The Use of Pigment Signatures to Assess Phytoplankton Assemblage Structure in Estuarine Waters

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

The Use of Pigment Signatures to Assess Phytoplankton Assemblage Structure in Estuarine Waters A. Ansotegui, J. M. Trigueros and E. Orivea a

Laboratorio de Ecologı´a, Facultad de Ciencias, Universidad del Paı´s 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 on live and fixed material were investigated in the Urdaibai estuary, Spain. Fucoxanthin was the dominant pigment during the peak in chlorophyll a, with which it was strongly correlated. Concentrations of fucoxanthin (81·30
g l
1) in the upper estuary were amongst the highest found in the literature, and were mainly associated with diatoms and symbiotic dinoflagellates. In the lower estuary, fucoxanthin showed values typical of coastal waters (<5
g l
1) and was mainly due to diatoms and prymnesiophytes. Chlorophyll b concentration was high along the estuary, followed the same seasonal pattern as chlorophyll a, and was associated with the presence of euglenophytes, chlorophytes and prasinophytes. High values of 19 -butanoyloxyfucoxanthin were often measured, but no organisms containing this pigment were observed in live or fixed samples. Alloxanthin and peridinin were found in low concentrations which was in agreement with cell counts of cryptophytes and peridinin-containing dinoflagellates. Two main patterns of phytoplankton assemblages were observed along the estuary. In the upper segments, during the chlorophyll a maximum fucoxanthin containing algae masked the other 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 phytoplankton assemblages 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

Introduction Photosynthetic pigments have been widely used as taxonomic markers in the marine environment (Jeffrey et al., 1997) to assess the relative importance of the most delicate and/or smallest component of the phytoplankton, which are frequently underestimated. Such is the case of the small cyanobacteria (genus Synechococcus) and small prochlorophytes, both of which are broadly distributed in the oligotrophic oceans and can be estimated by means of their pigment signatures. This technique has also been shown to be useful in the detection of fragile flagellates, which do not survive the fixative procedures necessary for microscopic observations. Only a few accessory chlorophylls and carotenoids show an unambiguous chemotaxonomic interpretation. Among these, divinyl chlorophylls can be used as pigment signatures for prochlorophytes (Goericke & Repeta, 1992), 19 -hexanoyloxyfucoxanthin for a

Corresponding author. E-mail: [email protected]

0272–7714/01/060689+15 $35.00/0

some prymnesiophytes (Jeffrey & Wright, 1994) while peridinin is the accessory pigment characteristic of some photosynthetic dinoflagellates. In many cases, care must be taken in assigning an accessory pigment to a certain algal group. Fucoxanthin, which is frequently associated with diatoms, occurs in all prymnesiophytes (Jeffrey & Wright, 1994), is present in chrysophytes (Withers et al., 1981), and raphydophytes (Fiksdahl et al., 1984). The fucoxanthin derivative 19 -butanoyloxyfucoxanthin has been assigned to pelagophytes (Bjørnland & Liaaen-Jensen, 1989), but it has also been found in some prymnesiophytes (Barlow et al., 1993; Jeffrey & Wright, 1994). Zeaxanthin appears in prochlorophytes, cyanobacteria, chlorophytes and prasinophytes, whilst chlorophyll b is present in euglenophytes, chlorophytes and prasinophytes, and these are, therefore, poor specific signature pigments. Furthermore, while euglenophytes and chlorophytes show a fixed pigment pattern through the group, prasinophytes exhibit some diversity.  2001 Academic Press

690 A. Ansotegui et al.

The occurrence of symbiosis, with the subsequent adoption of the symbiont pigment pattern by the host, can also lead to misinterpretation. Alloxanthin, the major carotenoid in cryptophytes, has been found in the ciliate Mesodinium rubrum (Hibberd, 1977), which possesses cryptomonad-like endosymbionts, and in the dinoflagellate Dinophysis norvegica (Meyer-Harms & Pollehne, 1998). In the same way, some dinoflagellates have diatoms, chrysophytes, green algae or prymnesiophytes as endosymbionts (Millie et al., 1993), making invalid the assumption that all photosynthetic dinoflagellates contain peridinin. Therefore, when dealing with natural communities, microscopic observations are still required to obtain a reliable interpretation of the information derived from pigment analyses. Although the pigment content of the cells varies with the physiological state of the algae, it has been stated that both chlorophyll a and accessory pigments co-vary. This makes the chlorophyll a:accessory pigment ratios more constant than the pigment content per cell in each phytoplankton species (Goericke & Montoya, 1998). These ratios can be used to assess the contribution of each algal group to total chlorophyll a (Gieskes et al., 1988; Everitt et al., 1990; Mackey et al., 1996). Previous studies in the Urdaibai estuary to determine the taxonomic composition of the phytoplankton by microscopy have revealed the dominance of diatoms and thecate dinoflagellates (Orive et al., 1998; Trigueros et al., 2000a, b). However, several studies on size-fractionation showed the relevance of the smallest organisms in terms of biomass and primary production (Franco, pers. comm; Revilla et al., 2000), denoting that these organisms might have been overlooked when observed at the microscope. In this work, accessory pigments complemented by microscopic observations were used to assess the seasonal trends in phytoplankton assemblages along the trophic gradient of the highly dynamic Urdaibai estuary. By means of both procedures, the relative importance of the smallest and more fragile component of the phytoplankton was evaluated, and an attempt was made to assign the correct taxa to ambiguous accessory pigments. Materials and methods Study site The Urdaibai Estuary drains into the Bay of Biscay in Northern Spain (4322 N; 240 W, Figure 1). The estuary is 12·5 km in length, covers 1·9 km2 with an average depth of 3 m and a maximum width of 1·2 km at the mouth. This estuary is dominated by river

2° 45'

2° 35'

43° 25'

Bay of Biscay

Mundaka

Lower estuary

1

2

3

N Upper estuary

4

0

1 km

2 Wastewater treatment plant

5

Gernika 43° 15'

F 1. Map of the study area showing the location of the sampling stations.

discharge in the upper reaches and by tidal inflow in the lower euhaline zone. The lower estuary is mostly well mixed as a consequence of tidal flushing. In contrast, the upper segment is partially mixed during low river flow but well mixed during enhanced river flows (Orive et al., 1995). The upper region received a high nutrient load from a wastewater treatment plant and industrial sources. In this region, high levels of chlorophyll a and primary production are common in spring and summer coinciding with periods of low to moderate river flow. In the lower estuary, factors controlling phytoplankton growth are typical of coastal waters (nutrients, light and grazing) and chlorophyll a concentration follows the typical seasonal succession of temperate coastal waters (Orive et al., 1995; Revilla et al., 2000).

