The Use of Pigment Signatures to Assess Phytoplankton ... et al 2001 Cont... · pigment analyses. Although the pigment content of the cells varies with the physiological state of

Estuarine, Coastal and Shelf Science (2001) 52, 689–703doi:10.1006/ecss.2001.0785, available online at http://www.idealibrary.com on

The Use of Pigment Signatures to AssessPhytoplankton Assemblage Structure in EstuarineWaters

A. Ansotegui, J. M. Trigueros and E. Orivea

aLaboratorio de Ecologıa, Facultad de Ciencias, Universidad del Paıs Vasco, Apdo. 644, 48080 Bilbao, Spain

Received 12 January 2001 and accepted in revised form 10 May 2001

The seasonal dynamics of chlorophyll a and the main accessory pigments accompanied by microscopic observations onlive and fixed material were investigated in the Urdaibai estuary, Spain. Fucoxanthin was the dominant pigment duringthe peak in chlorophyll a, with which it was strongly correlated. Concentrations of fucoxanthin (81·30 �g l�1) in theupper estuary were amongst the highest found in the literature, and were mainly associated with diatoms and symbioticdinoflagellates. In the lower estuary, fucoxanthin showed values typical of coastal waters (<5 �g l�1) and was mainly dueto diatoms and prymnesiophytes. Chlorophyll b concentration was high along the estuary, followed the same seasonalpattern as chlorophyll a, and was associated with the presence of euglenophytes, chlorophytes and prasinophytes. Highvalues of 19�-butanoyloxyfucoxanthin were often measured, but no organisms containing this pigment were observed inlive or fixed samples. Alloxanthin and peridinin were found in low concentrations which was in agreement with cell countsof cryptophytes and peridinin-containing dinoflagellates. Two main patterns of phytoplankton assemblages were observedalong the estuary. In the upper segments, during the chlorophyll a maximum fucoxanthin containing algae masked theother algal groups, which were relatively more abundant during or after enhanced river flows. In the lower estuary,although dominated by fucoxanthin-containing algae, the other algal groups were important all year around. In this study,the use of diagnostic pigments has provided considerable insight into the temporal and spatial dynamics of phytoplanktonassemblages by detecting phytoplankton taxa generally underestimated or overlooked by microscopy.

� 2001 Academic Press

Keywords: photosynthetic pigments; HPLC; CHEMTAX; phytoplankton; diatoms; dinoflagellates; small flagellates;estuarine waters

aCorresponding author. E-mail: [email protected]

Introduction

Photosynthetic pigments have been widely used astaxonomic markers in the marine environment (Jeffreyet al., 1997) to assess the relative importance of themost delicate and/or smallest component of thephytoplankton, which are frequently underestimated.Such is the case of the small cyanobacteria (genusSynechococcus) and small prochlorophytes, both ofwhich are broadly distributed in the oligotrophicoceans and can be estimated by means of their pig-ment signatures. This technique has also been shownto be useful in the detection of fragile flagellates,which do not survive the fixative procedures necessaryfor microscopic observations.

Only a few accessory chlorophylls and carotenoidsshow an unambiguous chemotaxonomic interpret-ation. Among these, divinyl chlorophylls can be usedas pigment signatures for prochlorophytes (Goericke& Repeta, 1992), 19�-hexanoyloxyfucoxanthin for

0272–7714/01/060689+15 $35.00/0

some prymnesiophytes (Jeffrey & Wright, 1994) whileperidinin is the accessory pigment characteristic ofsome photosynthetic dinoflagellates. In many cases,care must be taken in assigning an accessory pigmentto a certain algal group. Fucoxanthin, which isfrequently associated with diatoms, occurs in allprymnesiophytes (Jeffrey & Wright, 1994), is presentin chrysophytes (Withers et al., 1981), and raphydo-phytes (Fiksdahl et al., 1984). The fucoxanthinderivative 19�-butanoyloxyfucoxanthin has beenassigned to pelagophytes (Bjørnland & Liaaen-Jensen,1989), but it has also been found in some prymnesio-phytes (Barlow et al., 1993; Jeffrey & Wright, 1994).Zeaxanthin appears in prochlorophytes, cyano-bacteria, chlorophytes and prasinophytes, whilstchlorophyll b is present in euglenophytes, chloro-phytes and prasinophytes, and these are, therefore,poor specific signature pigments. Furthermore, whileeuglenophytes and chlorophytes show a fixed pigmentpattern through the group, prasinophytes exhibit somediversity.

