0.080 – – –

0.203 0.125 0.083 0.165 0.194 0.112 0.231 0.257 0.260 0.318 0.644 0.255 0.097 0.117 0.094 0.175 0.146 0.082 0.203 0.260 0.563 0.365 0.514 0.160 0.221 0.328 0.098 0.351 0.475 0.158

– – 0.033 – – 0.016 – – 0.032 – 0.025 0.044 – 0.005 0.027 0.024 0.040 0.078 – 0.017 0.066 0.017 0.017 0.024 – – 0.054 – 0.019 0.016

0.022 0.012 – 0.012 0.020 0.007 0.030 0.028 – 0.045 0.020 – 0.017 0.007 – 0.013 – – 0.028 0.025 – – – – 0.025 0.023 – – – –

0.037 0.022 0.022 0.025 0.040 0.013 0.015 0.030 0.035 0.055 0.068 0.013 0.020 0.023 0.028 0.037 0.033 0.033 0.043 0.052 0.045 0.093 0.061 0.043 0.035 0.102 0.072 0.103 0.105 0.033

chl c3 – – –

chl c2-Pg 0.057 0.035 0.012

MgDVP 0.009 0.013 0.015

chl c2 0.056 0.044 0.018

chl c1 0.119 0.075 0.054

– – –

– – –

– – –

0.735 0.603 0.181

– – –

– – –

0.355 0.594 0.098

0.027 0.104 0.030

– – –

0.035 0.092 0.025

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Fig. 3. Absorption chromatograms (440 nm) of six haptophyte species growing at 110 μmol photons m− 2 s− 1. Peak identification is as follows: 1 = chl c2-like P. gyrans-type; 2 = chl c3; 3 = MV chl c3; 4 = MgDVP; 5 = chl c2; 6 = chl c1; 7 = But-fuco; 8 = Fuco; 9 = 4-keto-Hex-fuco; 10 = Hex-fuco; 11= Diadino; 12 = Diato; 13 = Zea; 14= Unk 444.5; 15= Unk 440.9; 16: Unk 444.5; 17= np-chl c2-Eh; 18 = chl a; 19 = np-chl c2-Cp; 20 = β,β-car.

levels) and C. simplex (at 60 μmol photons m− 2 s− 1), though 4 k-Hexfuco derivative was detected. An unknown pigment spectrally similar to that observed in E. huxleyi (λmax = 444.5 nm) but eluting at min 29 was observed in all Chrysochromulina species (type 7). Imantonia rotunda (type 8) is the only species having But-fuco as an important component of the fucoxanthin pool (But-fuco/chl a ratio ranging from 0.099 to 0.119). But-fuco is a minor pigment in P. globosa (But-fuco/ chl a ratio 0.007–0.008) differing from other Phaeocystis species such as. P. antarctica or P. pouchetii. Hex-fuco is the major fucoxanthin pigment in I. rotunda while Fuco is the dominant for in P. globosa. Both species contain the unknown pigment (λmax = 444.5 nm) which elutes at min 29 such as in Chrysochromulina species. 3.3. Molar pigment ratios Chlorophyll and carotenoid values normalized to chl a (pigment ratios) for the eleven haptophytes growing at different light intensities are given in Table 2. Ratios between close related pigments and the total pools of chls c and fucoxanthin pigments are given in Table 3. The chl c2 to chl a ratio was nearly constant at the three light intensities tested: 0.225 ± 0.030 (mean ± SE) at 60 and 350 μmol photons m− 2 s− 1, and 0.217 ± 0.029 at 110 μmol photons m− 2 s− 1.

Moreover, the chl c3 to chl a ratio decreased with the increase of light intensity, from 0. 245 ± 0.014 at 60 μmol photons m− 2 s− 1 to 0.187 ± 0.010 at 350 μmol photons m− 2 s− 1. In C. pringsheimii, C. throndsenii, E. huxleyi and I. rotunda, the decrease of chl c3 ratios was paralleled with increased MV chl c3 ratios. The np-chl c2-Eh to chl a ratio was similar at 60 and 110 μmol photons m− 2 s− 1 (0.099 ± 0.011 and 0.100 ± 0.014, respectively) and lower values were detected at 350 μmol photons m− 2 s− 1 (0.071 ± 0.022). Both np-chl c2-Cp and MgDVP ratios showed higher values at 350 μmol photons m− 2 s− 1 (Table 2). The fucoxanthin to chl a molar ratios were similar at 60 and 110 μmol photons m− 2 s− 1 (0.495 ± 0.112 and 0.532 ± 0.134, respectively) and lower at 350 μmol photons m− 2 s− 1 (0.238 ± 0.064). The 4 k-Hex-fuco ratios matched with the pattern observed for Fuco, showing a decrease from 0.090 ± 0.019 at 60 μmol photons m− 2 s− 1 to lowest values (0.032 ± 0.006) at 350 μmol photons m− 2. However, the Hex-fuco to chl a ratios described an increase with light intensity in C. pringsheimii, C. throndsenii and P. globosa, and a decrease in E. huxleyi, I. rotunda y C. acantha. The Fucoxanthin index (Fi), which reflects the relative contribution of Fuco to the fucoxanthin pool, varied among species and growth light intensities (Table 3).

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291

Table 3 Pigment indexes of the haptophytes studied growing at different light intensities.

