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Nutrient limitation of autotrophic and
mixotrophic phytoplankton in a temperate
and tropical humic lake gradient
CARINA PÅLSSON* AND WILHELM GRANÉLI
LIMNOLOGY, DEPARTMENT OF ECOLOGY, LUND UNIVERSITY, ECOLOGY BUILDING, SE-22362 LUND, SWEDEN
*CORRESPONDING AUTHOR:
[email protected]
Received December 22, 2003; accepted in principle February 27, 2004; accepted for publication April 21, 2004; published online May 4, 2004
Nutrient enrichment experiments were carried out in three tropical (once) and three temperate (twice)
lakes differing in humic content in order to examine whether there was a relationship between the limiting
nutrient for algal growth [nitrogen (N) or phosphorus (P)] and humic content, and whether the
prevailing limitation was connected to the relative abundance of autotrophic and phagotrophic phytoplankton (mixotrophs). In both climatic regions, there was a stronger tendency for total phytoplankton
biomass accumulation to be N limited in lakes with a high humic content. However, in contrast to what
we expected, there was no tendency for the mixotrophs to be more favored by the addition of N than of
P. In the temperate lakes, the relative abundance of mixotrophs increased in the treatments receiving
N or P separately or no nutrients (control) when exposed to a high light availability. In the following
year, when the light availability was low, the mixotrophs increased relative to the obligate autotrophs
in all treatments, irrespective of nutrient addition. Possibly, this was a result of their ability to
supplement photosynthesis with the ingestion of prey. The results indicate that mixotrophy is an
advantageous strategy when the availability of light and/or nutrients is low.
INTRODUCTION
The study of limiting macronutrients for bacterial and,
in particular, algal growth has been a central theme of
modern limnology, with a peak during the 1970s as a
consequence of an increased awareness of eutrophication processes. Based on extended studies carried out in
primarily North American and European lakes, growth of
phytoplankton and bacteria is generally considered to be
phosphorus (P) limited in freshwater systems (Schindler,
1977; Elser et al., 1995). Although nutrient limitation
in lakes subjected to a high input of allochthonous carbon has been little examined, P could also be expected to
be the limiting nutrient for bacterial and phytoplankton
growth in these systems. This is a result of the strong
tendency of humic substances to associate with phosphate ions and thereby decrease P availability ( Jones,
1992). However, nutrient enrichment studies indicate
that the phytoplankton community in humic lakes
might be simultaneously limited by nitrogen (N) and P
( Jones, 1990) or even limited by N alone ( Jansson et al.,
1996, 2001). One possible explanation for this paradox
was proposed by Jansson et al. ( Jansson et al., 1996,
1999). They argued that the observed N limitation of
the phytoplankton community in humic Lake Örträsket
(northern Sweden) could be related to high densities of
mixotrophic phytoplankton grazing on P-rich bacteria.
Mixotrophy, i.e. the ability to combine phototrophy and
heterotrophy, includes by definition osmotrophic as well
as phagotrophic heterotrophy. Throughout this paper,
however, the term mixotrophy will be used to refer only
to phagotrophic mixotrophy, which has been suggested
to be an especially successful strategy in humic lakes
( Jones, 1992). This hypothesis is based on the impaired
light climate, the high bacteria:phytoplankton biomass
ratio and fairly low concentrations of inorganic nutrients
characteristic of these environments (Tranvik, 1989;
Jones, 1992).
There is a large nutritional diversity among mixotrophic phytoplankton; however, prey ingestion can be
a means of obtaining major nutrients (Caron et al., 1990;
doi:10.1093/plankt/fbh089, available online at www.plankt.oupjournals.org
Journal of Plankton Research Vol. 26 No. 9 Ó Oxford University Press 2004; all rights reserved
JOURNAL OF PLANKTON RESEARCH
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Nygaard and Tobiesen, 1993), specific growth factors or
essential vitamins (Kimura and Ishida, 1985; Skovgaard
2000), or to acquire carbon during periods of low photon
flux density or short photoperiod ( Jones et al., 1995).