� 2001 Academic Press

690 A. Ansotegui et al.

The occurrence of symbiosis, with the subsequentadoption of the symbiont pigment pattern by the host,can also lead to misinterpretation. Alloxanthin, themajor carotenoid in cryptophytes, has been found inthe ciliate Mesodinium rubrum (Hibberd, 1977), whichpossesses cryptomonad-like endosymbionts, and inthe dinoflagellate Dinophysis norvegica (Meyer-Harms& Pollehne, 1998). In the same way, some dino-flagellates have diatoms, chrysophytes, green algae orprymnesiophytes as endosymbionts (Millie et al.,1993), making invalid the assumption that all photo-synthetic dinoflagellates contain peridinin. Therefore,when dealing with natural communities, micro-scopic observations are still required to obtain areliable interpretation of the information derived frompigment analyses.

Although the pigment content of the cells varieswith the physiological state of the algae, it has beenstated that both chlorophyll a and accessory pigmentsco-vary. This makes the chlorophyll a:accessory pig-ment ratios more constant than the pigment contentper cell in each phytoplankton species (Goericke &Montoya, 1998). These ratios can be used to assessthe contribution of each algal group to total chloro-phyll a (Gieskes et al., 1988; Everitt et al., 1990;Mackey et al., 1996).

Previous studies in the Urdaibai estuary to deter-mine the taxonomic composition of the phytoplank-ton by microscopy have revealed the dominance ofdiatoms and thecate dinoflagellates (Orive et al., 1998;Trigueros et al., 2000a, b). However, several studieson size-fractionation showed the relevance of thesmallest organisms in terms of biomass and primaryproduction (Franco, pers. comm; Revilla et al., 2000),denoting that these organisms might have been over-looked when observed at the microscope. In this work,accessory pigments complemented by microscopicobservations were used to assess the seasonal trends inphytoplankton assemblages along the trophic gradientof the highly dynamic Urdaibai estuary. By means ofboth procedures, the relative importance of the small-est and more fragile component of the phytoplanktonwas evaluated, and an attempt was made to assign thecorrect taxa to ambiguous accessory pigments.

Materials and methods

43° 15

0

N

'

43° 25'

2° 45' 2° 35'

1 2km

1

2

3

4

5

Bay of Biscay

Mundaka

Lowerestuary

Upperestuary

Wastewatertreatment plant

Gernika

F 1. Map of the study area showing the location of thesampling stations.

Study site

The Urdaibai Estuary drains into the Bay of Biscay inNorthern Spain (43�22�N; 2�40�W, Figure 1). Theestuary is 12·5 km in length, covers 1·9 km2 with anaverage depth of 3 m and a maximum width of 1·2 kmat the mouth. This estuary is dominated by river

discharge in the upper reaches and by tidal inflow inthe lower euhaline zone. The lower estuary is mostlywell mixed as a consequence of tidal flushing. Incontrast, the upper segment is partially mixed duringlow river flow but well mixed during enhanced riverflows (Orive et al., 1995). The upper region received ahigh nutrient load from a wastewater treatment plantand industrial sources. In this region, high levels ofchlorophyll a and primary production are common inspring and summer coinciding with periods of low tomoderate river flow. In the lower estuary, factorscontrolling phytoplankton growth are typical ofcoastal waters (nutrients, light and grazing) andchlorophyll a concentration follows the typicalseasonal succession of temperate coastal waters (Oriveet al., 1995; Revilla et al., 2000).

Pigment signatures in estuarine waters 691

Sampling

Five permanent stations (Figure 1), located in thelower (station 1), middle (stations 2 and 3) and upperestuary (stations 4 and 5) were visited at high tide, 32times from May 1996 to January 1998. Samples weretaken near monthly, with increased frequency inspring and summer. At each site, vertical profiles ofsalinity and temperature were obtained with a WTWMicroprocessor Conductivity Meter. Water sampleswere collected from near the surface (0·5 m depth)and 0·5 m from the bottom, transferred to darkcarboys and kept cool and shaded. Samples wereprocessed within 3 h of collection. Subsamples fornutrient, pigment and microscopic analyses wereremoved from bulk water samples.

Pigment analysis by HPLC

For pigment determination 0·2–2 l of water werefiltered under gentle vacuum (<150 mm Hg) ontoGF/F filters, immediately frozen in liquid nitrogenand stored at �20 �C until analysis. Pigments wereextracted in buffered methanol (98% methanol+2%0·5 M ammonium acetate) and stored for 24 h at4 �C. An aliquot of 100 �l of extract was injected intoa HPLC system equipped with a Rheodyne 7125injector, two Waters (501 and 510) pumps, aNovapack C-18 (150�3·9 mm, 4-�m particle size)column and a UV/visible detector (Waters LambdaMax Model 481) set at 440 nm for pigment detection.

The method for pigment separation was basicallythat of Gieskes et al. (1988). It consisted of a binarylinear gradient programmed as follows (minutes,% solvent A, % solvent B):(0, 10, 90) (20, 10, 0) (29,100, 0). Solvent A consisted of 70:30 (v/v) methanol:ethyl acetate and solvent B 70:25:5 (v/v/v) methanol:buffered phosphate (KH2PO4 0·05 M): ethyl acetate.