Chrysochromulina acantha C. cf cymbium

C. pringsheimii

C. simplex

C. throndsenii

Emiliania huxleyi

Imantonia rotunda

Isochrysis galbana

Phaeocystis globosa Pleurochrysis roscoffensis

PAVLOVOPHYCEAE Pavlova gyrans

a

Light intensity μmol m− 2 s− 1

c3/c2

c1/c2

npc2 /c2

Tchls c/a

TFuco(s)/a

(Dd + Dt)/a

TXantho/a

Tchls c/ Tfucos

Fuco index

Dd + Dt/ Tfuco(s)

Dt/ (Dd + Dt)

βε-car/ ββ-car

60 110 350 60 110 350 60 110 350 60 110 350 60 110 350 60 110 350 60 110 350 60 110 350 60 110 350 60 110 350

0.96 1.00 0.98 1.33 1.13 0.90 0.84 0.62 0.59 0.72 0.64 0.64 1.05 0.77 0.67 0.67 0.59 0.45 0.78 1.00 0.69 – – – 1.19 1.15 0.79 – – –

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.04 0.04 0.09 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.24 0.21 0.36 0.00 0.00 0.00 1.65 1.59 1.54

0.44a 0.55a 0.55a 0.30a 0.31a 0.51a 0.45 0.49 0.47 0.91a 0.95a 1.06a 0.50a 0.34a 0.47a 0.29 0.31 0.15 0.50 0.40 0.31 0.19 0.18 0.09 0.36 0.40 0.18 – – –

0.55 0.59 0.57 0.53 0.48 0.50 0.70 0.69 0.71 0.70 0.70 0.82 0.60 0.41 0.59 0.69 0.62 0.53 0.56 0.57 0.54 0.52 0.51 0.48 0.69 0.65 0.59 0.18 0.16 0.18

1.02 0.59 0.63 1.16 1.03 0.45 1.09 0.95 0.88 1.17 1.23 0.77 0.70 0.65 0.64 0.98 0.83 0.52 1.19 1.19 0.93 0.78 0.80 0.28 1.49 1.41 0.58 0.47 0.42 0.24

0.20 0.13 0.08 0.17 0.19 0.13 0.23 0.26 0.29 0.32 0.67 0.30 0.10 0.12 0.12 0.20 0.19 0.16 0.20 0.28 0.63 0.38 0.53 0.18 0.22 0.33 0.17 0.35 0.49 0.17

1.22 0.72 0.71 1.33 1.22 0.58 1.32 1.21 1.17 1.49 1.90 1.07 0.80 0.77 0.74 1.18 1.02 0.68 1.39 1.47 1.56 1.16 1.33 0.46 1.71 1.74 1.75 0.82 0.91 0.41

0.54 1.00 1.01 0.46 0.47 1.12 0.64 0.73 0.81 0.59 0.63 1.07 0.86 0.63 0.92 0.71 0.75 1.02 0.47 0.48 0.58 0.67 0.63 1.74 0.47 0.46 1.02 0.38 0.38 0.77

0.29 0.17 0.06 0.91 0.91 0.92 0.20 0.14 0.10 0.98 0.97 0.92 0.14 0.10 0.08 0.32 0.26 0.09 0.12 0.14 0.14 1.00 1.00 1.00 0.94 0.88 0.80 1.00 1.00 1.00

0.20 0.21 0.18 0.14 0.19 0.29 0.21 0.27 0.33 0.27 0.55 0.39 0.14 0.19 0.19 0.20 0.22 0.31 0.17 0.23 0.68 0.49 0.66 0.66 0.15 0.23 0.26 0.75 1.19 0.74

0.00 0.00 0.28 0.00 0.00 0.13 0.00 0.00 0.11 0.00 0.04 0.15 0.00 0.04 0.22 0.12 0.22 0.49 0.00 0.06 0.11 0.05 0.03 0.13 0.00 0.00 0.36 0.00 0.04 0.09

0.60 0.55 0.00 0.48 0.50 0.54 2.00 0.93 0.00 0.82 0.29 0.00 0.85 0.30 0.00 0.35 0.00 0.00 0.65 0.48 0.00 0.00 0.00 0.00 0.71 0.23 0.00 0.00 0.00 0.00

60 110 350

– – –

2.13 1.71 1.71

c2Pg/c2 1.02 1.26 1.50

0.24 0.17 0.10

0.74 0.60 0.18

0.38 0.70 0.13

1.12 1.30 0.31

0.33 0.28 0.55

1.00 1.00 1.00

0.52 1.16 0.71

0.07 0.15 0.23

0.00 0.00 0.00

np-chl c2-Chrysochromulina polylepis-type to chl c2 ratio.

Diadinoxanthin to chl a ratios showed an increase from 60 to 110 μmol photons m− 2 s− 1, where the highest values were observed, and a subsequent decrease at 350 μmol photons m− 2 s− 1. The Diato to chl a ratio for all species was higher at 350 μmol photons m− 2 s− 1. The de-epoxidation index defined as Diato / (Diadino + Diato) showed values over 0.20 at 350 μmol photons m− 2 s− 1 and it is highly variable among species (Table 3). β,ε-carotene was only detected E. huxleyi, Chrysochromulina spp., I. rotunda and Phaeocystis globosa (types 6 to 8) The pigment was generally restricted to 60 and 110 μmol photons m− 2 s− 1. β,βcarotene to chl a ratio showed highest values at 110 μmol photons m− 2 s− 1. The carotene ratio (β,ε-car /β,β-car) was higher at 60 μmol photons m− 2 s− 1 (higher value of 2.00 in C. pringsheimii) and its variation pattern at higher light intensities was species-specific: only C. cf. cymbium showed β,ε-car at 350 μmol photons m− 2 s− 1 (β,ε-car/β,β-car ratio = 0.54), whereas in the other species there was a general decrease at 110 μmol photons m− 2 s− 1. 4. Discussion 4.1. Growth rates There is a limited understanding of the response of bloom-forming species to different physical conditions, this is a relevant question due to the marked increase of harmful algal blooms in coastal and estuarine waters (Hallegraeff, 2003). The ability to grow at relatively high rates makes a species a good competitor in environments of great variability as estuaries, where both physical and biotic factors can change on the order of a few days or even hours. Among bloomforming species, haptophytes are known for their capability to reach