Pigment signatures in estuarine waters 691

Sampling Five permanent stations (Figure 1), located in the lower (station 1), middle (stations 2 and 3) and upper estuary (stations 4 and 5) were visited at high tide, 32 times from May 1996 to January 1998. Samples were taken near monthly, with increased frequency in spring and summer. At each site, vertical profiles of salinity and temperature were obtained with a WTW Microprocessor Conductivity Meter. Water samples were collected from near the surface (0·5 m depth) and 0·5 m from the bottom, transferred to dark carboys and kept cool and shaded. Samples were processed within 3 h of collection. Subsamples for nutrient, pigment and microscopic analyses were removed from bulk water samples. Pigment analysis by HPLC For pigment determination 0·2–2 l of water were filtered under gentle vacuum (<150 mm Hg) onto GF/F filters, immediately frozen in liquid nitrogen and stored at
20 C until analysis. Pigments were extracted in buffered methanol (98% methanol+2% 0·5 M ammonium acetate) and stored for 24 h at 4 C. An aliquot of 100
l of extract was injected into a HPLC system equipped with a Rheodyne 7125 injector, two Waters (501 and 510) pumps, a Novapack C-18 (1503·9 mm, 4-
m particle size) column and a UV/visible detector (Waters Lambda Max Model 481) set at 440 nm for pigment detection. The method for pigment separation was basically that of Gieskes et al. (1988). It consisted of a binary linear 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 standards obtained commercially: chlorophylls a and b from Sigma, and carotenoids from the VKI Water Quality Institute (Hørsholm, Denmark). Pigment peaks were identified by comparison with retention times of the standards and with that of extracts of cultures of selected phytoplankton species belonging to the main algal classes. The analytical precision of the HPLC determination was assessed by analysing replicates (n=3) of standard mixtures. The coefficients of variation obtained were below 3%.

ammonium, phosphate and silicate) following Parsons et al. (1984). Phytoplankton communities For the identification of the most prominent members of the phytoplankton, live and glutaraldehyde fixed (final concentration 0·5%) samples were observed under inverted (Nikon) and direct (Leica) light microscopy. To estimate the contribution of the different algal classes to total chlorophyll a the matrix factorisation program CHEMTAX (Mackey et al., 1996, 1997) was applied. The program uses a steepestdescent algorithm to find the best fit to the data based on suggested pigment:chlorophyll a ratios of both diagnostic pigments and pigments present in several phytoplankton groups for the phytoplankton groups to be determined. This method estimates the abundance of the algal classes, not necessarily from the same taxonomic category, but characterized by a particular pigment fingerprint. Following Mackey et al. (1996), we divided the data set by stations and depth in order to obtain as homogeneous subsets as possible, based on both microscopic and pigment data. Based on these observations, the following groups of algae were taken into account when applying the CHEMTAX program: containing fucoxanthin, containing 19 -butanoyloxyfucoxanthin, dinoflagellates with peridinin, cryptophytes (alloxanthin), euglenophytes (chlorophyll b) and chlorophytes (chlorophyll b). For CHEMTAX purposes both Chlorophyceae and Prasinophyceae were considered as chlorophytes. Each group of algae was characterized by a main fingerprint pigment and by other accessory pigments like diadinoxanthin (for algae containing fucoxanthin, peridinin, 19 -butanoyloxyfucoxanthin and euglenophytes), violaxanthin and lutein (for chlorophytes) and neoxanthin (for euglenophytes and chlorophytes). Statistical analyses Relationships between pigments were determined using the non-parametric Spearman Rank correlation coefficient.

Results Hydrographic data

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

Maximum river discharge was observed in autumn and winter (data not shown). In spring and summer only a few events of enhanced river flow were recorded.

692 A. Ansotegui et al.

The main physical data obtained during the study period are summarized in Table 1. Water temperature experienced broader seasonal changes in the upper estuary (from 6·3 C to 25·8 C) than in the lower estuary (from 12·1 C to 22.5 C). Differences with depth were not observed at any location. During this study, the upper estuary (stations 4 and 5) was oligo-meso-polyhaline (0·1–24·9 salinity) whilst the middle (stations 2 and 3) was meso-poly-euhaline (18·8–34·8 salinity) and the lower (station 1) euhaline (>33). Nutrient concentrations decreased markedly towards the mouth of the estuary, where concentrations were frequently at the level of detection (Table 1). Phosphate and ammonium were positively correlated (r2 =0·95, P<0·01) sharing a common origin. In this estuary, both nutrients are mainly provided by the sewage treatment plant located at the head of the estuary.

Pigments distribution and abundance In the upper and middle segments, chlorophyll a was higher at salinities characteristic of periods of low river flow. Under these conditions, concentrations up to 120
g l
1 and 133
g l
1 were recorded at stations 4 and 5, respectively. In the middle segment, peaks of this pigment exceeded 20
g l
1 (Figure 2). Chlorophyll a followed a different seasonal pattern in the lower estuary where concentrations remained below 6
g l
1. Measured concentrations were highest in spring with minor peaks in early autumn. No clear differences in chlorophyll a concentration were found between surface and bottom waters, except during peaks in the upper estuary. Fifteen pigments were identified: chlorophyll c, peridinin, 19 -butanoyloxyfucoxanthin, fucoxanthin, neoxanthin, violaxanthin, diadinoxanthin, antheraxanthin, alloxanthin, diatoxanthin, lutein,
-carotene, chlorophyll b and occasionally, 19 hexanoyloxyfucoxanthin and echinenone. The major taxon-specific pigments were fucoxanthin, chlorophyll b, 19 -butanoyloxyfucoxanthin, alloxanthin and peridinin. Fucoxanthin was the most abundant accessory pigment and showed the same spatial and temporal trends as chlorophyll a, decreasing drastically from the upper to the lower estuary (Figure 2). In most cases, peak concentrations of fucoxanthin closely followed those of chlorophyll a and reached values of 80
g l
1 in the upper estuary during April. Fucoxanthin concentrations reached 10
g 1
1 in the middle estuary during spring and summer. In the lower estuary, fucoxanthin peaked in

spring with maximum concentrations of 4·8
g 1
1 in April. In this segment, some minor peaks of 2·0
g 1
1 were occasionally found in summer and autumn. Differences between surface and bottom waters were only noticeable in the upper estuary during some blooms. Values of chlorophyll b closely followed those of chlorophyll a in the upper and middle reaches (Figure 2). Concentrations of up to 14·5
g 1
1 were measured in July 1997 at station 5, and 8·4
g 1
1 at station 4 in September 1996. In the middle estuary peaks of more than 2·5
g 1
1 were recorded in July 1997. No clear temporal trend was observed in the lower estuary where chlorophyll b always remained below 0·4
g 1
1. The concentration of 19 -butanoyloxyfucoxanthin was high along the estuary, particularly in the upper and middle reaches (Figure 2). This pigment did not follow any clear seasonal pattern at any station, and the highest value (12·4 µg 1
1 was measured in the uppermost site in September 1997. This pigment also showed high concentrations in the middle estuary where several peaks of more than 1·0 µg 1
1 were measured. In the lower estuary values remained below 0·6 µg 1
1. Alloxanthin was generally present in levels below 1·0 µg 1
1, except for the upper estuary in summer when 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 the estuary, generally appearing in concentrations below 1·0
g 1
1 (Figure 2). Several peaks between 1·5– 2·5
g 1
1 were found in the upper estuary and occasionally in the middle estuary. In the lower estuary, the highest values (0·2–0·3 µg 1
1) were found during the summer-autumn transition. Other diagnostic pigments were found in low concentrations and data are not reported here. To establish relationships between the major pigments, correlation analyses were performed separately for each estuarine segment. For this exercise surface and bottom data were combined (Table 2). In the upper estuary, most pigments showed a significant positive correlation, except peridinin, which was not correlated with chlorophyll b and only weakly correlated to the other pigments. Similar results were obtained from the middle estuary, although in this case peridinin was not correlated with any other pigment. In the lower estuary, chlorophyll a was only correlated with fucoxanthin and 19 butanoyloxyfucoxanthin. The later pigment was moderately correlated with fucoxanthin and slightly with alloxanthin.