The system was calibrated with external standardsobtained commercially: chlorophylls a and b fromSigma, and carotenoids from the VKI Water QualityInstitute (Hørsholm, Denmark). Pigment peaks wereidentified by comparison with retention times of thestandards and with that of extracts of cultures ofselected phytoplankton species belonging to the mainalgal classes. The analytical precision of the HPLCdetermination was assessed by analysing repli-cates (n=3) of standard mixtures. The coefficients ofvariation obtained were below 3%.

Nutrient analysis

Samples filtered through GF/F filters were storedfrozen before analysis for dissolved nutrients (nitrate,

ammonium, phosphate and silicate) following Parsonset al. (1984).

Phytoplankton communities

For the identification of the most prominent membersof the phytoplankton, live and glutaraldehyde fixed(final concentration 0·5%) samples were observedunder inverted (Nikon) and direct (Leica) light mi-croscopy. To estimate the contribution of the differentalgal classes to total chlorophyll a the matrix factor-isation program CHEMTAX (Mackey et al., 1996,1997) was applied. The program uses a steepest-descent algorithm to find the best fit to the data basedon suggested pigment:chlorophyll a ratios of bothdiagnostic pigments and pigments present in severalphytoplankton groups for the phytoplankton groups tobe determined. This method estimates the abundanceof the algal classes, not necessarily from the sametaxonomic category, but characterized by a particularpigment fingerprint. Following Mackey et al. (1996),we divided the data set by stations and depth inorder to obtain as homogeneous subsets as possible,based on both microscopic and pigment data. Basedon these observations, the following groups of algaewere taken into account when applying theCHEMTAX program: containing fucoxanthin, con-taining 19�-butanoyloxyfucoxanthin, dinoflagellateswith peridinin, cryptophytes (alloxanthin), eugleno-phytes (chlorophyll b) and chlorophytes (chlorophyllb). For CHEMTAX purposes both Chlorophyceaeand Prasinophyceae were considered as chlorophytes.Each group of algae was characterized by a mainfingerprint pigment and by other accessory pigmentslike diadinoxanthin (for algae containing fucoxanthin,peridinin, 19�-butanoyloxyfucoxanthin and eugleno-phytes), violaxanthin and lutein (for chlorophytes)and neoxanthin (for euglenophytes and chlorophytes).

Statistical analyses

Relationships between pigments were determinedusing the non-parametric Spearman Rank correlationcoefficient.

Results

Hydrographic data

Maximum river discharge was observed in autumnand winter (data not shown). In spring and summeronly a few events of enhanced river flow wererecorded.


The main physical data obtained during the studyperiod are summarized in Table 1. Water temperatureexperienced broader seasonal changes in the upperestuary (from 6·3 �C to 25·8 �C) than in the lowerestuary (from 12·1 �C to 22.5 �C). Differences withdepth were not observed at any location. Duringthis study, the upper estuary (stations 4 and 5) wasoligo-meso-polyhaline (0·1–24·9 salinity) whilst themiddle (stations 2 and 3) was meso-poly-euhaline(18·8–34·8 salinity) and the lower (station 1) euhaline(>33).

Nutrient concentrations decreased markedlytowards the mouth of the estuary, where concen-trations were frequently at the level of detection(Table 1). Phosphate and ammonium were positivelycorrelated (r2=0·95, P<0·01) sharing a common ori-gin. In this estuary, both nutrients are mainly providedby the sewage treatment plant located at the head ofthe estuary.

Pigments distribution and abundance

In the upper and middle segments, chlorophyll a washigher at salinities characteristic of periods of low riverflow. Under these conditions, concentrations up to120 �g l�1 and 133 �g l�1 were recorded at stations 4and 5, respectively. In the middle segment, peaks ofthis pigment exceeded 20 �g l�1 (Figure 2). Chloro-phyll a followed a different seasonal pattern in thelower estuary where concentrations remained below6 �g l�1. Measured concentrations were highest inspring with minor peaks in early autumn. No cleardifferences in chlorophyll a concentration were foundbetween surface and bottom waters, except duringpeaks in the upper estuary.

Fifteen pigments were identified: chlorophyll c,peridinin, 19�-butanoyloxyfucoxanthin, fucoxanthin,neoxanthin, violaxanthin, diadinoxanthin, anther-axanthin, alloxanthin, diatoxanthin, lutein,�-carotene, chlorophyll b and occasionally, 19�-hexanoyloxyfucoxanthin and echinenone.