high densities in different environments, withstanding many of them a wide range of temperatures and salinities (Moestrup, 1994). Mixotrophy has been reported for some species (Jones et al., 1994), what makes of the estuaries, which generally carry great amounts of organic matter than other marine areas, a potential environment for the development of haptophyte blooms. Growth rates greater than 1 division d− 1 were reached for almost haptophytes isolated from the Nervion River estuary. Growth rates were close to those maxima found for Emiliania huxleyi, Isochrysis galbana and several species of Pavlova by Glover et al. (1987) under similar temperature and light conditions. However, these authors reported a growth rate of 2 divisions d− 1 for Imantonia rotunda, which in our study only reached a maximum of 1.4 divisions d− 1. The growth rates of Phaeocystis globosa in our study are close to those reported by Weisse and Scheffel-Möser (1990) and Peperzak et al. (2000). The isolates of Chrysochromulina showed growth rates within the range of those found for other haptophyte species, including the bloom-forming Chrysochromulina polylepis (Edvardsen, 1993). At least four of the isolated species including Emiliania huxleyi, Isochrysis galbana, Phaeocystis globosa, and Pleurochrysis roscoffensis have been reported to form blooms in diverse marine areas, though only two of these, Isochrysis galbana and the genus Phaeocystis, have been observed to bloom in the Nervion estuary (Orive, 1989; Seoane et al., 2005). The other isolates have always been found in relatively low numbers. From the observed growth rates, any of the isolates could form blooms in the estuary provided that other factors such as competition and grazing were of low intensity. Phaeocystis globosa is known as a bloom-forming species in nutrient-rich waters where it can outcompete other algae and avoids grazing by forming mucilaginous colonies (Lancelot et al., 1987; Cadée and Hegeman, 2002;

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Schoemann et al., 2005). In the case of Emiliania huxleyi, its success in the surface of oligotrophic waters has been explained by its ability to growth at high-light intensities without experiencing photoinhibition (Nanninga and Tyrrell, 1996; Harris et al., 2005, 2009) and by its high affinity for nutrients (Paasche, 2002; Iglesias-Rodríguez et al., 2002; Lessard et al., 2005). Pleurochrysis roscoffensis is a toxic haptophyte which use to bloom in coastal waters (Houdan et al., 2004), although to date it has a more limited distribution than Emiliania huxleyi. Its low density in the Nervion estuary could be due, as in the case of Emiliania huxleyi, to their preference for coastal areas where they have competitive advantage in high-light low nutrients waters. Several species of Chrysochromulina have been reported to form blooms although no regularly (Eikrem and Throndsen, 1993; Hansen et al., 1995; Edvardsen and Paasche, 1998). These blooms have been associated to enhanced river discharge, which leads to water column stratification, and high N: P ratios in coastal waters (Dahl et al., 2005). The abundance of Chrysochromulina in the Nervion river estuary remained always in low levels (Seoane et al., 2005, 2006) but the growth rates of most isolated species can be considered high enough as to promote rapid accumulation of cells in the estuary. 4.2. Pigment composition and pigments types In the last years, several polar and non-polar chls c have been isolated from haptophytes, although their specificity is still a matter of discussion (Zapata et al., 2006). Pavlova gyrans contains a red-shifted chl c2-like pigment, first detected in P. gyrans by Fawley (1989), which has also been found in some diatoms and chrysophyceans (Zapata, 2005), what precludes its use as marker for this species. More specific is the np-chl c1 from Prymnesium parvum present in Isochrysis galbana, although it appears in too low concentrations to be detected in natural samples of mixed populations. Chl c3, first detected in the pelagophycean Pelagococcus subviridis (Vesk and Jeffrey, 1997) and in the haptophyte Emiliania huxleyi (Jeffrey and Wright, 1987), has been used as marker for specific haptophytes such as Phaeocystis (Antajan et al., 2004; Muylaert et al., 2006). While this pigment is present in most of the haptophytes isolated from the estuary (8 of the 11 species), chl c3 is also present in bolidophyceans, some diatoms, dinoflagellates and chrysophyceans (see Zapata, 2005). Only after observations under the microscope could the relative importance of a particular species in the overall community be estimated by using chl c3 as marker. Non-polar chl c2 was initially observed in several haptophytes (Nelson and Wakeham, 1989; Bidigare et al., 1990; Zapata and Garrido, 1997) and misidentified as chl c2-phytolsubstituted. Mass spectrometry analyses unravel a high complex structure for non-polar chl c2 pigments consisting in galactoloipid moity (monogalactosyldiacylglycerol ) covalently linked to the acrylic acid of a chl c2 molecule, the pigment present in Emiliania huxleyi consist in MGDG (14:0/18:4)-chl c2 (Garrido et al., 2000). Further studies detected non-polar chl c2 pigments differing in the fatty acid composition (e.g. MGDG (14:0/14:0)-chl c2) in Chrysochromulina spp. (Zapata et al., 2001). Non-polar chls c are present in Haptophytes belonging to pigment types 3 to 8 (Zapata et al., 2004) and at trace levels in some dinoflagellates whose chloroplasts were acquired from a haptophyte by tertiary endosymbiosis (Zapata et al., 2004; Garcés et al., 2006). In our study, non-polar chl c2 was found in all isolated species but P. gyrans and P. roscoffensis, in agreement with previous reports on both species (Van Lenning et al., 2003a; Zapata et al., 2004). Non polar chl c2-Eh was present in I. galbana, E. huxleyi, I. rotunda, P. globosa and Chrysochromulina pringsheimii. Four of the five Chrysochromulina species studied contained the non-polar chl c2-Cp. It was a good marker for the genus Chrysochromulina (Zapata et al., 2001, 2004), despite it was detected in several coccolithophorid species (Van Lenning et al., 2003b) and as traces in Karenia mikimotoi (Zapata et al., 2004), Karlodinium armiger (Garcés et al., 2006) and Karlodinium decipiens (Laza-Martinez et al., 2007). In C. pringsheimii a less

