Aquatic Botany 67 (2000) 117–129

Pigment composition of freshwater charophyceae

Michael Schagerl∗ , Clemens Pichler
Department of Hydrobotany, Institute of Plant Physiology, University of Vienna, Althanstraße 14, A-1090
Vienna, Austria
Received 18 March 1999; received in revised form 13 July 1999; accepted 1 November 1999

Abstract
Twelve species of Charophyceae were collected at different locations in the eastern part of Austria
and photosynthetic pigments analyzed by means of rP-HPLC (reversed phase-high performance
liquid chromatography). Besides pigments typical of higher plants, also ␥-carotene was detected in
antheridia and for the first time in sterile specimens of Chara tomentosa. Probably there exists two
fractions of ß-carotene, one closely associated with ␥-carotene in separate compartments, the other
independent of ␥-carotene and located in the photosystems. Chlorophyll a on the basis of ash-free
dry mass was comparable to that of planktonic algae and submerged angiosperms (about 0.5%),
but pigment content varied in a wide range, suggesting adaptations to ecological conditions such
as light or temperature. Our results indicate that fresh and dry mass are not suitable as a reference
quantity in stoneworts due to high calcium carbonate precipitation. © 2000 Elsevier Science B.V.
All rights reserved.

Keywords: Charophyceae; Pigments; Carotenoids; Chlorophylls; Light adaptation

Abbreviations: Chlorophyll = chl; Carotene = car

1. Introduction

Large areas of the littoral zone are often covered with charophytes which hold an impor-
tant position in aquatic ecosystems. Charophytes may play a keyfactor in the appearance
of shallow meso/eutrophic lakes. Shallow lakes are generally characterized by abundant
submerged macrophytes and clear water at relatively low nutrient concentrations and by
high phytoplankton biomass and turbid water at higher nutrient concentrations. At in-
termediate nutrient concentrations lakes are dominated by either macrophytes or phyto-
plankton (Howard-Williams, 1978; Mitchell, 1989; Blindow et al., 1993; Scheffer et al.,

∗ Corresponding author. Tel.: +43-1-31336-1488; fax: +43-1-31336-776.

E-mail address: michael.schagerl@univie.ac.at (M. Schagerl)

0304-3770/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 3 7 7 0 ( 9 9 ) 0 0 0 9 5 - 9
118 M. Schagerl, C. Pichler / Aquatic Botany 67 (2000) 117–129

1994; Krolokowska, 1997). Charophytes compete with aquatic angiosperms, attached al-
gae and phytoplankton for nutrients and light. In eutrophic ecosystems, submerged macro-
phytes are often limited in growth because of bad light conditions. Seston and above all
the biofilm reduce light supply of macrophytes (Phillips et al., 1978; Krause and King,
1994).
Despite fluctuations in cellular content, chl-s and carotenoids are widely used for esti-
mations of phytoplankton and microphytobenthos biomass (Lehman, 1981; Gieskes and
Kraay, 1983, 1986; Roy, 1989; Latasa et al., 1992; Bianchi et al., 1993; Downes et al.,
1993; Millie et al., 1993a, b; Schagerl et al., 1996). Chl a also seems to be a valuable tool
for characterizing population structure of submerged macrophytes (Pokorny et al., 1984).
Ratios of carotenoids/chl-s are associated with nutrient supply of algae (Margalef, 1960,
1965; Riegman and Rowe, 1994) and appear to indicate stress of land plants (Ehrlich and
Grill, 1994).
As already known from land plants, water plants also adjust their photosynthetic units for
optimal utilization of irradiance (Spence and Chrystal, 1970; Sand-Jensen, 1978; Marcus,
1980; Barko and Filbin, 1983; Kunii, 1984; Pokorny et al., 1984; Spencer, 1986; Spencer
and Ksander, 1987; Dijk, 1991; Garcia et al., 1991; Frost-Christensen and Sand-Jensen,
1992). Adaptations of pigment quantities are realized as a protective measure against ex-
cessive light and for effective light harvesting at low light conditions. For many ecological
and physiological questions knowledge about pigment pattern and quantity is of great im-
portance, but only little attention was paid to pigments of stoneworts (Andrews et al., 1984;
Czezuga, 1986; Howard-Williams et al., 1995). In these few studies chl-s and carotenoids
were detected spectrophotometrically in the quality of total fractions. Rarely charophytes
were included in pigment studies of green algae (Seybold et al., 1941; Hager and Stran-
sky, 1970). Hager and Stransky (1970) analyzed pigment extracts by means of thin layer
chromatography.
This study was conducted to determine pigment patterns of various charophytes. We
analyzed pigments by means of rP-HPLC (reversed phase-high performance liquid chro-
matography) in order to create a basis for extended investigations. Specimens were collected
at different locations and various depths to get an insight into the ranges of the pigment
content. Special attention was paid to ␥-car (ß,9-carotene) which might be a marker for
sexual reproduction in various species with the exception of Chara tomentosa in which
␥-car occurs also in sterile plants.