The major taxon-specific pigments were fucox-anthin, chlorophyll b, 19�-butanoyloxyfucoxanthin,alloxanthin and peridinin. Fucoxanthin was the mostabundant accessory pigment and showed the samespatial and temporal trends as chlorophyll a, decreas-ing drastically from the upper to the lower estuary(Figure 2). In most cases, peak concentrations offucoxanthin closely followed those of chlorophyll aand reached values of 80 �g l�1 in the upper estuaryduring April. Fucoxanthin concentrations reached10 �g 1�1 in the middle estuary during spring andsummer. In the lower estuary, fucoxanthin peaked in

spring with maximum concentrations of 4·8 �g 1�1 inApril. In this segment, some minor peaks of2·0 �g 1�1 were occasionally found in summer andautumn. Differences between surface and bottomwaters were only noticeable in the upper estuaryduring some blooms.

Values of chlorophyll b closely followed thoseof chlorophyll a in the upper and middle reaches(Figure 2). Concentrations of up to 14·5 �g 1�1 weremeasured in July 1997 at station 5, and 8·4 �g 1�1 atstation 4 in September 1996. In the middle estuarypeaks of more than 2·5 �g 1�1 were recorded in July1997. No clear temporal trend was observed in thelower estuary where chlorophyll b always remainedbelow 0·4 �g 1�1.

The concentration of 19�-butanoyloxyfucoxanthinwas high along the estuary, particularly in the upperand middle reaches (Figure 2). This pigment did notfollow any clear seasonal pattern at any station, andthe highest value (12·4 µg 1�1 was measured in theuppermost site in September 1997. This pigment alsoshowed high concentrations in the middle estuarywhere several peaks of more than 1·0 µg 1�1 weremeasured. In the lower estuary values remained below0·6 µg 1�1.

Alloxanthin was generally present in levels below1·0 µg 1�1, except for the upper estuary in summerwhen a peak of 3·6 �g 1�1 was recorded (Figure 2).In the lower estuary, the highest concentration(0·14 µg 1�1) was detected in May.

Peridinin was the least abundant pigment in theestuary, generally appearing in concentrations below1·0 �g 1�1 (Figure 2). Several peaks between 1·5–2·5 �g 1�1 were found in the upper estuary andoccasionally in the middle estuary. In the lowerestuary, the highest values (0·2–0·3 µg 1�1) werefound during the summer-autumn transition.

Other diagnostic pigments were found in lowconcentrations and data are not reported here.

To establish relationships between the major pig-ments, correlation analyses were performed separatelyfor each estuarine segment. For this exercise surfaceand bottom data were combined (Table 2). In theupper estuary, most pigments showed a signifi-cant positive correlation, except peridinin, whichwas not correlated with chlorophyll b and onlyweakly correlated to the other pigments. Similarresults were obtained from the middle estuary,although in this case peridinin was not correlated withany other pigment. In the lower estuary, chlorophyll awas only correlated with fucoxanthin and 19�-butanoyloxyfucoxanthin. The later pigment wasmoderately correlated with fucoxanthin and slightlywith alloxanthin.


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F 2. Spatial and temporal changes in chlorophyll a, fucoxanthin, chlorophyll b, 19�-butanoyloxyfucoxanthin,alloxanthin and peridinin.


Relationships between signature pigments andphytoplankton taxa

In the upper estuary, microscopic observationsrevealed that the peaks of chlorophyll a and those offucoxanthin were mainly associated with the diatomsCyclotella atomus and Thalassiosira guillardii and thedinoflagellate Peridinium foliaceum. In the middleestuary, peaks in chlorophyll a and fucoxanthin corre-sponded with maximum concentrations of diatoms ofthe genera Chaetoceros and Thalassiosira, and the dino-flagellate Peridinium quinquecorne. Occasionally, smallflagellates like Prymnesium which contain fucoxanthinwere observed. In the lower estuary, the mostprominent peaks in fucoxanthin concentration corre-sponded to mixed assemblages of diatoms and to alesser extent prymnesiophytes. In this region, prymne-siophytes like Phaeocystis and the coccolithophoridEmiliania huxleyi were occasionally observed in livesamples.

Microscopic observations failed to recognise live orfixed algae associated with 19�-butanoyloxyfucoxanthin.Among the chlorophyll b containing groups observedalong the estuary, the most prominent were eugleno-phytes of the genera Eutreptia and Eutreptiella; chloro-phytes of the genus Chlamydomonas and prasinophytesof the genera Pyramimonas, Tetraselmis, Nephroselmisand Micromonas-like cells. Among peridinin contain-ing dinoflagellates the most important was the genusPeridiniopsis in the upper reaches and Heterocapsatowards the mouth of the estuary. Among crypto-

phytes, the most conspicuous were large Cryptomonas-like cells in the upper reaches, while smaller cells likeChroomonas or Hemiselmis were common in the lowerestuary. Low numbers of the alloxanthin containingciliate Mesodinium rubrum was observed in some livesamples.