34·4 (0·5) 34·6 (0·4)

29·3 (4·0) 29·5 (3·0)

20·9 (5·9) 24·4 (4·4)

10·6 (5·5) 15·8 (5·5)

6·6 (4·8) 15·8 (5·6)

Station 1 S B

Station 2 S B

Station 3 S B

Station 4 S B

Station 5 S B

Mean (SD)

0·2–18·6 0·1–19·9

0·7–19·4 2·2–24·9

5·7–28·5 13·2–33·3

18·8–34·6 23·2–34·8

33·2–35·0 33·5–35·0

Range

Salinity

18·6 (4·4) 18·6 (4·4)

18·8 (4·6) 18·8 (4·2)

19·2 (4·2) 18·8 (4·0)

18·8 (3·8) 18·6 (3·6)

18·3 (3·0) 18·2 (2·9)

Mean (SD)

2·5 (1·2) 1·4 (1·0)

Mean (SD)

9·1–99·7 4·8–74·2

6·3–25·6 89·0 (30·0) 42·8–153·8 6·3–25·8 74 (29·4) 21·5–143·9

6·2–26·5 63·5 (30·6) 13·2–125·2 6·9–25·3 45·5 (23·9) 9·6–117·6

7·3–25·1 38·9 (24·5) 7·3–24·4 23·7 (16·0)

2·0–39·9 1·4–32·2

0·5–5·0 0–4·2

Range

Silicate (
M)

7·9–24·7 14·2 (9·7) 8·6–24·0 9·7 (8·1)

12·1–22·5 12·3–22·4

Range

Temperature (C)

17·7 (9·0) 12·0 (6·9)

5·9 (3·6) 4·3 (2·4)

2·7 (1·3) 1·9 (1·4)

0·8 (0·6) 0·7 (0·5)

0·1 (0·1) 0·1 (0·1)

Mean (SD)

86·2 (45·2) 67·0 (36·8)

50·3 (26·1) 35·6 (19·4)

15·7 (13·2) 11·7 (8·6)

1·7 (2·1) 1·4 (2·2)

Mean (SD)

6·7 (6·7) 4·2 (4·4)

1·2 (1·4) 1·0 (1·2)

Mean (SD)

21·2–200·3 28·8 (19·7) 3·6–85·7 15·9–204·8 17·3 (13·6) 2·2–58·4

4·2–42·8 0·4–27·1

0–31·1 0–18·3

0–5·0 0–4·3

Range

Nitrate (
M)

8·1–138·9 16·1 (9·7) 1·0–86·6 10·2 (6·6)

0·7–48·6 0–26·5

0–10·0 0–10·0

Range

Ammonia (
M)

2·9–39·3 232·4 (142·7) 61·4–698·4 26·8 (18·4) 4·7–77·5 2·9–33·9 152·1 (75·1) 35·8–320·3 18·9 (14·0) 0·7–62·7

1·1–18·9 1·4–14·3

0·7–7·0 0·5–8·4

0·1–2·4 0·1–1·9

0–0·5 0–0·4

Range

Phosphate (
M)

T 1. Summary of surface (S) and bottom (B) water characteristics along the estuary of Urdaibai during the study period (May 1996–January 1998)

Pigment signatures in estuarine waters 693

Surface

Bottom

–1

Chlorophyll a (mg l )

M J J J J JLJLJL A A A S S O F M A A M M J J JLJL A A S S O N D J

Fucoxanthin (mg l–1)

M J J J J JLJLJL A A A S S O F M A A M M J J JLJL A A S S O N D J

1996

5

5

4

4

3

3

2

2

1

M J J J J JLJLJL A A A S S O F M A A M M J J JLJL A A S S O N D J

5

5

4

4

3

3

2

2

1

M J J J J JLJLJL A A A S S O F M A A M M J J JLJL A A S S O N D J

1997

1996

Month

1

1997 Month

<2

10

Chlorophyll b (mg l–1)

M J J J J JLJLJL A A A S S O F M A A M M J J JLJL A A S S O N D J

19' -Butanoyloxyfucoxanthin (mg l–1)

Alloxanthin (mg l–1)

M J J J J JLJLJL A A A S S O F M A A M M J J JLJL A A S S O N D J

Peridinin (mg l–1)

M J J J J JLJLJL A A A S S O F M A A M M J J JLJL A A S S O N D J

25

<50

5

5

4

4

3

3

2

2

1

M J J J J JLJLJL A A A S S O F M A A M M J J JLJL A A S S O N D J

1

5

5

4

4

3

3

2

2

1

M J J J J JLJLJL A A A S S O F M A A M M J J JLJL A A S S O N D J

1

5

5

4

4

3

3

2

2

1

M J J J J JLJLJL A A A S S O F M A A M M J J JLJL A A S S O N D J

1

5

5

4

4

3

3

2

2

1

M J J J J JLJLJL A A A S S O F M A A M M J J JLJL A A S S O N D J

1997

1996

Month

Stations

M J J J J JLJLJL A A A S S O F M A A M M J J JLJL A A S S O N D J

1996

1

1

1997 Month

<0.2

0.5

1.0

<1.5

F 2. Spatial and temporal changes in chlorophyll a, fucoxanthin, chlorophyll b, 19 -butanoyloxyfucoxanthin, alloxanthin and peridinin.

Pigment signatures in estuarine waters 695 T 2. Spearman rank correlation coefficients matrix for main pigment data set (*P<0·05, **P<0·01) (fuco, fucoxanthin; bfu, 19 -butanoyloxyfucoxanthin; allox, alloxanthin; per, peridinin) Lower estuary (n=64) fuco bfu Chl a 0·818** 0·414** fuco 0·523** bfu Middle estuary (n=128) fuco Chl b Chl a 0·894** 0·465** fuco 0·370** Chl b bfu Upper estuary (n=128) fuco Chl b Chl a 0·937** 0·627** fuco 0·512** Chl b bfu allox

allox

phytes, the most conspicuous were large Cryptomonaslike cells in the upper reaches, while smaller cells like Chroomonas or Hemiselmis were common in the lower estuary. Low numbers of the alloxanthin containing ciliate Mesodinium rubrum was observed in some live samples. Contribution of different groups of algae to total chlorophyll a

0·270** bfu 0·224* 0·262** 0·191*

allox 0·669** 0·535** 0·419** 0·211*

bfu 0·451** 0·454** 0·298**

allox 0·697** 0·606** 0·407** 0·428**

per 0·223* 0·302** 0·260** 0·238**

Relationships between signature pigments and phytoplankton taxa In the upper estuary, microscopic observations revealed that the peaks of chlorophyll a and those of fucoxanthin were mainly associated with the diatoms Cyclotella atomus and Thalassiosira guillardii and the dinoflagellate Peridinium foliaceum. In the middle estuary, peaks in chlorophyll a and fucoxanthin corresponded with maximum concentrations of diatoms of the genera Chaetoceros and Thalassiosira, and the dinoflagellate Peridinium quinquecorne. Occasionally, small flagellates like Prymnesium which contain fucoxanthin were observed. In the lower estuary, the most prominent peaks in fucoxanthin concentration corresponded to mixed assemblages of diatoms and to a lesser extent prymnesiophytes. In this region, prymnesiophytes like Phaeocystis and the coccolithophorid Emiliania huxleyi were occasionally observed in live samples. Microscopic observations failed to recognise live or fixed algae associated with 19 -butanoyloxyfucoxanthin. Among the chlorophyll b containing groups observed along the estuary, the most prominent were euglenophytes of the genera Eutreptia and Eutreptiella; chlorophytes of the genus Chlamydomonas and prasinophytes of the genera Pyramimonas, Tetraselmis, Nephroselmis and Micromonas-like cells. Among peridinin containing dinoflagellates the most important was the genus Peridiniopsis in the upper reaches and Heterocapsa towards the mouth of the estuary. Among crypto-