retained peak sharing absorption spectrum with np-chl c2 was detected as previously observed in other Chrysochromulina species (C. ericina, C. herdlensis, C.hirta) which lacked the major np-chl c2-Cp type. Acyloxy-fucoxanthins profile was highly specific within haptophytes. In consequence, they have been used as chemotaxonomic markers for the group. In that sense, the dominant fucoxanthin pigment observed in the Chrysochromulina species studied matched previous results from Chrysochromulina spp. isolated from different geographical areas (Zapata et al. 2001, 2004). However, Hex-fuco and its 4-keto derivate, the 4 k-Hex-fuco (Egeland et al., 2000), are found in some dinoflagellates (e.g. Karenia species). Both Hex-fuco and 4-kHex-fuco are not present in Isochrysis galbana, Pavlova gyrans and Pleurochrysis roscoffensis, as a consequence, the occurrence of these species in the estuary could not be revealed by using fucoxanthin derivatives as a single pigment marker. Though Phaeocystis spp. (mainly P. antarctica and P. pouchetii) were considered as pigmenttype 8 haptophytes, the lack of But-fuco in the Phaeocystis globosa strain isolated from Nervion estuary matched previous result obtained for P. globosa (CCMP627) from the Gulf of Mexico (Zapata et al. 2004). The pigment composition of P. globosa matched results obtained from Chrysochromulina kappa (Zapata et al., 2001, 2004) and the recently described species Chrysochromulina palpebralis (Seoane et al., 2009), both species did not match pigment type 6 (Emiliania huxleyi) nor pigment type 7 (Chrysochromulina spp.). The species analyzed belong to the two algal classes recognized within Haptophyta. The class Pavlovophyceae is represented by a single species, Pavlova gyrans, while the class Coccolithophyceae, new designation for the former class Prymnesiophyceae (Silva et al., 2007), is represented by 10 species distributed among the following orders: the Phaeocystales with Phaeocystis globosa, the Prymnesiales represented by 5 species of the genus Chrysochromulina and Imantonia rotunda, Isochrysidales, with Emiliania huxleyi and Isochrysis galbana and Coccolithales represented by Pleurochrysis roscoffensis. Considering the pigment composition, the eleven species isolated belong to six (1, 2, 3, 6, 7 and 8) of the eight pigment types observed in Haptophyta (Zapata et al. 2004). The pigment composition increases its complexity from type 1 to 8. However, the growth rate at different light intensities seems to be not related with the pigment complexity of light harvesting antenna, the faster growing species at the 3 light intensities, Pleurochrysis roscoffensis, belongs to pigment type 1, the simplest one, whereas the second and third faster species Phaeocystis globosa and Imantonia rotunda, respectively belong to pigment type 8, the more complex. 4.3. Pigment ratios variability Usually, published pigment ratios have been obtained from cultures growing under low to medium light intensities, ranging from 40 to 100 μmol photons m− 2 s− 1 (Mackey et al., 1996; Zapata et al., 2004; Laza-Martinez et al., 2007). Exceptions are specific studies of pigment ratios at different nutrient status and/or light intensities (Van Leeuwe and Stefels, 1998; Schlüter et al., 2000; Stolte et al., 2000; Leonardos and Harris, 2006; Lewitus et al., 2005; Rodríguez et al., 2006). The scarcity of pigment ratio values at high-light conditions constitutes a serious constraint for the use of chemotaxonomy especially when data are grouped in bins corresponding to different depths (Wright and van den Enden, 2000). Another limitation for comparative purposes is the use of HPLC methods unable to separate pigment pairs such as chl c1/chl c2, chl c3/MVchl c3, chl a /np-chl c2, 4 k-Hex-fuco/Hex-fuco as well as β,ε-car /β,β-car. The assessment of pigment diversity in chl c- and Fuco-pools is not only relevant to define pigment types but also to obtain accurate pigment ratios without interference of unresolved pigments. Since the discovery of new pigments in haptophytes few studies have been published on to their variability at different growth light intensities.