T 2. Spearman rank correlation coefficients matrix formain pigment data set (*P<0·05, **P<0·01) (fuco, fucox-anthin; bfu, 19�-butanoyloxyfucoxanthin; allox, alloxanthin;per, peridinin)

Lower estuary (n=64)fuco bfu allox

Chl a 0·818** 0·414**fuco 0·523**bfu 0·270**

Middle estuary (n=128)fuco Chl b bfu allox

Chl a 0·894** 0·465** 0·224* 0·669**fuco 0·370** 0·262** 0·535**Chl b 0·191* 0·419**bfu 0·211*

Upper estuary (n=128)fuco Chl b bfu allox per

Chl a 0·937** 0·627** 0·451** 0·697** 0·223*fuco 0·512** 0·454** 0·606** 0·302**Chl b 0·298** 0·407**bfu 0·428** 0·260**allox 0·238**

Contribution of different groups of algae to totalchlorophyll a

Fucoxanthin containing algae were the dominantgroup along the estuary during most of the studyperiod (Figure 3). In the lower region, this group ofalgae accounted for more than 75% of chlorophyll aduring biomass peaks. The high contribution (82%)was observed during the spring diatom bloom in1997. In the upper and middle estuary, the percentageof chlorophyll a attributed to fucoxanthin containingalgae was generally higher in spring and summer,being about 93% in April 1997 in the middle estuaryand almost 100% in July 1997 in the upper estuary.19�-butanoyloxyfucoxanthin containing algae consti-tuted one of the groups better represented in theestuary, showing their greatest contributions tochlorophyll a generally in summer and autumn. Inthe lower estuary, the highest contribution of 19�-butanoyloxyfucoxanthin to total chlorophyll a (39%)was found in December 1997 in bottom waters.Generally, 19�-butanoyloxyfucoxanthin containingalgae were proportionally more abundant in bottomwaters. In the upper estuary, the contribution of19�-butanoyloxyfucoxanthin increased coincidentwith the lowest values of total chlorophyll a. Chloro-phytes appeared in noticeable proportions in the lowerand middle estuary, being relatively less important inthe upper segment. In contrast, the contribution ofcryptophytes was higher in the upper segments, whereit peaked in summer. The contribution of eugleno-phytes was only occasionally important in summer inthe middle and upper estuary. Dinoflagellates withperidinin were a minor component of the community,reaching their highest contribution all along theestuary in summer and autumn.

In terms of the contribution of the different groupsof algae to total chlorophyll a, phytoplankton speciesdiversity was higher in the lower estuary. During mostof the year a mixed assemblage of diatoms, chloro-phytes, 19�-butanoyloxyfucoxanthin containing, andto a lesser extent, euglenophytes, cryptophytes anddinoflagellates with peridinin, was present. The con-centration of the signature pigments corresponding tosmall flagellates remained more constant through theyear in the lower estuary compared to the uppersegments, when the concentration of these pigments


showed strong fluctuations. In the upper estuary,occasional peaks of chlorophytes, euglenophytes andcryptophytes were observed, some coincided withpeaks in fucoxanthin. Others appeared after enhancedriver flows, when the upper estuary was recoveringfrom the wash out of cells.

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F 3. Spatial and temporal changes in the relative abundance of the different groups of algae as estimated by theCHEMTAX programme.

Pigment ratios

Differences in pigment ratio between the selectedinitial ratio and the ratio (final ratio) attributed by theCHEMTAX to each group of algae were found forsome of the groups. In addition, spatial and temporal


differences in the final ratio of each group of algaewere also observed for some clusters of algae. Table 3shows the pigment ratios attributed by theCHEMTAX program to the different groups of algae.While some pigment ratios remained constant for thewhole data sets, other exhibited marked changesbetween and within groups. Among the later, the ratiochlorophyll b:chlorophyll a for euglenophytes (0·406–1·239) and chlorophytes (0·330–0·572 and the ratiofucoxanthin:chlorophyll a (0·479–0·755) for algaewith fucoxanthin were the most variable.

Discussion

Signature pigments and phytoplankton assemblages

The analysis of algal pigments has proved to be usefulfor the determination of phytoplankton assemblagesand their dynamics in marine waters, revealing a closerelationship between the relative abundance of differ-ent signature pigments and the availability of nutri-ents. It is well established that small phytoplanktoncells are associated with areas of low nutrient concen-trations, whereas the importance of the larger species,mainly diatoms, increases with the availability of nu-trients. High levels of divinyl chlorophylls and zeaxan-thin are characteristic of oligotrophic areas dominatedby picoplanktonic prochlorophytes and cyanobacteria(e.g. Latasa & Bidigare, 1998). Pigments such aschlorophyll b, 19�-butanoyloxyfucoxanthin and 19�-hexanoyloxyfucoxanthin, corresponding to small flag-ellates, have more frequently been measured in eddiesand other moderately eutrophic areas (Bustillos-Guzmn et al., 1995; Barlow et al., 1997; Meyer-Harms et al., 1999). In productive areas such asupwelling, frontal and coastal regions, fucoxanthin,mainly from diatoms, is frequently the dominantpigment (Head et al., 1997; Peeken, 1997; Ahel &Terzic, 1998).