2. Materials and methods

2.1. Study sites and sampling

Plants were taken from six different locations in eastern Austria: (1) Pool with 30 cm depth
in the eastern part of Vienna, (2) Neufelder See (border lower Austria/Burgenland), a clear
oligotrophic lake with rich Chara vegetation, (3) Erlaufsee (border lower Austria/Styria), an
Alpine oligotrophic lake in the limestone region, (4) Hechtensee (Styria), a small dystrophic
lake beside a bog, (5) Hallstätter See (upper Austria), a deep Alpine lake in the limestone
 M. Schagerl, C. Pichler / Aquatic Botany 67 (2000) 117–129 119

region, (6) Irrsee (upper Austria), a shallower turbid lake. These sites cover a range of
typical water bodies inhabited by Charophyta in Austria.
Sampling was carried out in 1994 and 1995. Plants were taken at different depths with an
Ekman grab. After cleaning to get off detritus and epiphytes living material was determined
according to Wood and Imahori (1965). Because of their constant and high pigment contents
mainly younger thallus parts were analyzed (Andrews et al., 1984).

2.2. Light measurements

Light measurements were done with an underwater irradiance sensor (2␲ system, Li-Cor
LI 1000 and LI-192 SA). Light climate at different locations was expressed in percent of
surface irradiance.

2.3. Pigment analyses

Prior to extraction, harvested plants were stored at −20◦ C. Plant material was put
into 90% acetone and ground for homogenisation under dim light. The suspensions were
stored at 2◦ C for 12 h to achieve optimal extraction. After centrifugation for 10 min at
3000 rpm (Sigma-centrifuge) the supernatant was filled up to a constant volume and then
injected into the HPLC system (Merck-Hitachi, consisting of an autosampler (AS-4000),
a gradient-pump (L-6200) and an UV–VIS-detector (L-4250) set at 440 nm, rP-columns
Merck Superspher rP-18 250/4, Merck LiChroCart 125/4, Merck LiChrospher 100 rp-18/5,
precolumn Merck Lichrospher rp-18 endcapped).
Separation of the pigment extracts was carried out using a ternary low pressure gradient
program according to Wright et al. (1991). Identification of chl-s, ␣-car [(60 R)-␤,⑀-carotene],
␤-car (␤,␤-carotene) and lutein [(3R,30 R,60 R)- ␤,⑀-carotene-3,30 -diol] was carried out by
co-chromatography with authentic standards (Sigma). Other xanthophylls were identified
obtaining their specific absorption maxima, in comparison with values from the literature
and by co-chromatography with algal cultures (Foppen, 1971; Mantoura and Llewellyn,
1983; Wright and Shearer, 1984; Wright et al., 1991; Jeffrey et al., 1997). Quantification
of carotenoids was done after Mantoura and Llewellyn (1983) by calculating the ratio of
extinction coefficients.
Chl-s were quantified spectrophotometrically (Fa. LKB, Ultrospek K) after the formulae
of Lichtenthaler and Wellburn (1983) because chromatograms showed high amounts of
chl-ides which developed during extraction period.
Chl a which could be traced back to epiphytic diatoms was substracted from that of
total chl a. Calculation of diatoms’ chl a was done with a peakarea-ratio of fucoxanthin/chl
a = 0.743 (Schagerl, unpublished results).

2.4. Dry mass and ash mass determination

Residues from the extraction procedure were used for dry mass (24 h, 95◦ C) and ash mass
determination (3 h, 450◦ C). Ash-free dry mass was calculated by subtracting ash mass from
dry mass. Pigment contents were expressed on the basis of ash-free dry mass because of
highly varying lime coverings on the surface of the charophytes.
120 M. Schagerl, C. Pichler / Aquatic Botany 67 (2000) 117–129

Table 1
Charophytes used in this study from six locations in eastern Austria: (1) Pool in Vienna, (2) Lake Neufelder See,
(3) Lake Erlaufsee, (4) Lake Hechtensee, (5) Lake Hallstätter See, (6) Lake Irrseea
Species Depth Light 1 2 3 4 5 6
distribution supply
C. tomentosa var. tomentosa Wood 2.0–4.0 2.4–64.9 䊉 䊉
C. vulgaris var. vulgaris f. vulgaris Wood 7.0 17.8 䊉
C. vulgaris var. vulgaris f. contraria (A.Br. ex Kütz.) 0–0.3 <100.0 䊉
Wood
C. vulgaris var. gymnophylla f. gymnophylla Wood 12.0 5.2 䊉
C. hispida var. hispida f. hispida Wood 4.0–5.0 34.0–42.1 䊉
C. hispida var. hispida f. polyacantha (A.Br.) Wood 1.5–10.2 5.9–72.3 䊉
C. hispida var. major. f. rudis (A.Br.) Wood 0.5–6.7 16.1–87.2 䊉 䊉
C. hispida var. major. f. intermedia (A.Br.) Wood 10.0 8.5 䊉
C. globularis var. globularis f. globularis Wood 2.0–6.0 0.1–57.5 䊉 䊉
C. globularis var. aspera f. aspera Wood 1.5–6.7 4.1–52.3 䊉 䊉
C. globularis Thuillier (naked) 2.0 57.5 䊉
N. opaca Ag. 9.5–12.0 2.8–5.4 䊉
a Light supply is indicated as percent of surface irradiance, depth distribution in meters.