Thus, mixotrophy should be advantageous when nutrients are limited and/or the light regime poor; this is
consistent with the observed high proportion of mixotrophic species in humic and arctic oligotrophic lakes
(Salonen and Jokinen, 1988; Jansson et al., 1996; Hobbie
et al., 1999). Moreover, several studies have shown a
connection between nutrient limitation and prey ingestion rates among mixotrophs (Nygaard and Tobiesen,
1993; Urabe et al., 1999; Li et al., 2000; Smalley and
Coats, 2002). In bacterivorous mixotrophs, prey ingestion could be a means to remove nutrient competitors
(Thingstad et al., 1996) and simultaneously obtain P
(Nygaard and Tobiesen, 1993; Jansson et al., 1996;
Urabe et al., 1999). This hypothesis is based on bacteria
being superior competitors for inorganic P compared
with most phytoplankton as a result of their higher surface to volume ratio (Currie and Kalff, 1984; Bratbak
and Thingstad, 1985) and the often higher internal P:N
ratio of bacteria than of phytoplankton ( Jürgens and
Güde, 1990). According to Jansson et al., the ingestion
of bacteria will lead to a relative surplus of P over N
within the mixotrophs, which in combination with their
high abundance is suggested to account for the observed
N limitation of total phytoplankton biomass accumulation in humic lakes ( Jansson et al., 1996; Jansson, 1998).
In Lake Örträsket, this theory was supported by an
increase in mixotrophic and potentially mixotrophic
species when N was added ( Jansson et al., 1996).
We have examined the limiting nutrient for phytoplankton biomass accumulation in a temperate as well as
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a tropical humic lake gradient. In addition, we examined
the response of mixotrophic and obligate autotrophic
phytoplankton to nutrient enrichments. The aims were
to investigate (i) whether there was a stronger tendency
for N limitation in lakes with a higher humic content and
(ii) whether N limitation could be attributed to the mixotrophic part of the phytoplankton community, as proposed by Jansson et al. (Jansson et al., 1996).
METHOD
Study areas and field sampling
The study was carried out in three tropical lagoons
(Imboassica, Cabiunas and Comprida) situated in the
district of Macaé in the state of Rio de Janeiro, Brazil
(22 210 S), as well as in three temperate lakes (Skärlen,
Skärshult and Fräjen) in southern Sweden (57 070 N).
The lakes were chosen to represent a gradient in
humic content ranging from 0.17 to 2.27 and from
0.22 to 1.91 mmol dissolved organic carbon (DOC)
L1, respectively, in Brazil and Sweden; the three lakes
in each region are referred to as low, medium and high
humic (see Table I for further lake characteristics).
Imboassica is subjected to discharge of domestic sewage
from the surrounding urban areas, whereas Cabiunas
and Comprida are surrounded by coastal dune vegetation and little exposed to anthropogenic influence
(Panosso and Esteves, 1999). The temperate lakes are
all set in an area with coniferous forest. Nutrient enrichment experiments were carried out during summer
(November–December 1998) with water from the
Brazilian lagoons and during two subsequent summers
( June–July 1999 and 2000) in southern Sweden.
Table I: Lake characteristicsa and concentrations of DOC (mmol L1), inorganic nutrients (M),
TP b (M) and bacteria ( 106 cells mL1) on each sampling occasion
Area (km2)
Lake
Skärlen
1
3.30
Mean
DOC
NH4+ + NO3–
PO43–
Secchi
Bacteria
TP
depth (m)
(mmol L1)
(mM)
(mM)
depth (m)
( 106 cells mL1)
(mM)
8.7
0.22
2.64
0.10
6.0
0.95
0.16
0.37
3.16
0.16
7.5
0.63
1.39
4.78
0.16
0.7
1.42
1.25
5.78
0.07
1.1
1.03
2
Skärshult 1
0.30
3.8
2
Fräjen
DIN/TP
16.5
19.8
0.80
6.0
7.2
1
0.20
0.8
1.91
2
0.19
0.4
2.20
0.39
5.1
Imboassica
3.26
1.1
0.17
1.36
0.07
1.1
5.71
1.30
1.0
Cabiunas
0.34
2.4
0.52
1.36
0.10
1.0
6.51
0.39
3.5
Comprida
0.13
1.6
2.27
5.06
0.42
0.3
4.29
2.26
2.2
The numbers 1 and 2 denote the two separate years 1999 and 2000.
a
Area and mean depth from Bergvall (1992) and Panosso et al. (1998).
b
Concentration of TP from Panosso and Esteves (1999), Farjalla (1998), Tranvik (1988) and The Swedish Environmental database.