Estuaries display a wide range of trophic con-ditions linked to the supply of nutrients from naturaland anthropogenic sources and dilution of thenutrient-rich estuarine waters with coastal waters.Fucoxanthin, the pigment signature for diatoms,prymnesiophytes and chrysophytes, was the dominantpigment in the Urdaibai estuary. During peaks ofchlorophyll a, fucoxanthin was found in the upper andmiddle estuary in concentrations much higher thanthose reported for other estuarine or marine area(Table 4). The highest concentrations of this pigmentin the lower marine estuary are consistent with thosefound by Ahel and Terzic (1998) in the coastal watersof the Adriatic Sea, but much higher that thosereported in the literature for open waters. Although

there are only a few studies dealing with estuarinepigments, fucoxanthin has been reported as the domi-nant accessory pigment in other estuaries, beingattributed to diatoms (Ahel et al., 1996; Brotas &Plante-Cuny, 1998), chrysophytes and prymnesio-phytes (Tester et al., 1995). According to microscopicobservations, in the upper segments of the Urdaibaiestuary, diatoms and dinoflagellates accounted forfucoxanthin, while in the lower estuary this pigmentwas due to diatoms and prymnesiophytes.

In addition to pelagophytes, the accessory pigment19�-butanoyloxyfucoxanthin has been found in someprymnesiophytes (Jeffrey & Wright, 1994), and insome symbiont-bearing dinoflagellates (Bjørnland &Liaaen-Jensen, 1989). The relatively high amounts of19�-butanoyloxyfucoxanthin found in the Urdaibaiestuary could be accounted for by prymnesiophytes,widely distributed through the oceans (Andersenet al., 1996), or to pelagophytes. The later group ofalgae has been found in the open ocean and coastalecosystems, where they are responsible for browntides (Buskey et al., 1997). The small size ofthese groups precluded their identification by themicroscopic facilities used in this study. However,with the chromatographic method used, 19�-butanoyloxyfucoxanthin co-elutes with siphonaxan-thin, the principal accessory pigment in siphonal greenalgae (Anderson et al., 1985). Taking into accountthe absence of siphonal algae in the estuary due tothe soft nature of its bottom, we conclude that19�-butanoyloxyfucoxanthin was indicative of pel-agophytes in the estuary. The concentrations of19�-butanoyloxyfucoxanthin (up to 0·6 �g 1�1) inthe lower estuary are of the same order of magnitudeas the maxima found by Ahel and Terzic (1998) incoastal waters of the Adriatic. However, concen-trations of this pigment in the middle and upperestuary are much higher than those reported for otherestuarine or marine areas (see Table 4).

Other accessory pigments such as chlorophyll b,alloxanthin and peridinin appeared in quantities moresimilar to those obtained in other estuaries and coastalareas (see Table 4), except for some extraordinarilyhigh peaks recorded in the middle and upper estuary.The method used in this study does not separatelutein from zeaxanthin. Lutein is the major carotenoidin higher plants and in some members of the Chloro-phyta. Zeaxanthin is used as a signature pigment forcyanobacteria and prochlorophytes and takes partin the violaxanthin cycle in the chlorophytes andprasinophytes. We have not found any reference in theliterature reporting the presence of prochlorophytes inestuarine environments, although high abundance ofblue green algae had been found in some estuaries


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(e.g. Bianchi et al., 1993). In this study, we considerthat a peak corresponding to the mixture of lutein andzeaxanthin was mainly due to the former. Theassumption was based on microscopic observations,which showed a strong relationship between peaks oflutein-zeaxanthin and the abundance of chlorophytesin the samples. Filamentous or colonial blue-greenalgae were not observed in the samples. Furthermore,freshwater cyanobacteria that might have been flushedfrom the river can be characterized by carotenoidssuch as myxoxanthophyll and echinenone (Nichols,1973). Both are detectable by the chromatographicmethod used but were not detected. Finally, duringfreshets, the estuary is subject to inputs of vascularplant detritus, which represent another source oflutein.