Fucoxanthin containing algae were the dominant group along the estuary during most of the study period (Figure 3). In the lower region, this group of algae accounted for more than 75% of chlorophyll a during biomass peaks. The high contribution (82%) was observed during the spring diatom bloom in 1997. In the upper and middle estuary, the percentage of chlorophyll a attributed to fucoxanthin containing algae was generally higher in spring and summer, being about 93% in April 1997 in the middle estuary and almost 100% in July 1997 in the upper estuary. 19 -butanoyloxyfucoxanthin containing algae constituted one of the groups better represented in the estuary, showing their greatest contributions to chlorophyll a generally in summer and autumn. In the lower estuary, the highest contribution of 19 butanoyloxyfucoxanthin to total chlorophyll a (39%) was found in December 1997 in bottom waters. Generally, 19 -butanoyloxyfucoxanthin containing algae were proportionally more abundant in bottom waters. In the upper estuary, the contribution of 19 -butanoyloxyfucoxanthin increased coincident with the lowest values of total chlorophyll a. Chlorophytes appeared in noticeable proportions in the lower and middle estuary, being relatively less important in the upper segment. In contrast, the contribution of cryptophytes was higher in the upper segments, where it peaked in summer. The contribution of euglenophytes was only occasionally important in summer in the middle and upper estuary. Dinoflagellates with peridinin were a minor component of the community, reaching their highest contribution all along the estuary in summer and autumn. In terms of the contribution of the different groups of algae to total chlorophyll a, phytoplankton species diversity was higher in the lower estuary. During most of the year a mixed assemblage of diatoms, chlorophytes, 19 -butanoyloxyfucoxanthin containing, and to a lesser extent, euglenophytes, cryptophytes and dinoflagellates with peridinin, was present. The concentration of the signature pigments corresponding to small flagellates remained more constant through the year in the lower estuary compared to the upper segments, when the concentration of these pigments

696 A. Ansotegui et al. Surface

100

Bottom

Stn 5

75 50 25 0

M J J J J JL JL JL A A A S S O F M A A M M J J JL JL A A S S O N D J

M J J J J JL JL JL A A A S S O F M A A M M J J JL JL A A S S O N D J

M J J J J JL JL JL A A A S S O F M A A M M J J JL JL A A S S O N D J

M J J J J JL JL JL A A A S S O F M A A M M J J JL JL A A S S O N D J

M J J J J JL JL JL A A A S S O F M A A M M J J JL JL A A S S O N D J

M J J J J JL JL JL A A A S S O F M A A M M J J JL JL A A S S O N D J

Stn 4

100 75 50 25 0

Stn 3

100 75 50 25 0

Stn 2

100 75 50 25 0

M J J J J JL JL JL A A A S S O F M A A M M J J JL JL A A S S O N D J

M J J J J JL JL JL A A A S S O F M A A M M J J JL JL A A S S O N D J

M J J J J JL JL JL A A A S S O F M A A M M J J JL JL A A S S O N D J

M J J J J JL JL JL A A A S S O F M A A M M J J JL JL A A S S O N D J

Stn 1

100 75 50 25 0

1996

1996

1997 Month

1997 Month

Fucoxanthin-containing

Dinoflagellates

Cryptophytes

Chlorophytes

Euglenophytes

BFU-containing

F 3. Spatial and temporal changes in the relative abundance of the different groups of algae as estimated by the CHEMTAX programme.

showed strong fluctuations. In the upper estuary, occasional peaks of chlorophytes, euglenophytes and cryptophytes were observed, some coincided with peaks in fucoxanthin. Others appeared after enhanced river flows, when the upper estuary was recovering from the wash out of cells.

Pigment ratios Differences in pigment ratio between the selected initial ratio and the ratio (final ratio) attributed by the CHEMTAX to each group of algae were found for some of the groups. In addition, spatial and temporal

Pigment signatures in estuarine waters 697

differences in the final ratio of each group of algae were also observed for some clusters of algae. Table 3 shows the pigment ratios attributed by the CHEMTAX program to the different groups of algae. While some pigment ratios remained constant for the whole data sets, other exhibited marked changes between and within groups. Among the later, the ratio chlorophyll b:chlorophyll a for euglenophytes (0·406– 1·239) and chlorophytes (0·330–0·572 and the ratio fucoxanthin:chlorophyll a (0·479–0·755) for algae with fucoxanthin were the most variable. Discussion Signature pigments and phytoplankton assemblages The analysis of algal pigments has proved to be useful for the determination of phytoplankton assemblages and their dynamics in marine waters, revealing a close relationship between the relative abundance of different signature pigments and the availability of nutrients. It is well established that small phytoplankton cells are associated with areas of low nutrient concentrations, whereas the importance of the larger species, mainly diatoms, increases with the availability of nutrients. High levels of divinyl chlorophylls and zeaxanthin are characteristic of oligotrophic areas dominated by picoplanktonic prochlorophytes and cyanobacteria (e.g. Latasa & Bidigare, 1998). Pigments such as chlorophyll b, 19 -butanoyloxyfucoxanthin and 19 hexanoyloxyfucoxanthin, corresponding to small flagellates, have more frequently been measured in eddies and other moderately eutrophic areas (BustillosGuzmn´ et al., 1995; Barlow et al., 1997; MeyerHarms et al., 1999). In productive areas such as upwelling, frontal and coastal regions, fucoxanthin, mainly from diatoms, is frequently the dominant pigment (Head et al., 1997; Peeken, 1997; Ahel & Terzic, 1998). Estuaries display a wide range of trophic conditions linked to the supply of nutrients from natural and anthropogenic sources and dilution of the nutrient-rich estuarine waters with coastal waters. Fucoxanthin, the pigment signature for diatoms, prymnesiophytes and chrysophytes, was the dominant pigment in the Urdaibai estuary. During peaks of chlorophyll a, fucoxanthin was found in the upper and middle estuary in concentrations much higher than those reported for other estuarine or marine area (Table 4). The highest concentrations of this pigment in the lower marine estuary are consistent with those found by Ahel and Terzic (1998) in the coastal waters of the Adriatic Sea, but much higher that those reported in the literature for open waters. Although