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Water was collected from the central parts of each lake.
However, sampling depth and incubation procedures varied somewhat between the years. In 1998 and 1999, water
was collected from 0.2 m depth and incubated in a
temperature-controlled pool protected from direct sunlight
(40% of incident photosynthetically active radiation in
the incubation bottles; Brazil), or close to the surface in a
nearby lake (Sweden). In both instances, the light availability was high compared with that in the epilimnion. In
2000, water was collected from, and incubated at, 2.2 m
depth in Lake Skärlen as well as Lake Skärshult, Sweden.
The light climate at this depth was equivalent to 25% of
the surface light in the low humic lake (Skärlen) and <1%
in the high humic lake (Skärshult). On each sampling
occasion, Secchi depth and temperature were measured
and 40 mL of water from each lake were filtered
(Whatman GF/F) and stored frozen until analyzed for
dissolved inorganic nutrients (PO43–, NH4+, NO3–) and
DOC. In addition, samples for analyses of bacterial densities were preserved with formalin (2% final concentration).
Experimental design
Large zooplankton predators were removed by filtering,
using a 70 mm (1998 and 1999) or a 150 mm (2000) mesh
nylon net. The collected water was then transferred to 2 L
incubation bottles and subjected to the following treatments, each in three or four replicates depending on
year: nitrogen addition (N treatment), phosphorus addition (P treatment), nitrogen + phosphorus addition (NP
treatment) and no nutrients added (control). In the bottles receiving nutrients, the N content was increased by
140 mM by the addition of NH4NO3 (N and NP treatments), whereas the P concentration was increased by
3.23 mM by the addition of KH2PO4 (P and NP treatments). After nutrient addition, the bottles were incubated for 5–7 days. On a daily basis, the bottles were
aerated and gently shaken in order to prevent epiphytic
growth. As a means of surveying phytoplankton biomass,
analyses of chlorophyll (Chl) a were performed every
second day (1998 and 1999) or on the first and final
day (2000). In addition, samples from each treatment on
the first and last day of the incubation periods were
preserved with acid Lugol’s solution and examined to
determine phytoplankton species composition.
Abiotic and biotic measurements
DOC was analyzed by the Pt-catalyzed high-temperature
combustion method using a Shimadzu TOC-5000 total
carbon analyzer equipped with an ASI-5000 autosampler.
Concentrations of PO43–, NH4+ and NO3– were analyzed
on a Technicon Autoanalyzer II according to Technicon
protocols and Swedish standard methods [SS028126,
SS028134 and SS028133 (Bydén et al., 1996)]. For Chl a
analyses, aliquots from each replicate were filtered onto
GF/F filters (Whatman). The filters were stored frozen
until analyzed and the Chl a was extracted in ethanol
overnight ( Jespersen and Christoffersen, 1987). Chl a
fluorescence was measured on a Turner Designs TD-700
fluorometer and absorbance on a Beckman DU 650 spectrophotometer at 665 and 750 nm. Bacterial numbers
were obtained with a FACSort (Becton Dickinson) flow
cytometer according to del Giorgio et al. (del Giorgio et al.,
1996). Cells were stained with SYTO-13 (Molecular
Probes) at a final concentration of 2.5 mM using Fluoresbrite carboxylate microspheres (1.58 mm diameter) as the
reference. Taxonomic identifications and enumerations of
phytoplankton were performed using an inverted microscope. For estimates of phytoplankton biomass in the
sampled lakes, cell volumes were calculated from the formulae for solid geometric shapes according to Wetzel and
Likens (Wetzel and Likens, 1991).