Despite fucoxanthin being the dominant accessorypigment along the estuary, the phytoplankton com-munity was generally more diverse and included dino-flagellates with and without peridinin, cryptophytes,euglenophytes and chlorophytes. Indeed, a back-ground of mixed flagellates on which peaks of diatomswere superimposed was characteristic of the lowermarine estuary and this agrees well with results fromother coastal waters (Hallegraeff, 1981). In the upperestuary, fucoxanthin-containing algae generallymasked the other algal groups, except during somepeaks of euglenophytes, chlorophytes and crypto-phytes, most of which were recorded after freshets,coinciding with relatively low phytoplankton biomass.Based on microscopic observations, we presume thatin absence of mesozooplankton, which do not growefficiently in the upper region, heterotrophic micro-plankton (ciliates and heterotrophic dinoflagellatessuch as Protoperidinium achromaticum and Oxyrrhismarina), exert a stronger grazing pressure on smallflagellates than on diatoms and dinoflagellates, whichexperience enhanced growth during periods of highresidence time of the water.

Pigment ratios

A crucial step in the use of pigment signatures toestimate the contribution of different algal groups tototal chlorophyll a, is the selection of the correctaccessory pigment:chlorophyll a ratios as conversionfactors. The initial pigment ratios considered in thisstudy were obtained from Mackey et al. (1997) andmost of them were based on phytoplankton cultures.The same initial ratios were chosen for all the clustersof samples. However, whereas differences betweeninitial and final ratios were not found for some pig-ments, others experienced noticeable changes in theirfinal ratios respective to the initial ones. Nevertheless,

all ratios used were within the range reported in theliterature for other estuarine and marine areas.

The final fucoxanthin:chlorophyll a ratio for fucox-anthin containing algae varied along with the estuary.Ratios from the lower and middle estuary had valueswhich agree well with those reported for diatoms inmarine areas (Gieskes & Kraay, 1983; Barlow et al.,1995), estuaries (Meyer-Harms & von Bodungen,1997) and from cultures (Soma et al., 1993; Llewellyn& Gibb, 2000). However, in the upper estuary theratio was lower (0·479), although within the range ofreported values. Meyer-Harms et al., (1999) obtaineda similar ratio of 0·450 in the Norwegian Sea duringand after a spring diatom bloom and Letelier et al.,(1993) reported a value of 1·25 for shade adapteddiatoms. Based on cultures of the diatoms Phaeodac-tylum tricornutum and Ditylum brightwellii Schluteret al., (2000) obtained a broad range of ratios (0·485to 1·218 reflecting between and within species differ-ences in response to the light regime. In the Urdaibaiestuary, the presence of the fucoxanthin containingdinoflagellate Peridinium foliaceum which may havedifferent ratios than diatoms, could explain the differ-ences in the fucoxanthin:chlorophyll a ratio betweenthe upper and the lower estuary. The ratio of dia-dinoxanthin:chlorophyll a for fucoxanthin containingalgae ranged from 0·056 to 0·110, with highest valuesat the upper most turbid station. Based on cultures,Schluter et al., (2000) found that this ratio fluctuatedstrongly in response to the light regime and wasaffected by the physiological state of the algae.

Fucoxanthin:chlorophyll a and 19�-butanoyloxy-fucoxanthin:chlorophyll a ratios for 19�-butanoyl-oxyfucoxanthin containing algae (0·974 and 1·563,respectively), taken from a culture of Pelagococcussubviridis (Jeffrey & Wright, 1997), remained constantin all data sets. These ratios are similar to thoseobtained by Everitt et al., (1990) and Mackey et al.,(1998) for chrysophytes in the Equatorial Pacific,but are slightly higher than those reported by Meyer-Harms et al., (1999) for prymnesiophytes in theNorwegian Sea. The ratio of diadinoxanthin:chlorophyll a for 19�-butanoyloxyfucoxanthin-containing algae ranged from 0·119 to 0·800, beinghighest in the lower and middle estuary. The spatialdifferences can be interpreted as an adaptation of thealgae to the different light regime of the estuary. Theconcentration of diadinoxanthin, the epoxidated formof the xanthophyll cycle in chromophytes, increaseswith light intensity in the lower, less turbid regions ofthe estuary.

To estimate the contribution of peridinin contain-ing dinoflagellates to total chlorophyll a, an initialratio of 1·063, obtained by Jeffrey and Wright, (1997)


from a culture of Amphidinium carterae was used. Abroad range of final peridinin:chlorophyll a ratios werehowever obtained (1·063–1·295). Although many ofthese ratios were higher than those reported in theliterature (Schluter et al., 2000), Mackey et al., (1998)found a comparable final ratio (1·000) in deepsamples from the Western Equatorial Pacific, andPinckney et al. (1998) obtained a value of 1·176 forthe moderately eutrophic Neuse River Estuary. A ratioof 1·265 was however obtained from an extract of thedinoflagellate Heterocapsa rotundata from the estuaryof Urdaibai. The initial diadinoxanthin:chlorophyll aratio for dinoflagellates (0·241 remained unchangedafter the application of the CHEMTAX program.Most published values come from the cultures(Demers et al., 1991; Schluter et al., 2000) and arequite similar to those used here.