The error in determination of the organic content which is due to lipid extraction by
acetone appeared to be minimal. We could not observe any significant loss in ash-free dry
mass after the extraction procedure.

3. Results

In total, 12 species of charophytes were collected from six different locations (Table 1).
Pigment pattern analyzed corresponds for the most part to that of Embryophyta (Fig. 1,

Fig. 1. Chromatogram of C. aspera. 1 = chl-ide b, 2 = chl-ide a, 3 = chl c, 4 = fucoxanthin, 5 = neoxanthin,

6 = cis-fucoxanthin, 7 = violaxanthin, 8 = diadinoxanthin, 9 = antheraxanthin, 10 = lutein, 11 = zeaxanthin,
12 = chl b, 13 = chl a, 14 = phaeophytin a, 15 = ␥-car, 16 = all-trans-␤-car, 17 = cis-␤-car.
 M. Schagerl, C. Pichler / Aquatic Botany 67 (2000) 117–129 121

Table 2
Major pigments of charophytes and absorption maxima in the eluent
Pigment Retention time (min) Maxima in the eluent

chl-ide b 4.76 460, 650

chl-ide a 8.12 (414), 431, (580), (622), 665
Neoxanthin 13.35 (413), 438, 466
Violaxanthin 14.82 (415), 441, 469
Antheraxanthin 16.36 (420), 445, 474
Lutein 17.41 (422), 446, 474
Zeaxanthin 17.62 (425), 452, 478
chl b 19.01 (433), 456, (594), 646
chl a 19.98 (410), 428, (580), (617), 662
␥-car 22.06 (437–439), 460, 490
␣-car 22.75 (424), 446, 473
␤-car 22.91 (429), 452, 476

Table 2). Nitella opaca (Neufelder See, 18-8-95), Nitellopsis obtusa, C. tomentosa, C.
globularis (Irrsee, 9-9-1995), as well as C. hispida, N. opaca and C. globularis (naked)
(Neufelder See, 25-11-1995) possessed ␣-car, weight ratio of ␤/␣-car was between 5 and 50.
Chl c, fuco-, diadino- and diatoxanthin indicated epiphytic diatoms growing on charophytes
(Fig. 1).

Table 3
Major pigments on the basis of ash-free dry mass (mg g−1 ; means and extremes)a
Forma Neoxanthin Violaxanthin Lutein ␥-car ␤-car chl a chl b
C. tomentosa 0.17 0.19 0.79 1.55 1.56 5.44 2.24
n = 30 0.08–0.34 0.08–0.39 0.46–1.47 0.08–5.53 0.57–4.24 2.28–9.92 1.03–4.12
C. vulgaris 0.20 0.17 0.82 n.d. 0.35 4.04 1.97
n = 10 0.14–0.26 0.13–0.21 0.60–0.96 n.d. 0.27–0.43 2.40–5.48 1.24–3.02
C. contraria 0.29 0.37 1.36 2.13 2.49 6.49 2.56
n = 20 0.19–0.35 0.28–0.54 1.00–1.74 1.46–3.08 2.12–3.23 3.82–8.59 1.49–3.38
C. gymnophylla 0.38 0.25 1.45 n.d. 0.58 5.44 2.36
n = 10 0.30–0.48 0.19–0.36 1.11–2.04 n.d. 0.43–0.80 4.10–6.72 1.77–2.89
C. hispida 0.12 0.09 0.59 0.05 0.24 4.36 1.73
n=5 0.10–0.16 0.08–0.11 0.54–0.66 0.00–0.08 0.22–0.26 4.07–4.78 1.68–1.83
C. polyacantha 0.17 0.11 0.75 0.04 0.42 4.10 1.64
n = 27 0.09–0.34 0.04–0.27 0.40–1.33 0.00–0.26 0.24–0.63 2.13–7.93 0.87–3.03
C. rudis 0.14 0.10 0.68 0.26 0.45 4.49 1.86
n = 45 0.09–0.19 0.05–0.17 0.48–0.97 0.00–1.01 0.22–0.85 3.12–6.50 1.36–2.72
C. intermedia 0.22 0.15 0.98 n.d. 0.39 3.00 1.40
n = 10 0.10–0.36 0.09–0.21 0.43–2.33 n.d. 0.24–0.86 1.20–3.89 0.53–1.82
C. globularis 0.33 0.40 2.27 0.02 0.84 16.66 6.85
n=5 0.29–0.38 0.34–0.47 2.15–2.37 0.00–0.06 0.75–0.94 16.29–17.09 6.62–6.98
C. aspera 0.14 0.12 0.73 0.06 0.40 4.85 2.02
n = 45 0.08–0.24 0.04–0.20 0.41–1.19 0.00–0.28 0.16–0.78 2.64–7.12 1.15–3.05
N. opaca 0.35 0.21 1.54 <0.01 0.58 8.97 3.67
n = 27 0.21–0.58 0.14–0.30 0.91–2.32 0.00–0.01 0.33–0.83 5.69–11.93 2.44–4.78
a n.d. = not detected.
122 M. Schagerl, C. Pichler / Aquatic Botany 67 (2000) 117–129