Apart from determining taxonomic affiliation, species
reported to ingest bacteria or other algae were referred to
as mixotrophs, whereas unidentified pigmented flagellates
and species believed to have the capacity for ingestion
were referred to as potential mixotrophs. In this study,
Dinobryon spp., Ochromonas spp., Spiniferomonas spp., Cryptomonas spp., Gymnodinium spp. and Peridinium spp. were
referred to as mixotrophic (Hitchman and Jones, 2000;
Pålsson and Granéli, 2003), and Rhodomonas spp./Chroomonas spp. and small unidentified flagellates (monads <5 mm,
mainly believed to be Ochromonadales and prymnesiophytes) as potential mixotrophs. In the tropical lagoons,
Cryptomonas spp. were also treated as potential mixotrophs
due to there being no documentation of ingestion by this
genus in tropical environments.
To estimate differences in Chl a between treatments,
comparisons of the percentage change in Chl a during the
incubations were analyzed for each lake and year using
ANOVA followed by Tukey’s post-hoc test. Changes in
relative abundance of the major phytoplankton groups
(only temperate lakes) and mixotrophs (all lakes) in response
to the nutrient enrichment treatments were analyzed separately for each year. Nested ANOVA was used to examine
differences between lakes, whereas between-treatments
comparisons were made using ANOVA followed by
Tukey’s post-hoc test. The raw data were arcsin or log10
transformed when necessary. All statistical analyses were
performed using the SPSS 10 software (SPSS Inc.).
RESULTS
Concentrations of inorganic nutrients were low and
within the same range in both lake gradients (Table I).
Lake water temperatures were slightly higher in the
temperate lakes in the first summer (18–20 C) compared
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with the second one (16–17 C). Temperatures in the
tropical lagoons were 27–30 C. Irrespective of humic
content, bacterial densities were higher in the tropical
lakes (4.3–6.5 106 bacteria mL1) than in the temperate ones (0.6–2.2 106 bacteria mL1).
Initial phytoplankton biomass and
community composition
In the temperate lakes, the measured concentrations of
Chl a differed greatly between the years and were considerably higher in 1999, when they reached 22.4 and
42.1 mg Chl a L1 in Lake Skärshult and Lake Fräjen
(Figure 1a). This was due to mass occurrence of Gonyostomum semen, which was present at densities as high as
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460 and 970 cells mL1 in Lakes Skärshult and Fräjen,
respectively. As a result, G. semen (Raphidomonadida)
completely dominated the biomass in the medium
(Skärshult, 62%) as well as the high humic (Fräjen,
91%) lake during the first summer (Figure 1a). In the
second summer, there was a slight dominance of chlorophytes in Lake Skärshult. In the low humic lake
(Skärlen), the biomass distribution was similar during
both summers, with a slight dominance of chlorophytes
(40 and 27%). In the tropical lagoons, cyanophytes constituted the highest proportion of biomass in Lake
Imboassica (56%) as well as Lake Comprida (48%),
whereas cryptophytes (30%) and chlorophytes (36%) were
common in Lake Cabiunas (Figure 1a).
Fig. 1. Concentrations of Chl a (mg L1) and the proportion of (a) major phytoplankton taxonomic groups and (b) mixotrophs, potential
mixotrophs and obligate autotrophs as a percentage of the total phytoplankton biomass in the temperate (Skärlen, Skärshult and Fräjen) and
tropical (Imboassica, Cabiunas and Comprida) lakes.
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The biomass of mixotrophic and potentially mixotrophic phytoplankton differed greatly between the lakes,
and no consistent relationships were found between biomass and abiotic parameters or bacterial densities. In the
temperate lakes, the mixotrophic genera present were
Dinobryon spp., Ochromonas spp., Spiniferomonas spp., Cryptomonas spp., Gymnodinium spp. and Peridinium spp. Contrary
to bacterial densities, the mixotrophic biomass (absolute as
well as relative) in the temperate region was highest in the
low humic lake during both summers (28–41% of the total
biomass compared with 1–11% in the more humic lakes;
Figure 1b). In the tropical region, the mixotrophic biomass
was low in all lakes and constituted only 1–7% of the total
biomass (Figure 1b). The mixotrophic genera found in
these lakes, Dinobryon spp., Gymnodinium spp. and Ochromonas
spp., were present at low densities and Cryptomonas spp.,
which were classified as potentially mixotrophic, were
present at fairly high densities only in Lake Cabiunas.