Alloxanthin is the main pigment signature forcryptophytes, although it is also present in the ciliateMesodinium rubrum. The ciliate was observed in theestuary of Urdaibai in live samples, but not in greatnumbers. We may therefore assume that mostalloxanthin belonged to cryptophytes. The allox-anthin:chlorophyll a ratio remained unchanged(0·229) with respect to the initial ratio through theestuary. This ratio is within the values reported in theliterature, which range from 0·105 (Mackey et al.,1998 to 0·541 (Hager & Stransky, 1970). Values closeto those obtained in this study were found in theNorth Sea (0·234) Gieskes & Kraay, 1983), AlboranSea (0·278) (Barlow et al., 1995), Southern Ocean(0·186) (Wright et al., 1996) and New Port Estuary(0·329) (Tester et al., 1995).

The final chlorophyll b:chlorophyll a ratio foreuglenophytes varied between 0·406 and 1·239, beinghighest in bottom waters of the upper estuary wherelight availability is low. It has been suggested that ingreen algae the increase in chlorophyll b relative tochlorophyll a could mean a weak chromatic adaptation(Wood, 1979). In this sense, Mackey et al., (1998)found increasing values of this ratio with depth foreuglenophytes in the Equatorial Pacific. Our resultshowever, disagree with those of Schluter et al., (2000)who found that this ratio increases with light intensity.The same author observed that this ratio also increasesduring the stationary phase of the culture, whichmakes it difficult to explain the field data. The rangeof diadinoxanthin:chlorophyll a (0·042–0·230) andneoxanthin:chlorophyll a (0·015–0·030) ratios ob-tained in this study for euglenophytes are comparableto those obtained by Mackey et al., (1998).

The final ratios of chlorophytes fall within the rangeof those found by several authors in different systems,for example Gieskes et al., (1998) in the Banda Sea

and Tester et al., (1995) in New Port Estuary. How-ever, whereas the CHEMTAX program left a finalchlorophyll b:chlorophyll a ratio similar to the initialone (0·569) in the upper estuary, the final ratiodecreased to values as low as 0·330 towards themiddle and lower segments. These spatial differencesappear to be a consequence of the different lightregime of the different estuarine segments rather thancaused by taxonomic differences. Chlorophytes thusdominate the upper estuary while prasinophytes arerelatively more abundant in the lower segments.Several studies (e.g. Brown & Jeffrey, 1992, Wood,1979, and Schluter et al., 2000) have shown thatprasinophytes generally contain higher chlorophyllb:chlorophyll a ratios than chlorophytes. A broadrange of final lutein:chlorophyll a ratios (0·186–0·390) were obtained for chlorophytes and werehigher than those reported by Wright et al., (1996) forthe Southern Ocean (0·127) and by Mackey et al.,(1998) for the Equatorial Pacific (0·042–0·120). Theincrease in this ratio towards the upper estuary may beexplained by the presence of a higher amount ofdetritus of vascular plants, which contain more luteinper gram of biomass than non-vascular plants(Bianchi et al., 1993). The final neoxanthin:chloro-phyll a (0·047–0·191) and violaxanthin:chlorophyll a(0·042–0·055 ratios for chlorophytes obtained in thisstudy are within the range found in the EquatorialPacific by Mackey et al., (1998) and in cultures ofboth chlorophytes and prasinophytes by Jeffrey andWright (1997).

The use of diagnostic pigments accompanied bymicroscopic observations of live and fixed phytoplank-ton samples has thus provided considerable insightinto the seasonal dynamic of phytoplankton assem-blages along the trophic and salinity gradient of theUrdaibai estuary. By means of specific carotenoid pig-ments, the relative importance of small or fragile cellshas been assessed whereas microscopic observationshave been of great help to identify the taxa contribu-ting to ambiguous accessory pigments. The combi-nation of both methods enabled identification of themain taxonomic groups contributing to fucoxanthincontaining algae, alloxanthin containing and chloro-phytes, as well estimating their relative contribution.Further research is still needed to prove the presenceof pelagophytes in the estuary as well as to understandbetter the partitioning of 19�-butanoyloxyfucoxanthinwithin the different algal groups.

Acknowledgements

The University of the Basque Country (projectUPV 118.310-EB124/97) and the Department of


Education, Universities and Investigation of theBasque Government (project GV PI-1998-67) sup-ported this work. A. Ansotegui was also funded by agrant from the Spanish Ministry of Education andScience and J. M. Trigueros by a grant fromthe Department of Education, Universities andInvestigation of the Basque Government.

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The Use of Pigment Signatures to Assess Phytoplankton ... et al 2001 Cont... · pigment analyses. Although the pigment content of the cells varies with the physiological state of