there are only a few studies dealing with estuarine pigments, fucoxanthin has been reported as the dominant accessory pigment in other estuaries, being attributed to diatoms (Ahel et al., 1996; Brotas & Plante-Cuny, 1998), chrysophytes and prymnesiophytes (Tester et al., 1995). According to microscopic observations, in the upper segments of the Urdaibai estuary, diatoms and dinoflagellates accounted for fucoxanthin, while in the lower estuary this pigment was due to diatoms and prymnesiophytes. In addition to pelagophytes, the accessory pigment 19 -butanoyloxyfucoxanthin has been found in some prymnesiophytes (Jeffrey & Wright, 1994), and in some symbiont-bearing dinoflagellates (Bjørnland & Liaaen-Jensen, 1989). The relatively high amounts of 19 -butanoyloxyfucoxanthin found in the Urdaibai estuary could be accounted for by prymnesiophytes, widely distributed through the oceans (Andersen et al., 1996), or to pelagophytes. The later group of algae has been found in the open ocean and coastal ecosystems, where they are responsible for brown tides (Buskey et al., 1997). The small size of these groups precluded their identification by the microscopic facilities used in this study. However, with the chromatographic method used, 19 butanoyloxyfucoxanthin co-elutes with siphonaxanthin, the principal accessory pigment in siphonal green algae (Anderson et al., 1985). Taking into account the absence of siphonal algae in the estuary due to the soft nature of its bottom, we conclude that 19 -butanoyloxyfucoxanthin was indicative of pelagophytes in the estuary. The concentrations of 19 -butanoyloxyfucoxanthin (up to 0·6
g 1
1) in the lower estuary are of the same order of magnitude as the maxima found by Ahel and Terzic (1998) in coastal waters of the Adriatic. However, concentrations of this pigment in the middle and upper estuary are much higher than those reported for other estuarine or marine areas (see Table 4). Other accessory pigments such as chlorophyll b, alloxanthin and peridinin appeared in quantities more similar to those obtained in other estuaries and coastal areas (see Table 4), except for some extraordinarily high peaks recorded in the middle and upper estuary. The method used in this study does not separate lutein from zeaxanthin. Lutein is the major carotenoid in higher plants and in some members of the Chlorophyta. Zeaxanthin is used as a signature pigment for cyanobacteria and prochlorophytes and takes part in the violaxanthin cycle in the chlorophytes and prasinophytes. We have not found any reference in the literature reporting the presence of prochlorophytes in estuarine environments, although high abundance of blue green algae had been found in some estuaries

fuco-containing bfu-containing chlorophytes euglenophytes cryptophytes dinoflagellates

— — — — — 1·063–1·295

per — 1·563 — — — —

bfu 0·479–0·755 0·974 — — — —

fuco — — 0·047–0·191 0·015–0·030 — —

neo — — 0·042–0·055 — — —

viol

0·056–0·110 0·119–0·800 — 0·042–0·230 — 0·241

ddx

— — — — 0·229 —

allox

— — 0·186–0·390 — — —

lut

— — 0·330–0·572 0·406–1·239 — —

Chl b

T 3. Range of the accessory pigment Chl a ratios calculated by CHEMTAX for the different subsets considered (per, peridinin; bfu, 19 -butanoyloxyfucoxanthin; fuco, fucoxanthin; neo, neoxanthin; viol, violaxanthin; ddx, diadinoxanthin; allox, alloxanthin; lut, lutein)

698 A. Ansotegui et al.

Polar

Temperate

Subtropics

Open ocean Tropics

Coastal areas

Estuaries

Western Equatorial Pacific Pacific Ocean, Hawaii Gulf of Carpentaria Gulf of Mexico Northeastern Atlantic Norwegian Sea Southern Ocean Bellingshausen Sea

Krka River Chesapeake Bay Hudson River Krka River St. Lawrence Sabine-Neches Urdaibai French coastal waters Adriatic Sea

Locality

0·34 0·43 5·70 1·40 3·70 2·86 0·49 2·40

26·34 22·96 44·80 4·30 10·00 16·30 133·70 5·00 8·00

chl a

0·02 0·02 1·70 0·19 1·70 0·98 0·15 1·50

6·00 7·58 4·20 1·60 13·80 0·70 81·30 0·15 4·00

fuco

0·16 0·19 0·20 0·30 0·20 0·19 0·15 0·01

0·79 0·44 3·40 0·20 0·36 2·50 14·50 0·08 0·50

chl b

0·07

0·08

0·08 0·07

0·21 0·01

0·02

3·60 0·05

12·50 0·30

0·17

1·39 1·65 0·90

allox

0·08

bfu

0·01 0·05 0·01 0·08

0·01

1·00

0·77 0·90 2·60

1·01

per

Everitt et al., 1990 Letelier et al., 1993 Burford et al., 1995 Lambert et al., 1999 Barlow et al., 1993 Meyer-Harms et al., 1999 Wright et al., 1996 Barlow et al., 1998

Denant et al., 1991 McManus & Ederington-Cantrell 1992 Bianchi et al., 1993 Ahel et al., 1996 Roy et al., 1996 Bianchi et al., 1997 This study Klein & Sournia 1987 Ahel & Terzic 1998

Reference

T 4. Maximum concentration (
g l
1) of pigments found in the estuary of Urdaibai compared with those found in the literature (fuco, fucoxanthin; bfu, 19 -butanoyloxyfucoxanthin; allox, alloxanthin; per, peridinin)

Pigment signatures in estuarine waters 699

700 A. Ansotegui et al.

(e.g. Bianchi et al., 1993). In this study, we consider that a peak corresponding to the mixture of lutein and zeaxanthin was mainly due to the former. The assumption was based on microscopic observations, which showed a strong relationship between peaks of lutein-zeaxanthin and the abundance of chlorophytes in the samples. Filamentous or colonial blue-green algae were not observed in the samples. Furthermore, freshwater cyanobacteria that might have been flushed from the river can be characterized by carotenoids such as myxoxanthophyll and echinenone (Nichols, 1973). Both are detectable by the chromatographic method used but were not detected. Finally, during freshets, the estuary is subject to inputs of vascular plant detritus, which represent another source of lutein. Despite fucoxanthin being the dominant accessory pigment along the estuary, the phytoplankton community was generally more diverse and included dinoflagellates with and without peridinin, cryptophytes, euglenophytes and chlorophytes. Indeed, a background of mixed flagellates on which peaks of diatoms were superimposed was characteristic of the lower marine estuary and this agrees well with results from other coastal waters (Hallegraeff, 1981). In the upper estuary, fucoxanthin-containing algae generally masked the other algal groups, except during some peaks of euglenophytes, chlorophytes and cryptophytes, most of which were recorded after freshets, coinciding with relatively low phytoplankton biomass. Based on microscopic observations, we presume that in absence of mesozooplankton, which do not grow efficiently in the upper region, heterotrophic microplankton (ciliates and heterotrophic dinoflagellates such as Protoperidinium achromaticum and Oxyrrhis marina), exert a stronger grazing pressure on small flagellates than on diatoms and dinoflagellates, which experience enhanced growth during periods of high residence time of the water. Pigment ratios A crucial step in the use of pigment signatures to estimate the contribution of different algal groups to total chlorophyll a, is the selection of the correct accessory pigment:chlorophyll a ratios as conversion factors. The initial pigment ratios considered in this study were obtained from Mackey et al. (1997) and most of them were based on phytoplankton cultures. The same initial ratios were chosen for all the clusters of samples. However, whereas differences between initial and final ratios were not found for some pigments, others experienced noticeable changes in their final ratios respective to the initial ones. Nevertheless,