Besides chl a and chl b, lutein, neoxanthin and violaxanthin are the xanthophylls to be
found in large quantities (Table 3). Antheraxanthin and zeaxanthin which are part of the
xanthophyll-cycle appear only in low concentrations. As proportions of ␤-car depend on the
condition of sexual reproduction, approximate values should be taken from green, sterile
plants without incidence of ␥-car (as explained further). For sterile specimens, ␤-car was
found in lower contents than lutein (Table 3).
Extreme low light conditions were responsible for the much highest chl a concentrations
in C. globularis, which was collected from the Irrsee. Also N. opaca showed chl a contents
above average. These specimens were gathered from deeper sites of Neufelder See. With the
exception of antheraxanthin and zeaxanthin, other carotenoids displayed larger amounts too.
The observed increase of the pigment content per unit biomass can be seen as physiological
adaptation to low light conditions.
Lowest chl a amounts were detected in C. intermedia (mean was 0.3% of ash-free dry
mass). C. contraria possessed highest quantities of the xanthophyll-cycle pigments viola-,
anthera- and zeaxanthin (Tables 3 and 4). This can be seen as a protective measure against
high light intensities. In fact, this species was found in small, shallow pools in the east of
Vienna. C. contraria was the most fertile and therefore maximal amounts of ␤- and ␥-car
were detected too.
␥-car was observed mainly in fertile charophytes, eluting immediately before ß-car. It was
identified by comparing its absorption properties in the eluent with the absorption maxima in
various solvents (Table 5). Pigment extraction of antheridia confirmed the exclusive location
in these sexual organs. The only exception was C. tomentosa, in which ␥-car was found
also in sterile specimens. In this species red coloured parts (tops of shoots, end cells of
side branches, spine cells) contained above all ␥-car, ␤-car was of minor importance [␥-car:
4.74 mg g−1 ash-free dry mass (SD = 0.52, n = 5), ␤-car: 3.69 mg g−1 ash-free dry mass
(SD = 0.37, n = 5), ␥/␤-car: 1.28 (SD = 0.07, n = 5)], whereas in green thallus parts ß-car
dominated [␥-car: 1.03 mg g−1 ash-free dry mass (SD = 0.39, n = 5), ß-car: 1.23 mg g−1
ash-free dry mass (SD = 0.29, n = 5), ␥/ß-car: 0.81 (SD = 0.12, n = 5)].
Weight ratio of chl a/chl b was between 2.0 and 2.5, sterile charophytes showed chl/
carotenoid-ratios of about 4.2 and xanthophyll/␤-car-ratios of approximately 3.3. Substan-
tially smaller xanthophyll/␤-car-ratios indicated the presence of ␥-car.
The mean chl/xanthophyll-ratios of the species varied between 3.4 and 7.8 depending
on light supply. C. globularis collected from Irrsee contained highest chl quantities, this
location showed less than 1% of incoming surface irradiance. In these specimens highest
pigment contents could be observed (Fig. 2). Among xanthophylls, lutein showed high-
est concentrations (57–75% of total xanthophylls). Quantities of pigments involved in the
xanthophyll-cycle (viola-, anthera-, zeaxanthin) were between 13 and 45%. Maximal values
were analyzed in C. contraria originating from ponds in the east of Vienna with light supply
of nearly 100%.

4. Discussion

Photosynthetic pigment concentrations found in other studies in comparison with our

results are listed in Table 6. Phytoplankton biomass contains for the most part, 0.1 to
M. Schagerl, C. Pichler / Aquatic Botany 67 (2000) 117–129 123
124 M. Schagerl, C. Pichler / Aquatic Botany 67 (2000) 117–129

Table 5
Absorption maxima of ␥-car in different solvents according to Rawen (1989) and in the eluent (this study)
Solvent Max 1 Max 2 Max 3

Acetone 439 461 491

Ethanol 438–440 460–462 489–495
Hexan 431–437 460–462 489–494
Petrolether 431–437 456–62 486–494
Eluent, peak 439 461 491
Eluent, shoulder 437 460 488

2.0% chl a (Donabaum, 1992). The situation in aquatic angiosperms is comparable to that
of planktonic algae, but fresh weight contains rarely more than 1.0% chl a. Larger cell
volumes of macrophytes (higher performance of absorption) may contribute to reduced chl
a/biomass ratios. Chl a portion of dry mass in aquatic angiosperms and phytoplankton is
between 0.1 and 1.0%.