Although the highest absolute biomass of potential mixotrophs in the temperate region was found in the high
humic lake, the proportion of the total biomass that
could be attributed to the potential mixotrophs was higher
in the low humic lake (16–18%) than in the lakes with a
higher humic content (3–6%; Figure 1b). In the tropical
lagoons, the potential mixotrophs constituted 2–33% of
the biomass and primarily followed the distribution of
Cryptomonas spp.
Response to nutrient additions
In the examined lakes, there was no consistent relationship between humic content and the prevailing limiting
nutrient for the phytoplankton community. In the tropical region, the total phytoplankton biomass increased
significantly in all lakes in the NP and N treatments
compared with the controls (ANOVA followed by
Tukey, P < 0.05; Figure 2). However, the response to
N addition was most marked in the high humic lake. In
the temperate lakes, the limiting nutrient varied between
lakes and years. In the first summer, the phytoplankton
biomass in the low as well as the high humic lake
increased significantly compared with the control in
response to N and NP additions (ANOVA followed by
Tukey, P < 0.05; Figure 2). As in the tropical lakes, the
response to N addition was considerably stronger in the
high humic lake (Figure 2). In the medium humic lake,
the phytoplankton biomass reacted as if P limited and
increased in response to NP and P additions (ANOVA
followed by Tukey, P < 0.05; Figure 2). In the nutrient
enrichment experiments performed during the second
summer, the light availability was low due to a deeper
incubation depth. In the low humic lake, phytoplankton
biomass accumulation was limited by P and increased in
response to P and NP additions (ANOVA followed by
Tukey, P < 0.05). In the medium humic lake, the phytoplankton biomass decreased somewhat in all treatments
(Figure 2), presumably as a result of the low light
availability.
In the temperate lakes, the response to nutrient additions differed between taxonomic groups and years. In
1999, the proportion of chrysophytes increased in all
treatments except when N and P were added simultaneously (ANOVA, P < 0.05; Figure 3a). In 2000, the
chrysophytes increased in relative abundance in all treatments, and no differences were observed between them
(ANOVA, P > 0.05). Neither did the response of the
chrysophytes differ between the examined lakes in 1999
Fig. 2. Relative concentrations of Chl a in the nutrient enrichment treatments (N, P and NP) and control on the final day of incubation,
presented as a percentage of concentrations on day 1 (100%). Each value is the mean (n = 2–4) SD (asterisks denote the ANOVA betweentreatments significance level).
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Fig. 4. Mean changes (%) in the relative abundances of mixotrophic
phytoplankton in response to nutrient additions in the temperate
(1999 and 2000) as well as tropical lakes (n = 6 treatment1 year1).
Fig. 3. Mean changes (%) in the relative abundances of (a) chrysophytes,
(b) chlorophytes and (c) cyanophytes in response to nutrient additions in
the temperate lakes in 1999 and 2000 (n = 6 treatment1 year1).
or 2000 (nested ANOVA, P > 0.05). The chlorophytes,
on the other hand, decreased in relative abundance in all
treatments in the second year (Figure 3b), whereas they
increased significantly in proportion in the NP treatment
in 1999 (ANOVA, P < 0.05). The response towards nutrient additions did not vary between the lakes (nested
ANOVA, P > 0.05). For the cyanophytes, no significant
differences in relative abundance were found between
lakes or treatments (ANOVA, P > 0.05); however, the
proportion of cyanophytes decreased in all treatments in
2000 (Figure 3c). The relative abundance of cryptophytes
decreased in all treatments during both years, independent
of light regime.