all ratios used were within the range reported in the literature for other estuarine and marine areas. The final fucoxanthin:chlorophyll a ratio for fucoxanthin containing algae varied along with the estuary. Ratios from the lower and middle estuary had values which agree well with those reported for diatoms in marine 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 the ratio was lower (0·479), although within the range of reported values. Meyer-Harms et al., (1999) obtained a similar ratio of 0·450 in the Norwegian Sea during and after a spring diatom bloom and Letelier et al., (1993) reported a value of 1·25 for shade adapted diatoms. Based on cultures of the diatoms Phaeodactylum tricornutum and Ditylum brightwellii Schlu¨ ter et al., (2000) obtained a broad range of ratios (0·485 to 1·218 reflecting between and within species differences in response to the light regime. In the Urdaibai estuary, the presence of the fucoxanthin containing dinoflagellate Peridinium foliaceum which may have different ratios than diatoms, could explain the differences in the fucoxanthin:chlorophyll a ratio between the upper and the lower estuary. The ratio of diadinoxanthin:chlorophyll a for fucoxanthin containing algae ranged from 0·056 to 0·110, with highest values at the upper most turbid station. Based on cultures, Schlu¨ ter et al., (2000) found that this ratio fluctuated strongly in response to the light regime and was affected by the physiological state of the algae. Fucoxanthin:chlorophyll a and 19 -butanoyloxyfucoxanthin:chlorophyll a ratios for 19 -butanoyloxyfucoxanthin containing algae (0·974 and 1·563, respectively), taken from a culture of Pelagococcus subviridis (Jeffrey & Wright, 1997), remained constant in all data sets. These ratios are similar to those obtained by Everitt et al., (1990) and Mackey et al., (1998) for chrysophytes in the Equatorial Pacific, but are slightly higher than those reported by MeyerHarms et al., (1999) for prymnesiophytes in the Norwegian Sea. The ratio of diadinoxanthin: chlorophyll a for 19 -butanoyloxyfucoxanthincontaining algae ranged from 0·119 to 0·800, being highest in the lower and middle estuary. The spatial differences can be interpreted as an adaptation of the algae to the different light regime of the estuary. The concentration of diadinoxanthin, the epoxidated form of the xanthophyll cycle in chromophytes, increases with light intensity in the lower, less turbid regions of the estuary. To estimate the contribution of peridinin containing dinoflagellates to total chlorophyll a, an initial ratio of 1·063, obtained by Jeffrey and Wright, (1997)

Pigment signatures in estuarine waters 701

from a culture of Amphidinium carterae was used. A broad range of final peridinin:chlorophyll a ratios were however obtained (1·063–1·295). Although many of these ratios were higher than those reported in the literature (Schlu¨ ter et al., 2000), Mackey et al., (1998) found a comparable final ratio (1·000) in deep samples from the Western Equatorial Pacific, and Pinckney et al. (1998) obtained a value of 1·176 for the moderately eutrophic Neuse River Estuary. A ratio of 1·265 was however obtained from an extract of the dinoflagellate Heterocapsa rotundata from the estuary of Urdaibai. The initial diadinoxanthin:chlorophyll a ratio for dinoflagellates (0·241 remained unchanged after the application of the CHEMTAX program. Most published values come from the cultures (Demers et al., 1991; Schlu¨ ter et al., 2000) and are quite similar to those used here. Alloxanthin is the main pigment signature for cryptophytes, although it is also present in the ciliate Mesodinium rubrum. The ciliate was observed in the estuary of Urdaibai in live samples, but not in great numbers. We may therefore assume that most alloxanthin belonged to cryptophytes. The alloxanthin:chlorophyll a ratio remained unchanged (0·229) with respect to the initial ratio through the estuary. This ratio is within the values reported in the literature, which range from 0·105 (Mackey et al., 1998 to 0·541 (Hager & Stransky, 1970). Values close to those obtained in this study were found in the North Sea (0·234) Gieskes & Kraay, 1983), Alboran Sea (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 for euglenophytes varied between 0·406 and 1·239, being highest in bottom waters of the upper estuary where light availability is low. It has been suggested that in green algae the increase in chlorophyll b relative to chlorophyll a could mean a weak chromatic adaptation (Wood, 1979). In this sense, Mackey et al., (1998) found increasing values of this ratio with depth for euglenophytes in the Equatorial Pacific. Our results however, disagree with those of Schlu¨ ter et al., (2000) who found that this ratio increases with light intensity. The same author observed that this ratio also increases during the stationary phase of the culture, which makes it difficult to explain the field data. The range of diadinoxanthin:chlorophyll a (0·042–0·230) and neoxanthin:chlorophyll a (0·015–0·030) ratios obtained in this study for euglenophytes are comparable to those obtained by Mackey et al., (1998). The final ratios of chlorophytes fall within the range of 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. However, whereas the CHEMTAX program left a final chlorophyll b:chlorophyll a ratio similar to the initial one (0·569) in the upper estuary, the final ratio decreased to values as low as 0·330 towards the middle and lower segments. These spatial differences appear to be a consequence of the different light regime of the different estuarine segments rather than caused by taxonomic differences. Chlorophytes thus dominate the upper estuary while prasinophytes are relatively more abundant in the lower segments. Several studies (e.g. Brown & Jeffrey, 1992, Wood, 1979, and Schlu¨ ter et al., 2000) have shown that prasinophytes generally contain higher chlorophyll b:chlorophyll a ratios than chlorophytes. A broad range of final lutein:chlorophyll a ratios (0·186– 0·390) were obtained for chlorophytes and were higher than those reported by Wright et al., (1996) for the Southern Ocean (0·127) and by Mackey et al., (1998) for the Equatorial Pacific (0·042–0·120). The increase in this ratio towards the upper estuary may be explained by the presence of a higher amount of detritus of vascular plants, which contain more lutein per gram of biomass than non-vascular plants (Bianchi et al., 1993). The final neoxanthin:chlorophyll a (0·047–0·191) and violaxanthin:chlorophyll a (0·042–0·055 ratios for chlorophytes obtained in this study are within the range found in the Equatorial Pacific by Mackey et al., (1998) and in cultures of both chlorophytes and prasinophytes by Jeffrey and Wright (1997). The use of diagnostic pigments accompanied by microscopic observations of live and fixed phytoplankton samples has thus provided considerable insight into the seasonal dynamic of phytoplankton assemblages along the trophic and salinity gradient of the Urdaibai estuary. By means of specific carotenoid pigments, the relative importance of small or fragile cells has been assessed whereas microscopic observations have been of great help to identify the taxa contributing to ambiguous accessory pigments. The combination of both methods enabled identification of the main taxonomic groups contributing to fucoxanthin containing algae, alloxanthin containing and chlorophytes, as well estimating their relative contribution. Further research is still needed to prove the presence of pelagophytes in the estuary as well as to understand better the partitioning of 19 -butanoyloxyfucoxanthin within the different algal groups. Acknowledgements The University of the Basque Country (project UPV 118.310-EB124/97) and the Department of

