Journal of
Plankton Research
plankt.oxfordjournals.org
J. Plankton Res. (2013) 35(5): 1141 – 1153. First published online June 23, 2013 doi:10.1093/plankt/fbt061
Effects of growth condition on succession
patterns in tropical phytoplankton
assemblages subjected to experimental
eutrophication
ELISA M. SOARES1, CLEBER C. FIGUEREDO2, BJÖRN GÜCKER3 AND IOLA G. BOËCHAT3*
1
2
GRADUATE PROGRAM OF BIOENGINEERING, FEDERAL UNIVERSITY OF SÃO JOÃO DEL-REI, SÃO JOÃO DEL-REI, MG, BRAZIL, DEPARTMENT OF BOTANY, FEDERAL
3
UNIVERSITY OF MINAS GERAIS, BELO HORIZONTE, MG, BRAZIL AND DEPARTMENT OF BIOSYSTEMS ENGINEERING, FEDERAL UNIVERSITY OF SÃO JOÃO DEL-REI, SÃO
JOÃO DEL-REI, MG, BRAZIL
*CORRESPONDING AUTHOR: [email protected] or [email protected]
Received January 13, 2013; resubmitted on May 2, 2013; accepted May 27, 2013
Corresponding editor: Beatrix E. Beisner
We isolated phytoplankton assemblages from five tropical lakes differing in environmental conditions and incubated them under different extreme growth conditions.
Conditions were: under light with nutrient addition (L þ N), under light with nutrient
and organic carbon addition (L þ N þ OC), and in the dark with nutrient and organic
carbon addition (N þ OC). These were intended to simulate phytoplankton succession
during early, intermediate and late eutrophication stages, respectively. Decreases in
species diversity and richness in N þ OC assays from all lakes, except the most eutrophic one, suggested consistent diversity and richness declines during late eutrophication
stages. Declines in species diversity were generally caused by a stronger reduction in
species richness than evenness, indicating that species exclusion is a more probable response to eutrophication than co-existence and dominance of strong competitors.
Under N þ OC conditions, the osmotrophic chlorophyte Chlamydomonas was generally
dominant, indicating that this flagellate may prevail in late eutrophic communities in
the tropical lakes studied. The L þ N and L þ N þ OC assays did not differ from each
other in species diversity, but exhibited differences in species composition and richness.
This suggests that the changing role of the phytoplankton community in whole-lake
matter fluxes during early eutrophication stages could be a more important research
question, than predicting the community’s diversity response in tropical lakes.
available online at www.plankt.oxfordjournals.org
# The Author 2013. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]
JOURNAL OF PLANKTON RESEARCH
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PAGES 1141 – 1153
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KEYWORDS: lake eutrophication; trophic mode; mixotrophy; plankton succession
I N T RO D U C T I O N
As a result of increasing worldwide urban activities and
the transformation of natural areas for agriculture, cultural
eutrophication often leads to diversity loss and the dominance of few opportunistic species in freshwater lakes
(Smith et al., 2006). During nutrient enrichment and subsequent light limitation, several ecological processes drive
changes in lake phytoplankton communities. These processes include bottom-up (Tilman, 1977, 1982) and
top-down control (Porter, 1973; Sommer, 1988; Sarnelle,
1993) and interspecific competition (Hutchinson, 1961;
Sommer, 1985; Lagus et al., 2004). However, the fate of
phytoplankton community composition and diversity is
often difficult to predict, as complex community interactions also depend on the original community composition
as well as the intensity and frequency of disturbances.
Accordingly, chaotic oscillation dynamics is often observed
(Huisman et al., 2001; Huisman and Weissing, 2002).
Moreover, chaotic dynamics may be established before
perturbations by external stimuli are effective, because of
the short generation times of small microorganisms (Becks
et al., 2005).
In this context, predicting the outcome of biological
succession has been an important challenge for limnologists interested in comprehending and mediating the
effects of cultural eutrophication. Understanding the ecological mechanisms involved in generating final community composition and diversity requires elucidation of the
combined effects of environmental conditions and the
physiological abilities of interacting species (Sommer
et al., 1986; Tilman, 1990).
According to the resource-based competition theory,
changing environmental conditions alter species interactions from competition for nutrients to competition for
light as lake eutrophication advances (Tilman, 1982,
1985; Passarge et al., 2006). This occurs because high nutrient levels can support high phytoplankton biomass,
and thus high turbidity and light limitation in the water
column. Hence, competition for light often plays a more
important role in advanced eutrophication stages
(Huisman et al., 2004; Hautier et al., 2009). However, the
factors involved in the biological succession that follows
inorganic nutrient and/or organic carbon enrichment,
and subsequent decreases in light availability are still a
matter of debate.