Fig. 2. Pigment content of charophycean species analyzed in this study.

 M. Schagerl, C. Pichler / Aquatic Botany 67 (2000) 117–129 125

Table 6
Ranges/means of pigment contents of aquatic angiosperms and charophyceae cited and values obtained in this
studya
Species chl-s chl a chl b Carotenoids chl a/ chl-s/ x/
chl b carotenoids car-s
Charophytes
C. corallina Kl. ex
Willd., em. Rdw. (7) 2.00–9.00c 1.71–2.03
C. delicatula
Agardh (3) 0.36–0.64b 0.13–0.22b 0.31–0.57b
C. foetida A.Braun (5) 2.049c 0.69c 2.98
C. fragilis Desveux (3) 0.31–0.67b 0.10–0.24b 0.17–0.48b
C. hispida L. (1) 0.49–0.81b 0.19–0.37b 0.10–0.15b 2.10–2.70
C. hispida L. (1) 1.89–6.66c 0.72–3.01c 0.46–1.16c
C. rudis A.Braun (10) 0.28–0.73b
C. sp. (12) 2.34 2.41 2.48
C. tomentosa L. (8) 0.09–0.28b
C. vulgaris L. (3) 0.48–0.63b 0.17–0.24b 0.29–0.46b
Nitella sp. (13) 5.30 2.10 6.00
Others
E. canadensis
Michx. (2) 0.92 (0.08)b 3.52 (0.07)
E. canadensis 0.42–0.69b 0.309–0.52b 0.11–0.181b
Michx. (11)
E. canadensis 4.76–7.38c 3.51–5.91c 1.25–2.06c
Michx. (11)
Littorella uniflora
(L.) Aschers (13) 5.56–10.79c 4.19–7.94c 1.33–2.86c 0.79–1.23c 2.50–3.40
Myriophyllum
spicatum L. (9) 0.26–0.30b 0.11–0.15b 1.79–2.27
Potamogeton
nodosus Poiret (4) 0.58 (0.16)b 0.16 (0.04)b
P. nodosus Poiret (2) 1.91 (0.11)b 3.79 (0.09)
P. pectinatus L. (6) 0.00–0.26b 0.00–0.08b 1.60–5.40 0.77–3.33
P. pectinatus L. (4) 1.01 (0.22)b 0.24 (0.048)b
This study 1.73–23.96d 1.20–17.09d 0.53–6.98d 0.74–4.05d 1.82–3.21d 1.52–6.66d 1.73–
4.58d
a Data in mg pigment per g weight unit. Standard deviations in parentheses, x = xanthophylls. (1) Andrews

et al. (1984), (2) Barko and Filbin (1983), (3) Czezuga (1986), (4) Spencer and Ksander (1987), (5) Hager and
Stransky (1970), (6) Spencer (1986), (7) Howard-Williams et al. (1995), (8) Libbert and Walter (1985), (9) Marcus
(1980), (10) Pereyra-Ramos (1981), (11) Pokorny et al. (1984), (12) Seybold et al. (1941), (13) Sodergaard and
Bonde (1988).
b Fresh weight.
c Dry mass.
d Ash-free dry mass.

Charophytes showed distinct lower ratios of chl a/fresh weight and chl a/dry mass due
to calcium carbonate precipitation. In comparison with other submerged plants, the results
indicate that fresh and dry mass are not suitable as reference quantities. Chl a of stoneworts
on the basis of ash-free dry mass was comparable to that of planktonic algae and submerged
126 M. Schagerl, C. Pichler / Aquatic Botany 67 (2000) 117–129

Fig. 3. Regression line of ␥- and ␤-car in C. tomentosa.

angiosperms. Calculated ratios were about 0.5% chl a/ash-free dry mass which is surpassed
by many planktonic algae. But charophytes or aquatic angiosperms may compensate lower
chl contents with bigger surfaces for light absorption (Schoas, 1994; Kuchar, 1995).
Ratios of chl a/chl b were low, but lutein showed high concentrations. In comparison
with land plants (Maslova and Popova, 1993) sterile stoneworts also exhibit high ratios
of xanthophylls/␤-car. This can be noted as pre-adaptation in principle to low-light con-
ditions. According to Egle (1960) water plants display low chl a/chl b ratios but high
xanthophyll/ß-car ratios. This fact indicates an efficient formation of the light harvesting
complexes II.
The question arises whether car-s are located only in photosystems or in separate com-
partments, too. To solve this problem investigations were carried out on C. tomentosa,
which has pigment quantities like other charophytes with the exception of distinct higher
concentrations of ␥- and ␤-car. ␥- and ␤-car correlate to 97%, regression line intersects
the y-axis (␥-car = 0) at 0.56 mg ␤-car g−1 ash-free dry mass (Fig. 3). This corresponds
to the mean content of ß-car located in the photosystems in other stoneworts (Table 7). A