702 A. Ansotegui et al.

Education, Universities and Investigation of the Basque Government (project GV PI-1998-67) supported this work. A. Ansotegui was also funded by a grant from the Spanish Ministry of Education and Science and J. M. Trigueros by a grant from the Department of Education, Universities and Investigation of the Basque Government. References Ahel, M. & Terzic, S. 1998 Pigment signatures of phytoplankton dynamics in the northern Adriatic. Croatica Chemica Acta 71, 199–215. Ahel, M., Barlow, R. G. & Mantoura, R. F. C. 1996 Effect of salinity gradients on the distribution of phytoplankton pigments in a stratified estuary Marine Ecology Progress Series 143, 289–295. Andersen, R. A., Bidigare, R. R., Keller, M. D. & Latasa, M. 1996 A comparison of HPLC pigment signatures and electron microscopic observations for oligotrophic waters of the North Atlantic and Pacific Oceans. Deep-Sea Research II 43, 517–537. Anderson, J. M. 1985 Chlorophyll-protein complexes of marine alga, Codium species (Siphonales). Biochimica et Biophysica Acta 806, 39–50. Barlow, R. G., Mantoura, R. F. C., Gough, M. A. & Fileman, T. W. 1993 Pigment signatures of the phytoplankton composition in the northeastern Atlantic during the 1990 spring bloom. Deep-Sea Research II 40, 459–477. Barlow, R. G., Mantoura, R. F. C., Peinert, R. D., Miller, A. E. J. & Fileman, T. W. 1995 Distribution, sedimentation and fate of pigment biomarkers following thermal stratification in the western Alboran Sea. Marine Ecology Progress Series 125, 279–291. Barlow, R. G., Mantoura, R. F. C., Cummings, D. G. & Fileman, T. W. 1997 Pigment chemotaxonomic distributions of phytoplankton during summer in the western Mediterranean. Deep-Sea Research II 44, 833–850. Barlow, R. G., Mantoura, R. F. C. & Cummings, D. G. 1998 Phytoplankton pigment distributions and associated fluxes in the Bellingshausen Sea during the austral spring 1992. Journal of Marine Systems 17, 97–113. Bianchi, T. S., Findlay, S. & Dawson, R. 1993 Organic matter sources in the water column and sediments of the Hudson River Estuary: the use of plant pigments as tracers. Estuarine, Coastal and Shelf Science 36, 359–376. Bianchi, T. S., Baskaran, M., DeLord, J. & Ravichandran, M. 1997 Carbon cycling in a shallow turbid estuary of Southeast Texas: the use of plant pigment biomarkers and water quality parameters. Estuaries 20, 404–415. Bjørnland, T. & Liaaen-Jensen, S. 1989 Distribution patterns of carotenoids in relation to chromophyte phylogency and systematics. In The Chromophyte Algae: Problems and Perspectives (Green, J. C., Leadbeater, B. S. C. & Diver, W. L., eds). Clarendon Press, Oxford, pp. 37–61. Brotas, V. & Plante-Cuny, M. R. 1998 Spatial and temporal patterns of microphytobenthic taxa of estuarine tidal flats in the Tagus Estuary (Portugal) using pigments analysis by HPLC. Marine Ecology Progress Series 171, 43–57. Brown, M. R. & Jeffrey, S. W. 1992 Biochemical composition of microalgae from the green algal classes Chlorophyceae and Prasinophyceae. 1. Amino acids, sugars and pigments. Journal of Experimental Marine Biology and Ecology 161, 91–113. Burford, M. A., Rothlisberg, P. C. & Wang, Y. G. 1995 Spatial and temporal distribution of tropical phytoplankton species and biomass in the Gulf of Carpentaria, Australia. Marine Ecology Progress Series 118, 255–266. Buskey, E. J., Montagna, P. A., Amos, A. F. & Whitledge, T. E. 1997 Disruption of grazer populations as a contributing factor to the initiation of the Texas brown tide algal bloom. Limnology and Oceanography 42, 1215–1222.

Bustillos-Guzma´ n, J., Claustre, H. & Marty, J. C. 1995 Specific phytoplankton signatures and their relationship to hydrographic conditions in the coastal northwestern Mediterranean Sea. Marine Ecology Progress Series 124, 247–258. Demers, S., Roy, S., Gagnon, R. & Vignault, C. 1991 Rapid light-induced changes in cell fluorescence and in xanthophyllcycle pigments of Alexandrium excavatum (Dinophyceae) and Thalassiosira pseudonana (Bacillariophyceae): a photo-protection mechanism. Marine Ecology Progress Series 76, 185–193. Denant, V., Saliot, A. & Mantoura, R. F. C. 1991 Distribution of algal chlorophyll and carotenoid pigments in a stratified estuary: the Krka River, Adriatic Sea. Marine Chemistry 32, 285–297. Everitt, D. A., Wright, S. W., Volkman, J. K., Thomas, D. P. & Lindstrom, E. J. 1990 Phytoplankton community compositions in the western equatorial Pacific determined from chlorophyll and carotenoid pigment distributions. Deep-Sea Research 37, 975–997. Fiksdahl, A., Withers, N., Guillard, R. R. L. & Liaaen-Jenson, S. 1984 Carotenoids in the Raphidophyceae – a chemosystematic contribution. Comparative Biochemistry and Physiology 78, 265–271. Gieskes, W. W. C. & Kraay, G. W. 1983 Dominance of Cryptophyceae during the phytoplankton spring bloom in the central North Sea detected by HPLC analysis of pigments. Marine Biology 75, 179–185. Gieskes, W. W. C., Kraay, G. W., Nontji, A., Setiapermana, D. & Sutomo. 1988 Monsoonal alternation of a mixed and a layered structure in the phytoplankton of the euphotic zone of the Banda Sea (Indonesia): a mathematical analysis of algal pigment fingerprints. Netherlands Journal of Sea Research 22, 123–137. Goericke, R. & Montoya, J. P. 1998 Estimating the contribution of microalgal taxa to chlorophyll a in the field – variations of pigment ratios under nutrient- and light-limited growth. Marine Ecology Progress Series 169, 97–112. Goericke, R. & Repeta, D. J. 1992 The pigments of Prochlorococcus marinus: the presence of divinyl chlorophyll a and b in a marine procaryote. Limnology and Oceanography 37, 425–433. Hager, A. & Stransky, H. 1970 Das Carotenoidmuster und die Verbreitung des lichtinduzierten Xanthophyll-cyclus in Verschiedenen Algen Klassen. V. Einzelne Vertreter der Cryptophyceae, Euglenophyceae, Bacillariophyceae, Chrysophyceae und Phaeophyceae. Archiv fu¨ r Mikrobiologie 73, 77–89. Hallegraeff, G. M. 1981 Seasonal study of phytoplankton pigments and species at a coastal station off Sydney: importance of diatoms and nanoplankton. Marine Biology 61, 107–118. Head, E. J. H., Harrison, W. G., Irwin, B. I., Horne, E. P. W. & Li, W. K. W. 1996 Plankton dynamics and carbon flux in an area of upwelling off the coast of Morocco. Deep-Sea Research I 43, 1713–1738. Hibberd, D. J. 1977 Observations on the ultrastructure on the cryptomonad endosymbiont of the red-water ciliate Mesodinium rubrum. Journal of the Marine Biological Association of the United Kingdom 57, 45–61. Jeffrey, S. W. & Wright, S. W. 1994 Photosynthetic pigments in the Haptophyta. In The Haptophyte Algae (Green, J. C. & Leadbeater, B. S. C., eds). Clarendon Press, Oxford, pp. 111–132. Jeffrey, S. W. & Wright, S. W. 1997 Qualitative and quantitative HPLC analysis of SCOR reference algal cultures. In Phytoplankton pigments in oceanography (Jeffrey, S. W., Mantoura, R. F. C. & Wright, S. W., eds). UNESCO, pp. 343–360. Jeffrey, S. W., Llewellyn, C. A., Barlow, R. G. & Mantoura, R. F. C. 1997 Pigment processes in the sea: a selected bibliography. In Phytoplankton pigments in oceanography (Jeffrey, S. W., Mantoura, R. F. C. & Wright, S. W., eds). UNESCO, pp. 167–178. Klein, B. & Sournia, A. 1987 A daily study of the diatom spring bloom at Roscoff (France) in 1985. II. Phytoplankton pigment composition studied by HPLC analysis. Marine Ecology Progress Series 37, 265–275. Lambert, C. D., Bianchi, T. S. & Santschi, P. H. 1999 Cross-shelf changes in phytoplankton community composition in the Gulf of