The relative abundance of mixotrophs also differed
between treatments and years. In 1999, the response of
the mixotrophs to nutrient enrichment did not differ
between lakes (nested ANOVA, P > 0.05), but did
between treatments (ANOVA, P < 0.05). The mixotrophs increased significantly in the P treatment and
control (Tukey’s post-hoc test, P < 0.05) and somewhat
less also in the N treatment relative to the NP treatment
(Figure 4). In the following year, the relative abundance
of mixotrophs increased in all treatments and no differences were found between them. When comparing the
two lakes, the increase was more marked in the low
humic than the high humic lake (nested ANOVA, P <
0.01). In the tropical lakes, the relative abundances of
mixotrophs were relatively unchanged and no statistically significant differences were observed between the
lakes (nested ANOVA, P > 0.05) or treatments
(ANOVA, P > 0.05). For the potential mixotrophs, the
overall response towards nutrient enrichment was less
consistent and their relative abundance decreased in the
tropical as well as the temperate lakes in 1999, whereas
they increased in relative abundance in 2000. No statistically significant differences were observed between the
treatments.
DISCUSSION
Although the limiting nutrient(s) for phytoplankton biomass accumulation varied between the sampled lakes,
we found a stronger tendency for N limitation in high
humic lakes than in lakes with a lower DOC content in
both climatic regions. However, in contrast to the theory
proposed by Jansson et al. ( Jansson et al., 1996), there
was no tendency for the mixotrophs to be more favored
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by the addition of N than of P. In the temperate lakes,
the mixotrophs increased in relative abundance over the
obligate autotrophs when N, P or no nutrients (control)
were added in the first year. The following year, the
mixotrophic phytoplankton increased in relative abundance in all treatments, possibly as a result of the low light
availability.
Nutrient limitation
Although phytoplankton growth in freshwater systems is
generally considered to be P limited, algal growth limitation by N is also commonly observed (Elser et al., 1990).
While some studies show no general relationship
between limiting nutrient (N or P) and humic content
(Nürnberg and Shaw, 1999), other studies have indicated that N limitation of algal growth might be particularly common in humic-rich systems ( Jansson et al.,
1996, 2001), which is in agreement with the results
from our study. In addition, N is thought to be more
likely limiting for phytoplankton growth in tropical compared with temperate systems (Elser et al., 1990). In the
tropical lakes included in this study, phytoplankton biomass accumulation was limited by N, which was also
supported by the low dissolved inorganic nitrogen:total
phosphorus (DIN:TP) ratio. However, the N limitation
was more pronounced in the lake with the highest humic
content, in which the increase in phytoplankton biomass
was close to that in the NP treatment. The same trend
was observed in the temperate lakes, which are situated
in an area with a higher atmospheric N-deposition (and
hence higher total nitrogen concentrations) than the
lakes studied by Jansson et al. ( Jansson et al., 1996). Also
in this instance, the pronounced N limitation of phytoplankton biomass accumulation in the high humic lake
was supported by the DIN:TP ratio. However, a fairly
high humic content was not necessarily associated with
N limitation, as phytoplankton biomass accumulation in the
temperate medium humic lake (DOC 1.25 mmol L–1)
was clearly P limited in 1999. Nitrogen limitation of
algal growth in humic lakes has been suggested to be a
result of high densities of mixotrophic algae grazing on
bacteria rich in P (Jansson et al., 1996). Although the
relative abundance of mixotrophs in the temperate lakes
increased in response to N addition, this hypothesis was
not supported by our results, as the mixotrophs also
appeared to be favored over the obligate autotrophs in
the P and control treatments. As bacterial biomass accumulation is often P limited (Elser et al., 1995), the mixotrophs might have been stimulated by an increased
bacterial growth in the P treatment. In the tropical
lakes, the mixotrophic biomass was low and no differences in relative abundances were observed between the
treatments. Thus, in both climatic regions, the biomass
of obligate autotrophs also increased in response to N
addition, which is in agreement with the results from a
whole-lake nutrient-enrichment experiment performed
in northern Sweden ( Jansson et al., 2001).