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Data here shown reflect pigment variability at 3 light intensities among haptophytes belonging to 6 pigment types as well as among species belonging to a single genus (Chrysochromulina) and pigment type (7). General trends point to the inter-conversion of distinct chl c pigments as a response to light intensity; chl c3, when present, is the dominant form of the chl c-pool at 60 and 110 μmol photons m− 2 s− 1 being chl c2 the major chl c pigment at 350 μmol photons m− 2 s− 1. Even though a fraction of chl c3 could be converted into MV chl c3, the decay is also observed in chl c3-containing species unable to synthesize MV chl c3. The non-polar chl c2 contribution to chl c pool is higher at low and medium light and it decreased at 350 μmol photons m− 2 s− 1. Total chl c to chl a ratios depend on the chl c pigment diversity, so higher values are observed within pigment types 3, 6, 7 and 8 and low values in pigment types 1 and 2. The fucoxanthin pigments depict similar behaviour with a Hex-fuco increase at expense of Fuco and 4 k-Hex-fuco. Total Fuco to chl a ratio shows a higher variability than chl c to chl a. When Fuco is the only pigment in the pool its pigment ratio to chl a is close to species having a complex fuco pool. In such a case the marked decrease in pigment ratios at 350 μmol photons m− 2 s− 1 affects to light harvesting pigments without a concomitant increase in photoprotective carotenoids (Table 3). An increase in Diadino to chl a ratio was observed in several species at 110 μmol photons m− 2 s− 1 with a parallel synthesis of diatoxanthin (C. simplex, Isochrysis galbana, Pavlova gyrans, Pleurochrysis roscoffensis), in other cases Diadino/chl a ratio reach higher values at 350 μmol photons m− 2 s− 1 together with maxima in Diato/chl a ratios (e.g. Imantonia rotunda). There is no a common light threshold over that Diato is synthesized in the haptophyte species studied. Only 3 species showed Diato when growing at 60 μmol photons m− 2 s− 1: E. huxleyi, I. galbana, P. gyrans; on the other hand, Diato was only synthesized at 350 μmol photons m− 2 s− 1 in 4 species: C. acantha, C. cf. cymbium, C. pringsheimii, Phaeocystis globosa, while 110 μmol photons m− 2 s− 1 seems to be the light threshold for Diato occurrence in C. simplex, C. throndsenii, I. rotunda and Pleurochrysis roscoffensis. Diatoxanthin never exceeded Diadino concentration, in consequence the deepoxidation index (Table 3) showed low values. Higher indexes are restricted to cultures growing at 350 μmol photons m− 2 s− 1: 0.49 in Emiliania huxleyi and 0.36 in I. rotunda. The ratio of photoprotective carotenoids (Diadino + Diato) to light harvesting carotenoids (fucoxanthin pool) was nearly stable in species belonging to pigment types 6, 7 and 8 (the more complex fucoxanthin pool). In species having only fucoxanthin (pigment types 1, 2 and 3) the ratios were more variable, pigment ratios increased at 110 and 350 μmol photons m− 2 s− 1. The changes in pigment ratios at different light intensity were species-specific probably reflecting both differences in the pigment composition of the different components of the light harvesting protein complexes (LHCs) as well as the balance between LHCs and reaction centre complexes (Harris et al., 2009). 5. Conclusion A great diversity of haptophyte species can be found in estuarine waters showing relatively high growth rates when cultured under conditions of light, temperature and salinity typical of these environments in temperate areas. The knowledge of the values of the pigment ratios under different light conditions and the use of pigment types, taking into account not only major but also minor non conventional markers, should be of great utility to detect accurately initial stages of haptophyte blooms or to establish the contribution of haptophytes in the overall phytoplankton assemblages. The potential of the chemotaxonomic approach should be based on the combined use of chlorophylls and carotenoids to define pigment types within algal classes. Another approach could be to define pigment-based chloroplast types irrespective of taxonomic