Pigment signatures in estuarine waters 703 Mexico (Texas shelf/slope): the use of plant pigments as biomarkers. Continental Shelf Research 19, 1–21. Latasa, M. & Bidigare, R. R. 1998 A comparison of phytoplankton populations of the Arabian Sea during the Spring Intermonsoon and Southwest Monsoon of 1995 as described by HPLCanalysed pigments. Deep-Sea Research II 45, 2133–2170. Letelier, R. M., Bidigare, R. R., Hebel, D. V., Ondrusek, M., Winn, C. D. & Karl, D. M. 1993 Temporal variability of phytoplankton community structure based on pigment analysis. Limnology and Oceanography 38, 1420–1437. Llewellyn, C. A. & Gibb, S. W. 2000 Intra-class variability in the carbon, pigment and biomineral content of prymnesiophytes and diatoms. Marine Ecology Progress Series 193, 33–44. Mackey, D. J., Higgins, H. W., Mackey, M. D. & Holdsworth, D. 1998 Algal class abundances in the western equatorial Pacific: estimation from HPLC measurements of chloroplast pigments using CHEMTAX. Deep-Sea Research 45, 1441–1468. 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. Marine Ecology Progress Series 144, 265–283. Mackey, M. D., Higgins, H. W., Mackey, D. J. & Wright, S. W. 1997 CHEMTAX user’s manual: A program for estimating class abundances from chemical markers – application to HPLC measurements of phytoplankton pigments. CSIRO, Hobart, Australia (Mar. Lab. Rep. No. 229), 41 pp. McManus, G. B. & Ederington-Cantrell, M. C. 1992 Phytoplankton pigments and growth rates, and microzooplankton grazing in a large temperate estuary. Marine Ecology Progress Series 87, 77–85. Meyer-Harms, B., Irigoien, X., Head, R. & Harris, R. 1999 Selective feeding on natural phytoplankton by Calanus finmarchicus before, during, and after the 1997 spring bloom in the Norwegian Sea. Limnology and Oceanography 44, 154–165. Meyer-Harms, B. & Pollehne, F. 1998 Alloxanthin in Dinophysis norvegica (Dinophysiales, Dinophyceae) from the Baltic Sea. Journal of Phycology 34, 280–285. Meyer-Harms, B. & von Bodungen, B. 1997 Taxon-specific ingestion rates of natural phytoplankton by calanoid copepods in an estuarine environment (Pomeranian Bight, Baltic Sea) determined by cell counts and HPLC analyses of marker pigments. Marine Ecology Progress Series 153, 181–190. Millie, D. F., Paerl, H. W. & Hurley, J. P. 1993 Microalgal pigment assessments using high-performance liquid chromatography: a synopsis of organismal and ecological applications. Canadian Journal of Fisheries and Aquatic Sciences 50, 2513–2527. Nichols, B. W. 1973 Lipid composition and metabolism. In The Biology of Blue-green Algae (Carr, N. G. & Whitton, B. A., eds). Blackwells, Oxford, pp. 144–161. Orive, E., Franco, J. & Ruiz, A. 1995 Importancia del fitoplancton en estuarios meso-macromareales someros: el ejemplo del estuario de Urdaibai. In Urdaibai: investigacio´ n ba´ sica y aplicada (Angulo, E., ed.). Gobierno Vasco, pp. 57–74. Orive, E., Iriarte, A., de Madariaga, I. & Revilla, M. 1998 Phytoplankton blooms in the Urdaibai estuary during summer:

physico-chemical conditions and taxa involved. Oceanologica Acta 21, 293–305. Parsons, T. R., Maita, Y. & Lalli, C. M. 1984 A manual of chemical and biological methods for sea water analysis. Pergamon Press, Oxford, 173 pp. Peeken, I. 1997 Photosynthetic pigment fingerprints as indicators of phytoplankton biomass and development in different water masses of the Southern Ocean during austral spring. Deep-Sea Research II 44, 261–282. Pinckney, J. L., Paerl, H. W., Harrington, M. B. & Howe, K. E. 1998 Annual cycles of phytoplankton community-structure and bloom dynamics in the Neuse River Estuary, North Carolina. Marine Biology 131, 371–381. Revilla, M., Iriarte, A., de Madariaga, I. & Orive, E. 2000 Bacterial and phytoplankton dynamics along a trophic gradient in a shallow temperate estuary. Estuarine, Coastal and Shelf Science 50, 297–313. Roy, S., Chanut, J. P., Gosselin, M. & Sime-Ngando, T. 1996 Characterization of phytoplankton communities in the lower St. Lawrence Estuary using HPLC-detected pigments and cell microscopy. Marine Ecology Progress Series 142, 55–73. Schlu¨ ter, L., Møhlenberg, 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. Marine Ecology Progress Series 192, 49–63. Soma, Y., Imaizumi, T., Yagi, K. & Kasuga, S. 1993 Estimation of algal succession in lake water using HPLC analysis of pigments. Canadian Journal of Fisheries and Aquatic Sciences 50, 1142–1146. Tester, P. A., Geesey, M. E., Guo, C., Paerl, H. W. & Millie, D. F. 1995 Evaluating phytoplankton dynamics in the Newport River estuary (North Carolina, USA) by HPLC-derived pigment profiles. Marine Ecology Progress Series 124, 237–245. Trigueros, J. M., Ansotegui, A., Orive, E. & No´ , M. L. 2000a Morphology and distribution of two brackish diatoms (Bacillariophyceae): Cyclotella atomus Hustedt and Thalassiosira guillardii Hasle in the estuary of Urdaibai (northern Spain). Nova Hedwigia 70, 431–450. Trigueros, J. M., Ansotegui, A. & Orive, E. 2000b Remarks on morphology and ecology of recurrent dinoflagellate species in the estuary of Urdaibai (northern Spain). Botanica Marina 43, 93–103. Withers, N. W., Fiksdahl, A., Tuttle, R. C. & Liaaen-Jensen, S. 1981 Carotenoids of the Chrysophyceae. Comparative Biochemistry and Physiology 68, 345–349. Wood, A. M. 1979 Chlorophyll a:b ratios in marine planktonic algae. Journal of Phycology 15, 330–332. Wright, S. W., Thomas, D. P., Marchant, H. J., Higgins, H. W., Mackey, M. D. & Mackey, D. J. 1996 Analysis of phytoplankton of the Australian sector of the Southern Ocean: comparisons of microscopy and size frequency data with interpretations of pigment HPLC data using the ‘ CHEMTAX ’ matrix factorisation program. Marine Ecology Progress Series 144, 285–298.