Apart from a stronger N limitation of algal growth in
the high humic lakes, the limiting nutrient(s) varied considerably between lakes and, for the temperate lakes,
between the two years. These inconsistencies may be
explained by the method (i.e. nutrient enrichment bioassay) used for examining the prevailing nutrient limitation, as the outcome mirrors the situation at a precise
occasion and depth, and no consideration is given to
spatial and seasonal variations. Thus, the variations in
nutrient limitation observed in this study are consistent
with the view that both N and P are in relatively short
supply in oligotrophic lakes, and that the short-term
limiting nutrient of a natural multi-species phytoplankton community might vary over time as a result of
ambient nutrient availability and specific physiological
requirements of the various species (Hecky and Kilham,
1988; Elser et al., 1990). It could be argued that the
results of the incubations were affected by the small
incubation volumes and the inability of algae to migrate
in the water column and thereby access nutrients in
deeper layers ( Järvinen and Salonen, 1998). The confinement could affect some species more than others,
and might explain the decreasing abundances of cryptophytes in all treatments, as these algae are known to
perform extensive vertical migrations ( Jones, 1988).
Mixotrophic phytoplankton
During the first summer in the temperate lakes, the extremely high phytoplankton biomasses in the medium and
high humic lakes were mainly a result of high abundances
of G. semen, a species that thrives in humic lakes and often
forms high-density blooms (Lepistö et al., 1994). This
species has been referred to as mixotrophic (Lepistö and
Saura, 1998), but, as far as we know, this has not been
verified experimentally. Mixotrophy has been suggested to
be an especially favorable strategy in humic environments
and high mixotrophic biomasses have been reported from
humic lakes (Salonen and Jokinen, 1988; Jansson et al.,
1996). In the temperate lakes examined, the mixotrophic
proportion of the phytoplankton biomass was highest in
Lake Skärlen (low humic content). Thus, our results show
that mixotrophic phytoplankton can at times also be
important in terms of biomass in oligotrophic clear-water
lakes. In Lake Skärlen, the high mixotrophic biomass was
mainly due to fairly high densities of large dinoflagellate
species and Dinobryon spp., species that are known to be
primarily phototrophic, and for which mixotrophy could
be expected to be particularly useful in oligotrophic waters
with a high light availability (Arenowski et al., 1995). In the
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tropical lakes, mixotrophic genera constituted only a small
proportion of total biomass. As the lakes were only
sampled once, we cannot exclude the possibility that the
mixotrophs might be of higher importance on another
occasion. However, the fairly low biomass of mixotrophs
could be related to the general characteristics of tropical
lakes (e.g. higher lake temperature and incident solar
radiation), or simply be related to lake trophy. Instead,
cyanobacteria were common in these lakes. High densities
of cyanobacteria are in agreement with them being known
to have their maximal competitive advantage at high
temperatures (Tilman et al., 1986).
In 1999, when the incubation procedure resulted in a
higher light availability, the mixotrophic proportion of
the phytoplankton community was overall significantly
smaller in the NP treatment compared with the treatments receiving one (N or P) or no nutrients. Hence,
mixotrophy appeared to be an advantageous strategy
when the concentrations of either N or P, or both,
were low. A similar response to nutrient additions was
reported in the marine mixotroph Chrysochromulina sp.
(Samuelsson et al., 2002). In microcosm experiments,
Chrysochromulina sp. was found at high densities in the
control treatments, whereas the obligate autotrophs and
heterotrophs increased comparatively more in the treatments receiving N and P simultaneously. Thus, the
results indicate that the ability to exploit an alternative
nutrient source such as bacteria may give the mixotrophs
a competitive advantage over the obligate autotrophs
when concentrations of inorganic nutrients are low.
Mixotrophy has been suggested to incur higher basal
metabolic costs than obligate auto- or heterotrophy
(Holen and Boraas, 1995; Raven, 1995, 1997). The
most apparent cost is that of synthesizing both the
photosynthetic and the phagotrophic apparatus. In addition, some mixotrophic species, such as Ochromonas spp.
and Poterioochromonas malhamensis, might have a less
efficient photosynthetic apparatus compared with that
of obligate autotrophs (Myers and Graham, 1956;
Anderson et al., 1989). Few studies have examined the
trade-offs associated with mixotrophic nutrition, or their
effects on the competitive relationships between mixotrophs and obligate autotrophs or heterotrophs. However, mixotrophs may have lower growth rates
compared with specialized competitors (Rothhaupt,
1996; Raven, 1997). As a result, mixotrophy cannot be
expected to be an advantageous strategy when nutrient
and light availability are high, as in the NP treatments in
1999. This is because the cost of maintaining two different functional modes of nutrition under these circumstances may be higher than the benefits.