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groups as proposed by Zapata (2005). In this case algal groups sharing similar chloroplast pigment composition could be grouped as a functional group assuming similar light harvesting strategies. Acknowledgments This research was funded by the University of the Basque Country (project 9/UPV00118.310-15339/2003), by the Basque Government (Project GIC07/111-417-07) and by a postdoctoral contract to S.S. References Antajan, E., Chretiennot-Dinet, M.J., Leblanc, C., Daro, M.H., Lancelot, C., 2004. 19′hexanoyloxyfucoxanthin may not be the appropriate pigment to trace occurrence and fate of Phaeocystis: the case of P. globosa in Belgian coastal waters. J. Sea Res. 52, 165–177. Barlow, R.G., Aiken, J., Holligan, P.M., Cummings, D.G., Maritorena, S., Hooker, S., 2002. Phytoplankton pigment and absorption characteristics along meridional transects in the Atlantic Ocean. Deep Sea Res. I 49, 637–660. Bidigare, R.R., Kennicutt II, M.C., Ondrusek, M.E., Keller, M.D., Guillard, R.R.L., 1990. Novel chlorophyll-related compounds in marine phytoplankton: distributions and geochemical implications. Energy Fuels 4, 653–657. Carreto, J.I., Montoya, N.G., Benavides, H.R., Guerrero, R., Carignan, M.O., 2003. Characterization of spring phytoplankton communities in the Rio de la Plata maritime front using pigment signatures and cell microscopy. Mar. Biol. 143, 1013–1027. Cadée, G.C., Hegeman, J., 2002. Phytoplankton in the Marsdiep at the end of the 20th century, 30 years monitoring biomass, primary production, and Phaeocystis blooms. J. Sea Res. 48, 97–110. Dahl, E., Edvarsen, B., Eikrem, W., 1998. Chrysochromulina blooms in the Skagerrak after 1988. In: Reguera, B., Blanco, J., Fernandez, M.L., Wyatt, T. (Eds.), Harmful algae: Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, pp. 104–105. Dahl, E., Bagoien, E., Edvardsen, B., Stenseth, N.C., 2005. The dynamics of Chrysochromulina species in the Skagerrak in relation to environmental conditions. J. Plankton Res. 54, 15–24. Edvardsen, B., 1993. Toxicity of Chrysochromulina species (Prymnesiophyceae) to the brine shrimp, Artemia salina. In: Smayda, T.J., Shimizu, Y. (Eds.), Toxic Blooms in the Sea. Elsevier, Amsterdam, pp. 681–686. Edvardsen, B., Paasche, E., 1998. Bloom dynamics and physiology of Prymnesium and Chrysochromulina. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. Springer-Verlag, Berlin, pp. 193–208. Egeland, E.S., Garrido, J.L., Zapata, M., Maestro, M.A., Liaaen-Jensen, S., 2000. Algal carotenoids. Part 64. Structure and chemistry of 4-keto-19′-hexanoyloxyfucoxanthin with a novel carotenoid end group. J. Chem. Soc. Perkin Trans. 1, 1223–1230. Eikrem, W., Throndsen, J., 1993. Toxic prymnesiophytes identified from Norwegian coastal waters. In: Smayda, T.J., Shimizu, Y. (Eds.), Toxic Blooms in the Sea. Elsevier, Amsterdam, pp. 687–692. Fawley, M.W., 1989. A new form of chlorophyll c involved in light harvesting. Plant Physiol. 91, 727–732. Garcés, E., Fernandez, M., Penna, A., Van Lenning, K., Gutierrez, A., Camp, J., Zapata, M., 2006. Characterization of NW Mediterranean Karlodinium spp. (Dinophyceae) strains using morphological, molecular, chemical, and physiological methodologies. J. Phycol. 42, 1096–1112. Garrido, J.L., Zapata, M., 1998. Detection of new pigments from Emiliania huxleyi (Prymnesiophyceae) by high-performance liquid chromatography, liquid chromatography-mass spectrometry, visible spectroscopy and fast atom bombardment mass spectrometry. J. Phycol. 34, 70–78. Garrido, J.L., Zapata, M., 2006. Chlorophyll analysis by new HPLC methods. In: Grimm, B., Porra, R.J., Rüdiger, W., Scheer, U. (Eds.), Chlorophylls and Bacteriochlorophylls: biochemistry, biophysics, functions and applications. Springer, Dordrecht, pp. 109–121. Garrido, J.L., Zapata, M., Muñiz, S., 1995. Spectral characterization of new chlorophyll c pigments isolated from Emiliania huxleyi (Prymnesiophyceae) by high-performance liquid chromatography. J. Phycol. 31, 761–768. Garrido, J.L., Otero, J., Maestro, M.A., Zapata, M., 2000. The main non-polar chlorophyll c from Emiliania huxleyi (Prymnesiophyceae) is a chlorophyll c2-monogalactosyldiacylglyceride ester: a mass spectrometry study. J. Phycol. 36, 497–505. Gibb, S.W., Cummings, D.G., Irigoien, X., Barlow, R.G., Fauzi, R., Mantoura, C., 2001. Phytoplankton pigment chemotaxonomy of the northeastern Atlantic. Deep-Sea Res. 48, 795–823. Gieskes, G.G., Kraay, G.W., 1986. Floristic and physiological differences between the shallow and the deep nanophytoplankton community in the euphotic zone of the open tropical Atlantic revealed by HPLC analysis of pigments. Mar. Biol. 91, 567–576. Gieskes, W.W., Kraay, G.W., 1989. Analysis of phytoplankton pigments by HPLC before, during and after mass occurrence of the microflagellate Corymbellus aureus during the spring bloom in the open northern North-Sea in 1983. Mar. Biol. 92, 45–52. Glover, H.E., Keller, M.D., Spinrad, R.W., 1987. The effects of light quality and intensity on photosynthesis and growth of marine eukariotic and prokariotic phytoplankton clones. J. Exp. Mar. Biol. Ecol. 105, 137–159.