Table 7
Comparison of measured and calculated (thylakoids) chl-a/␤-car weight ratios of C. tomentosa (n = 30) vs values
obtained from other sterile charophycean species (n = 67)
C. tomentosa γ /β chl-a/␤-car chl-a/␤-car thylakoids chl-a/␤-car sterile charophytes

Minimum 0.13 0.89 4.11 4.51

Minimum 1.48 11.78 17.85 21.46
Mean 0.80 5.15 9.79 11.13
Standard deviation 0.41 3.44 3.60 3.27
 M. Schagerl, C. Pichler / Aquatic Botany 67 (2000) 117–129 127

further part of ␤-car is positioned in other compartments together with ␥-car. It becomes
apparent that there may exist two fractions of ␤-car. The first displays highest correlation
with ␥-car and is probably located in separate units, the second fraction is independent of
␥-car and can be found in thylakoids. Jimenez and Pick (1994) observed separated pools of
␤-car in Dunaliella bardawil Ben-Amotz and Avron. In D. salina Teodorescu, low temper-
atures led to the formation of ␥-car containing globules inside the plastids. In C. tomentosa,
light microscopic examination demonstrated that there exists pigment globules inside the
chloroplasts which are responsible for the red colouration of shoot tops sprouted quite
recently, end cells of side branches and spine cells. The green colouration of older plant
parts indicates a metabolization of secondary carotenoids during the ontogenesis of the
plastids.
The biological function of ␥-car is still unclear. ␥-car also occurs in shield cells of
antheridia (Karrer et al., 1943). Karrer et al. (1943) proposed a sexual function which was
not defined clearly. A further explanation could be a protective role against high light or
UV-radiation. In contrast to antheridia, egg cells are corticated with filaments containing
chloroplasts and guarded in this way, whereas antheridial shield cells lack intact chloroplasts
but possibly protect spermatozoids by means of ␥-car containing globules. The occurrence
of ␥-car in sterile specimens of C. tomentosa can also be seen as a protective measure
against excessive light supply but further investigations on this subject are necessary.
In limnological standard series ␥-car may be used as a pigment marker of the reproduction
state of characean meadows because ␥-car content can probably be traced back to the number
of antheridia.

5. Conclusion

In principle stoneworts contain the same chloroplast pigments as higher plants with the
exception of ␥-car. ␥-car was found in antheridia and is also mainly responsible for the red
colouration of sterile specimens of C. tomentosa. It is assumed to have a protective function
against excessive light supply.
Variations in pigment content between the different taxa were substantial but there seemed
to be no specific differences. The main part of variation can certainly be attributed to
adaptations to low light or high light conditions in the habitat.
A detailed analyses of the relationship between pigment composition and light supply
within Charophyta will be published separately.

References

Andrews, M., Box, R., McInroy, S., Raven, J.A., 1984. Growth of Chara hispida II. Shade adaptation. J. Ecol. 72,
885–895.
Czezuga, B., 1986. The effect of light on the content of photosynthetically active pigments in species of the genus
Chara. Aquat. Bot. 24, 397–401.
Barko, J.W., Filbin, G.J., 1983. Influences of light and temperature on chlorophyll composition in submersed
freshwater macrophytes. Aquat. Bot.15, 249–255.
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. Estuar. Coast. Shelf Sci. 36, 359–376.
128 M. Schagerl, C. Pichler / Aquatic Botany 67 (2000) 117–129