The outcome of the competition between mixotrophs
and obligate autotrophs could be expected to change
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when the light conditions are unfavorable for phototrophic growth ( Jones, 2000). Mixotrophic phytoplankton (Dinobryon spp., Ochromonas spp. and Spiniferomonas
spp.) increased in relative abundance in all treatments
in 2000. As the light availability this year was low compared with 1999 due to deeper incubation, the higher
proportion of mixotrophs may have been a result of their
ability to supplement photosynthesis with the ingestion
of prey and thus have access to an alternative carbon/
energy source. Light is known to be one of the main
factors affecting ingestion rates in primarily phototrophic
mixotrophs ( Jones, 1995). In some of these species, the
ingestion rate is inversely proportional to light intensity,
whereas in others, it appears to be dependent on photosynthesis and thereby directly proportional to light intensity ( Jones, 1997). In the former group, phagotrophy is
believed to primarily provide carbon, whereas in the
latter group, it is mainly a means of obtaining limiting
nutrients or growth factors ( Jones, 1997). In primarily
heterotrophic mixotrophs, the light regime has little
impact on ingestion rates (Caron et al., 1990). Owing to
their primarily heterotrophic mode of nutrition, it is of
no surprise that Ochromonas spp. increased in relative
abundance in the low-light treatments. For Dinobryon
spp., prior studies have shown that the influence of
light varies within the group and both of the above
described relationships between light conditions and
ingestion rates have been found ( Jones, 1997). Little is
known regarding the probable mixotrophic capacity of
Spiniferomonas spp.; however, Spiniferomonas spp. increased
in relative abundance in the light-limited treatments.
Usually, the advantage of mixotrophic nutrition is
explained in terms of acquiring one element or the other,
depending on species. However, recent studies suggest that
phagotrophy may provide the same species with more
than one element, depending on the prevailing abiotic
conditions. The ability to use the ingested food as a source
of organic carbon or growth factor, as well as to supplement nutrition in a nutrient-limited environment, has been
supported by several studies on marine dinoflagellates (Li
et al., 2000; Skovgaard, 2000; Smalley and Coats, 2002). It
also agrees with the results from this study, in which the
same group of mixotrophs increased in what appeared to
be a response to low light availability as well as in the
treatments exposed to a high light availability but low
concentrations of N, P or both. The results stress how
complicated the regulation of phagotrophy may be in
natural, ever changing, environments.
We conclude that although total phytoplankton biomass accumulation was limited by N in some of the
investigated lakes with a high humic content, N limitation was not associated specifically with the mixotrophic
part of the community, as suggested by Jansson et al.
1012
C. PÅLSSON AND W. GRANÉLI
j
NUTRIENT LIMITATION OF PHAGOTROPHIC MIXOTROPHS
( Jansson et al., 1996). When exposed to a high light
availability, the mixotrophic phytoplankton increased
in relative abundance in the treatments receiving no
nutrients or N and P separately. In the following year,
the mixotrophs appeared to be favored over the obligate
autotrophs independently of treatment. Possibly, this was
a result of the low light availability. The results suggest
that mixotrophy is an advantageous strategy when the
availability of light and/or nutrients is low.
ACKNOWLEDGEMENTS
We thank Bias Marcal de Faria and personnel at Universidade Federal do Rio de Janeiro for their assistance in
Brazil. Johan Birgersson, Emma Kritzberg and Karin
Wessman for their assistance in the field, and Getrud
Cronberg for her help with taxonomic analyses. We also
thank Karin Rengefors and E. Kritzberg for valuable
comments on an earlier version of this manuscript, and
Per Nyström for statistical advice. This work was supported by grants from the Swedish Foundation for International Cooperation in Research and Higher Education
(STINT) and the Swedish Research Council (VR) to W.G.
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