294

S. Seoane et al. / Journal of Sea Research 62 (2009) 286–294

Graneli, E., Moreira, M.O., 1990. Effects of river water of different origin on the growth of marine dinoflagellates and diatoms in laboratory cultures. J. Exp. Mar. Biol. Ecol. 136, 89–106. Guillard, R.R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hutedt and Detonula confervacea Cleve. Can. J. Microbiol. 8, 229–239. Hallegraeff, G.M., 2003. Harmful algal blooms: A global overview. In: Hallegraeff, G.M., Anderson, D.M., Cembella, A.D. (Eds.), Manual on Harmful Marine Microalgae. UNESCO, Paris, pp. 25–50. Hansen, P.J., Nielsen, T.G., Kaas, H., 1995. Distribution and growth of protists and mesozooplankton during a bloom of Chrysochromulina spp. (Prymnesiophyceae, Prymnesiales). Phycologia 34, 409–416. Harris, G.N., Scanlan, D.J., Geider, R.J., 2005. Acclimation of Emiliania huxleyi (Prymnesiophyceae) to photon flux density. J. Phycol. 41, 851–862. Harris, G.N., Scanlan, D.J., Geider, R.J., 2009. Responses of Emiliania huxleyi (Prymnesiophyceae) to step changes in photons flux density. Eur. J. Phycol. 44, 31–48. Houdan, A., Fresnel, B., Fouchard, S., Billard, C., Probert, I., 2004. Toxicity of coastal coccolithophores (Prymnesiophyceae, Haptophyta). J. Plankton Res. 26, 875–883. Iglesias-Rodríguez, M.D., Brown, C.W., Doney, S.C., Kleypas, J., Kolber, D., Kolber, Z., Hayes, P.K., Falkowski, P.G., 2002. Representing key phytoplankton functional groups in ocean carbon cycle models: Coccolithophorids. Glob. Biogeochem. Cycles 16, 1100. doi:10.1029/2001GB001454. Jeffrey, S.W., 1997. Chlorophyll and carotenoid extinction coefficients. In: Jeffrey, S.W., Mantoura, R.F.C., Wright, S.W. (Eds.), Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO Publishing, Paris, pp. 595–596. Jeffrey, S.W., Wright, S.W., 1987. A new spectrally distinct component in preparations of chlorophyll c from the micro-alga Emiliania huxleyi (Prymnesiophyceae). Biochim. Biophys. Acta 894, 180–188. Jeffrey, S.W., Wright, S.W., 1994. Photosynthetic pigments in the Haptophyta. In: Green, J.C., Leadbeater, B.S.C. (Eds.), The Haptophyte Algae. Clarendon Press, Oxford, pp. 111–132. Jeffrey, S.W., Wright, S.W., Zapata, M., 1999. Recent advances in HPLC pigment analysis of phytoplankton. Mar. Freshw. Res. 50, 879–896. Jones, H.L.J., Leadbeater, B.S.C., Green, J.C., 1994. Mixotrophy in haptophytes. In: Green, J.C., Leadbeater, B.S.C. (Eds.), The Haptophyte Algae. Clarendon Press, Oxford, pp. 247–264. Lancelot, C., Billen, G., Sournia, A., Weisse, T., Colijn, F., Veldhuis, M., Davies, A., Wassman, P., 1987. Phaeocystis blooms and nutrient enrichment in the continental coastal zones of the North Sea. Ambio 16, 38–46. Laza-Martinez, A., Seoane, S., Zapata, M., Orive, E., 2007. Phytoplankton pigment patterns in a temperate estuary: from unialgal cultures to natural assemblages. J. Plankton Res. 29, 913–929. Leonardos, N., Harris, G.N., 2006. Comparative effects of light on pigments of two strains of Emiliania huxleyi. J. Phycol. 42, 1217–1224. Lessard, E.J., Merico, A., Tyrrell, T., 2005. Nitrate:phosphate ratios and Emiliania huxleyi blooms. Limnol. Oceanogr. 50, 1020–1024. Lewitus, A.J., White, D.L., Tymowski, R.G., Geesey, M.E., Hymel, S.N., Noble, P.A., 2005. Adapting the CHEMTAX method for assessing phytoplankton taxonomic composition in southeastern U.S. estuaries. Estuaries 28, 160–172. Llewellyn, C.A., Fishwick, J.R., Blackford, J.C., 2005. Phytoplankton community assemblage in the English Channel: a comparison using chlorophyll a derived from HPLC-CHEMTAX and carbon derived from microscopy cell counts. J. Plankton Res. 27, 103–119. Mackey, M.D., Mackey, D.J., Higgins, H.W., Wright, S.W., 1996. CHEMTAX — a program for estimating class abundances from chemical markers: application to HPLC measurements of phytoplankton. Mar. Ecol. Prog. Ser. 144, 1441–1468. Moestrup, Ø., 1994. Economic aspects: “blooms”, nuisance species, and toxins. In: Green, J.C., Leadbeater, B.S.C. (Eds.), The Haptophyte Algae. Clarendon Press, Oxford, pp. 265–285. Moestrup, Ø., Thomsen, H.A., 2003. Taxonomy of toxic haptophytes (Prymnesiophytes). In: Hallegraeff, G.M., Anderson, D.M., Cembella, A.D. (Eds.), Manual of harmful marine microalgae. IOC Manual and Guides UNESCO, Paris, pp. 433–463. Moon-van der Staay, S.Y., van der Staay, G.W.M., Guillou, L., Vaulot, D., Claustre, H., Medlin, L.K., 2000. Abundance and diversity of prymnesiophytes in the picoplankton community from the equatorial Pacific Ocean inferred from 18 S rDNA sequences. Limnol. Oceanogr. 45, 98–109. Muylaert, K., Gonzales, R., Franck, M., Lionard, M., Van der Zee, C., Cattrijsse, A., Sabbe, K., Chou, L., Vyverman, W., 2006. Spatial variation in phytoplankton dynamics in the Belgian Coastal zone of the North Sea studied by microscopy, HPLC-CHEMTAX and underway fluorescence recordings. J. Sea Res. 55, 253–265. Nanninga, H.J., Tyrrell, T., 1996. The importance of light for the formation of algal blooms by Emiliania huxleyi. Mar. Ecol. Prog. Ser. 136, 195–203. Nelson, J.R., Wakeham, S.G., 1989. A phytol-substituted chlorophyll c from Emiliania huxleyi (Prymnesiophyceae). J. Phycol. 25, 761–766. Obayashi, Y., Tanoue, E., Suzuki, K., Handa, N., Nojiri, Y., Wong, C.S., 2001. Spatial and temporal variabilities of phytoplankton community structure in the northern North Pacific as determined by phytoplankton pigments. Deep-Sea Res. I 48, 439–469. Orive, E., 1989. Differences in phytoplankton abundance and distribution between the Abra of Bilbao and the adjacent shelf waters. Hydrobiologia 182, 121–135.