Blindow, I., Andersson, G., Hargeby, A., Johansson, S., 1993. Long-term pattern of alternative stable states in two
shallow eutrophic lakes. Freshwater Biol. 30, 159–167.
Dijk, G.M. van, 1991. Light climate and its impact on Potamogeton pectinatus L. in a shallow eutrophic lake.
Thesis, Wageningen.
Donabaum, K., 1992. Der Chlorophyll-a-Gehalt von Planktonalgen. Thesis, Vienna.
Downes, M.T., Hrstich, L., Vincent, W.F., 1993. Extraction of chlorophyll and carotenoid pigments from Antarctic
benthic mats for analysis by HPLC. J. Appl. Phycol. 5, 623–628.
Egle, K., 1960. Menge und Verhältnis der Pigmente. In: Ruhland, W. (Ed.), Handbuch der Pflanzenphysiologie,
Vol. V/1. Springer Verlag, Berlin, Göttingen, Heidelberg, pp. 444–496.
Ehrlich, U., Grill, D., 1994. Vitalitätsbeurteilung von Eichen eines steirischen Standortes im Vergleich zu
Standorten in Niederösterreich und Burgenland. Mitt. naturwiss. Ver. Steiermark 124, 219–236.
Foppen, F.H., 1971. Tables for the identification of carotenoid pigments. Chromatogr. Rev. 14, 133–298.
Frost-Christensen, H., Sand-Jensen, K., 1992. The quantum efficiency of photosynthesis in macroalgae and
submerged angiosperms. Oecologia 91, 377–384.
Garcia, C.M., Perez-Llorens, J.I., Niell, F.X., Lucena, J., 1991. Pigment estimations and photosynthesis of Ruppia
drepanensis Tin. ex Guss. in a hypersaline environment. Hydrobiologia 220, 147–153.
Gieskes, W.W., Kraay, G.W., 1983. Dominance of Cryptophyceae during the phytoplankton spring bloom in the
central North Sea detected by HPLC analysis of pigments. Mar. Biol. 75, 179–185.
Gieskes, W.W., Kraay, G.W., 1986. Analysis of phytoplankton pigments by HPLC before, during and after mass
occurence of the microflagellate Corymbellus aureus during the spring bloom in the open Northern Sea in
1983. Mar. Biol. 92, 45–52.
Hager, A., Stransky, H., 1970. Das Carotinoidmuster und die Verbreitung des lichtinduzierten Xanthophyllcyclus
in verschiedenen Algenklassen III. Grünalgen. Arch. Mikrobiol. 72, 68–83.
Howard-Williams, C., 1978. Growth and production of aquatic macrophytes in a south temperate saline lake. Verh.
Int. Verein. Limnol. 20, 1153–1158.
Howard-Williams, C., Schwarz, A.-M., Vincent, W.F., 1995. Deep-water aquatic plant communities in an
oligotrophic lake: physiological responses to variable light. Freshwater Biol. 33, 91–102.
Jeffrey, S.W., Mantoura, R.F.C., Wright, S.W. (Eds.), 1997. Phytoplankton Pigments in Oceanography.
Monographs on Oceanographic Methodology 10. Unesco Publishing, France.
Jimenez, C., Pick, U., 1994. Differential stereoisomer compositions of ␤, ␤-carotene in thylakoids and in pigment
globules in Dunaliella. J. Plant Physiol. 143, 257–263.
Karrer, P., Fatzer, W., Favarger, M., Jucker, E., 1943. Die Antheridienfarbstoffe von Chara-Arten
(Armleuchtergewächse). Helv. Chim. Acta 26, 2121–2122.
Krause, W., King, J.J., 1994. The ecological status of Lough Corrib, Ireland, as indicated by physiographic factors,
water chemistry and macrophytic flora. Vegetatio 110, 149–161.
Krolokowska, J., 1997. Eutrophication process in a shallow, macrophyte-dominated lake. Species differentiation,
biomass and the distribution of submerged macrophytes in Lake Luknajno (Poland). Hydrobiologia 342, 411–
416.
Kuchar, F., 1995. Die vertikale Verteilung von Licht, Biomasse und Struktur in Makrophytenbeständen von
Ranunculus fluitans und Groenlandia densa. Thesis, Vienna.
Kunii, H., 1984. Seasonal growth and profile structure development of Elodea nuttallii (Planch.) St. John in Pond
Ojaga-Ike, Japan. Aquat. Bot. 18, 239–247.
Latasa, M., Estrada, M., Delgado, M., 1992. Plankton-pigment relationships in the Northwestern Mediterranean
during stratification. Mar. Ecol. Prog. Ser. 88, 61–73.
Lehman, P.W., 1981. Comparison of chlorophyll-a and carotenoid pigments as predictors of phytoplankton
biomass. Mar. Biol. 65, 237–244.
Libbert, E., Walter, T., 1985. Photosynthetic production of a brackish water community of Chara tomentosa L.
and its dependence on environmental conditions. Int. Rev. ges. Hydrobiol. 70, 359–368.
Lichtenthaler, H.K., Wellburn, A.R., 1983. Determinations of total carotenoids and chlorophylls a and b of leaf
extracts in different solvents. Biochem. Soc. T. 11, 591–592.
Mantoura, R.F.C., Llewellyn, C.A., 1983. The rapid determination of algal chlorophyll and carotenoid pigments
and their breakdown products in natural waters by reverse-phase high-performance liquid chromatography.
Anal. Chim. Acta 151, 297–314.
 M. Schagerl, C. Pichler / Aquatic Botany 67 (2000) 117–129 129

