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 Ecologı́a, Facultad de Ciencias, Universidad del Paı́s 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 (4322N; 240W, 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, 19hexanoyloxyfucoxanthin 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 19butanoyloxyfucoxanthin. 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 19butanoyloxyfucoxanthin 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 19hexanoyloxyfucoxanthin, corresponding to small flagellates, have more frequently been measured in eddies
and other moderately eutrophic areas (BustillosGuzmń 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, 19butanoyloxyfucoxanthin 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 Schlüter
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,
Schlüter 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 (Schlüter 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; Schlüter 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 Schlüter 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 Schlüter 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-Guzmán, 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 für 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: investigación básica 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.
Schlüter, 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. & Nó, 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.