Paasche, E., 2002. A review of the coccolithophorid Emiliania huxleyi (Prymnesiophyceae), with particular reference to growth, coccolith formation, and calcificationphotosynthesis interactions. Phycologia 40, 503–529. Peperzak, L., Duin, R.N.M., Colijn, F., Gieskes, W.W.C., 2000. Growth and mortality of flagellates and non-flagellate cells of Phaeocystis globosa (Prymnesiophyceae). J. Plankton Res. 22, 107–119. Qian, Y.R., Jochens, A.E., Kennicutt, M.C., Biggs, D.C., 2003. Spatial and temporal variability of phytoplankton biomass and community structure over the continental margin of the northeast Gulf of Mexico based on pigment analysis. Cont. Shelf Res. 23, 1–17. Rodríguez, F., Pazos, Y., Maneiro, J., Zapata, M., 2003. Temporal variation in phytoplankton assemblages and pigment composition at a fixed station of the Ría of Pontevedra (NW Spain). Est. Coast. Shelf Sci. 58, 499–515. Rodríguez, F., Chauton, M., Johnsen, G., Andresen, K., Olsen, L.M., Zapata, M., 2006. Photoacclimation in phytoplankton: implications for biomass estimates, pigment functionality and chemotaxonomy. Mar. Biol. 148, 963–971. Schlüter, L., Mohlenberg, F., Havskum, H., Larsen, S., 2000. The use of phytoplankton pigments for identifying and quantifying phytoplankton groups in coastal areas: testing the influence of light and nutrients on pigment/chlorophyll a ratios Mar. Ecol. Prog. Ser. 192, 49–63. Schoemann, W., Becquevort, S., Stefels, J., Rousseau, W., Lancelot, C., 2005. Phaeocystis blooms in the global ocean and their controlling mechanisms: a review. J. Sea Res. 53, 43–66. Seoane, S., Laza, A., Urrutxurtu, I., Orive, E., 2005. Phytoplankton assemblages and their dominant pigments in the Nervion River estuary. Hydrobiologia 549, 1–13. Seoane, S., Laza, A., Orive, E., 2006. Monitoring phytoplankton assemblages in estuarine waters: The application of pigment analysis and microscopy to size-fractionated samples. Est. Coast. Shelf Sci. 67, 343–354. Seoane, S., Eikrem, W., Pienaar, R., Edvardsen, B., 2009. Chrysochromulina palpebralis sp. nov. (Prymnesiophyceae): a haptophyte, possessing two alternative morphologies. Phycologia 48, 166–176. Silva, P.C., Throndsen, J., Eikrem, W., 2007. Revisiting the nomenclature of haptophytes. Phycologia 46, 471–475. Stolte, W., Kraay, G.W., Noordeloos, A.A.M., Riegman, R., 2000. Genetic and physiological variation in the composition of Emiliania huxleyi (Prymnesiophyceae) and the potential use of its pigment ratios as a quantitative physiological marker. J. Phycol. 36, 529–539. Tengs, T., Dahlberg, O.J., Schalchian-Tabrizi, K., Klaveness, D., Rudi, K., Delwiche, C.F., Jakobsen, K.S., 2000. Phylogenetic analyses indicate that 19´hexanoyloxyfucoxanthin-containing dinoflagellates have tertiary plastids of haptophyte origin. Mol. Biol. Evol. 17, 718–729. Van Leeuwe, M.A., Stefels, J., 1998. Effects of iron and light stress on the biochemical composition of Antarctic Phaeocystis sp. (Prymnesiophyceae). II. Pigment composition. J. Phycol. 34, 496–503. Van Lenning, K., Latasa, M., Estrada, M., Saez, A.G., Medlin, L., Probert, I., Veron, B., Young, J., 2003a. Pigments signatures phylogenetic relationships of the Pavlophyceae (Haptophyta). J. Phycol. 39, 379–389. Van Lenning, K., Probert, I., Latasa, M., Estrada, M., Young, J.R., 2003b. Pigment diversity of coccolithophores in relation to taxonomy, phylogeny and ecological preferences. In: Thierstein, H.R., Young, J.R. (Eds.), coccolithophores. Springer, Berlin, pp. 51–73. Weisse, T., Scheffel-Möser, U., 1990. Growth and grazing loss rates in single-celled Phaeocystis sp. (Prymnesiophyceae). Mar. Biol. 106, 153–158. Wright, S.W., van den Enden, R.L., 2000. Phytoplankton community structure and stocks in the East Antarctic marginal ice zone (BROKE survey, January–March 1996) determined by CHEMTAX analysis of HPLC pigment signatures. Deep-Sea Res. 47, 2363–2400. Zapata, M., 2005. Recent advances in pigment analysis as applied to picophytoplankton. Vie Milieu 55, 233–248. Zapata, M., Garrido, J.L., 1991. Influence of injection conditions in reversed-phase highperformance liquid chromatography of chlorophylls and carotenoids. Chromatographia 31, 589–594. Zapata, M., Garrido, J.L., 1997. Occurrence of phytylated chlorophyll c in Isochrysis galbana and Isochrysis sp. (CLONE T-ISO) (Prymnesiophyceae). J. Phycol. 33, 209–214. Zapata, M., Rodríguez, F., Garrido, J.L., 2000. Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containing mobile phases. Mar. Ecol. Prog. Ser. 195, 29–45. Zapata, M., Edvardsen, B., Rodríguez, F., Maestro, M.A., Garrido, J.L., 2001. Chlorophyll c2 monogalactosyldiacylglyceride ester (chl c2-MGDG). A novel marker pigment for Chrysochromulina species (Haptophyta). Mar. Ecol. Prog. Ser. 219, 85–98. Zapata, M., Jeffrey, S.W., Wright, S.W., Rodríguez, F., Garrido, J.L., Clementson, L., 2004. Photosynthetic pigments in 37 species (65 strains) of Haptophyta: implications for oceanography and chemotaxonomy. Mar. Ecol. Prog. Ser. 270, 83–102. Zapata, M., Garrido, J.L., Jeffrey, S.W., 2006. Chlorophyll c pigments: current status. In: Grimm, B., Porra, R.J., Rüdiger, W., Scheer, U. (Eds.), Chlorophylls and Bacteriochlorophylls: biochemistry, biophysics, functions and applications. Springer, Dordrecht, pp. 39–53.