Marcus, B.A., 1980. Relationship between light intensity and chlorophyll content in Myriophyllum spicatum L.
in Canadice Lake (New York, USA). Aquat. Bot. 9, 169–172.
Margalef, R., 1960. Valeur indicatrice de la composition des pigments du phytoplankton sur la productivitè,
composition taxonomique et propriètès dynamiques des populations. Rapp. Process-Verbaux C.I.E.S.M. 15,
274–281.
Margalef, R., 1965. Ecological correlations and the relationship between primary productivity and community
structure. In: Goldman, C.R. (Ed.), Primary Productivity in Aquatic Environments. Mem. Ist Ital. Idrobiol.
(Suppl. 18), 355–364.
Maslova, T.G., Popova, I.A., 1993. Adaptive properties of the plant pigment systems. Photosynthetica 29, 195–203.
Millie, D.F., Paerl, H.W., Hurley, J.P., 1993a. Microalgal pigment assessments using High-Performance Liquid
Chromatography: a synopsis of organismal and ecological applications. Can. J. Fish. Aquat. Sci. 50, 2513–2527.
Millie, D.F., Paerl, H.W., Hurley, J.P., Kirkpatrick, G.J., 1993b. Algal pigment determinations in aquatic
ecosystems: analytical evaluations, application, and recommendations. Curr. Topics Bot. Res. 1, 1–13.
Mitchell, S.F., 1989. Primary production in a shallow eutrophic lake dominated alternatively by phytoplankton
and by submerged macrophytes. Aquat. Bot. 33, 101–110.
Pereyra-Ramos, E., 1981. The ecological role of Characeae in the lake littoral. Ekologia polska 29, 167–209.
Phillips, G.L., Eminson, D., Moss, B., 1978. A mechanism to account for macrophyte decline in progressively
eutrophicated freshwaters. Aquat. Bot. 4, 103–126.
Pokorny, J., Kvet, J., Ondok, J.P., Toul, Z., Ostry, I., 1984. Production-ecological analysis of a plant community
dominated by Elodea canadensis Michx. Aquat. Bot. 19, 263–292.
Rawen, K.S., 1989. Photosynthetic pigments of algae. Cambridge University Press, Cambridge.
Riegman, R., Rowe, A., 1994. Nutritional status and pigment composition of phytoplankton during spring and
summer Phaeocystis blooms in dutch coastal waters (Marsdiep area). Neth. J. Sea Res. 32, 13–21.
Roy, S., 1989. HPLC-measured chlorophyll-type pigments during a phytoplankton spring bloom in Bedford Basin
(Canada). Mar. Ecol. Prog. Ser. 55, 279–290.
Sand-Jensen, K., 1978. Metabolic adaptation and vertical zonation of Littorella uniflora (L.) Aschers. and Isoetes
lacustris L. Aquat. Bot. 4, 1–10.
Schagerl, M., Krbec, H., Nairz, S., Wieltschnig, C., 1996. Pelagische Primärproduktion in einem Donaualtarm bei
Regelsbrunn (Niederösterreich). Verh. Zool. Bot. Ges. Österreich 133, 201–216.
Scheffer, M., van den Berg, M., Breukelaar, A., Breukers, C., Coops, H., Doef, R., Meijer, M.-L., 1994. Vegetated
areas with clear water in turbid shallow lakes. Aquat. Bot. 49, 193–196.
Schoas, R., 1994. Vertikale Verteilung von Photosynthesepigmenten in zwei verschieden strukturierten
Makrophytenbeständen. Thesis, Vienna.
Seybold, A., Egle, K., Hülsbruch, W., 1941. Chlorophyll- und Carotinoidbestimmungen von Süßwasseralgen. Bot.
Arch. 42, 239–253.
Sodergaard, M., Bonde, G., 1988. Photosynthetic characteristics and pigment content and composition in Littorella
uniflora (L.) Aschers. in a depth gradient. Aquat. Bot. 32, 307–319.
Spence, D.H.N., Chrystal, J., 1970. Photosynthesis and zonation of freshwater macrophytes. New Phytol. 69,
217–227.
Spencer, D.F., 1986. Early growth of Potamogeton pectinatus L. in response to temperature and irradiance:
morphology and pigment composition. Aquat. Bot. 26, 1–8.
Spencer, D.F., Ksander, G.G., 1987. Comparison of three methods for extracting chlorophyll from aquatic
macrophytes. J. Freshwater. Ecol. 4, 201–208.
Wood, R.D., Imahori, K., 1965. A Revision of the Characeae, Vols. 1 and 2. Cramer, Weinheim.
Wright, S.W., Shearer, J.D., 1984. Rapid extraction and high-performance liquid chromatography of chlorophylls
and carotenoids from marine phytoplankton. J. Chromatogr. 294, 281–295.
Wright, S.W., Jeffrey, S.W., Mantoura, R.F.C., Llewellyn, C.A., Bjornland, T., Repeta, D., Welschmeyer, N., 1991.
Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar.
Ecol. Prog. Ser. 77, 183–196.