The prevalent nutritional strategy of an organism has
profound implications for its physiology and biochemical
composition and may affect trophic transfer of energy
and essential compounds in aquatic food webs
(Katechakis et al., 2005; Boëchat et al., 2007). Recent ecological studies have focused on determining the effects of
changing environmental conditions on the trophic mode
of aquatic protists, in order to predict the consequences
of such changes for community diversity patterns and organism spatial distribution (Tittel et al., 2003; Jost et al.,
2004; Kamjunke et al., 2004).
The prevailing trophic mode, auto-, hetero- or mixotrophy, seems to be a result of both environmental limitation and species-specific metabolic abilities (Sanders,
1991; Jones, 2000). In temperate acidic mining lakes and
deep oligotrophic lakes, as well as in the pelagic oceanic
zone, light and/or nutrient limitation are key factors
leading to shifts in the main energy and carbon flux pathways among algae and protozoa (Jones et al., 1993;
Wollmann et al., 2000; Tittel et al., 2003; Schmidtke et al.,
2006). Under such limiting conditions, mixotrophic abilities (combined photosynthesis and phagotrophy and/or
osmotrophy) represent an alternative metabolic strategy
that results in additional energy uptake and the ecological success of protist species, such as many euglenophytes,
chrysophytes and chlamydomonads (Rothhaupt, 1996;
Nixdorf et al., 1998; Stoecker, 1998; Schmidtke et al.,
2006). Thus, mixotrophic species may outcompete strict
auto- and heterotrophs under limiting growth conditions
(Rothhaupt, 1996).
Recently, laboratory studies have shown that mixotrophy can also offer energetic and ecological advantages
under non-limiting conditions, because synergetic effects
arising from the combination of autotrophic and heterotrophic metabolism may outweigh the higher metabolic
costs of maintaining both systems (Boëchat et al., 2007).
This finding challenges the traditional view that mixotrophy puts an organism at a competitive disadvantage with
autotrophs or heterotrophs under non-limiting conditions
(Rothhaupt, 1996; Stickney et al., 2000). Consequently, the
question arises as to whether the prevalent trophic mode
could be a relevant factor driving competition and succession in natural communities under non-limiting resource
conditions.
By isolating phytoplankton assemblages of lakes differing in environmental conditions and incubating them
under (i) light and nutrient sufficiency (L þ N), (ii) light,
nutrient sufficiency and organic carbon sufficiency (L þ
N þ OC), and (iii) nutrient and organic carbon sufficiency, but without light (N þ OC), we aimed to simulate
different eutrophication scenarios in order to identify,
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respectively, key autotrophic, mixotrophic and heterotrophic players in succession outcomes. This experimental design allowed us to evaluate how different
physiological abilities of phytoplankton species may
affect succession outcomes as eutrophication advances.
We tested the hypothesis that the simulated growth condition will affect physiological abilities differently within
natural phytoplankton assemblages and lead to differences in species composition and diversity. Moreover,
within the same growth condition, we expected the original assemblage composition to affect the succession
outcome.
Specifically, we hypothesized that prevalent heterotrophic nutrition, which may represent late eutrophication stages (simulated in N þ OC assays), will force the
assemblage towards the strongest changes in composition
with only a few dominant opportunistic species and,
therefore, a lower final diversity compared with assemblages under growth conditions that may favor autotrophy (simulated in L þ N assays) and mixotrophy
(simulated in L þ N þ OC assays). In order to test this
hypothesis, we carried out a series of experiments using
natural phytoplankton assemblages isolated from five
tropical lakes differing in nutrient status and light conditions. The isolated assemblages were inoculated in a nutrient rich medium and kept for 22 days. During the
experiments, we quantified changes in phytoplankton
species composition and diversity.
METHOD
Sampling and preparation of natural
phytoplankton communities
We selected five small tropical lakes in the Federal State of
Minas Gerais, Brazil, differing in environmental conditions (Table I): one lake with low light availability (CTAN
Lake, whose surface was completely covered by the macrophyte Salvinia sp.), three lakes with low nutrient and high
light availability (Casa de Pedra, Azul, and Boa Vista
Lakes) and one lake with high nutrient and light availability, receiving untreated sewage discharge (Ferros Lake). We
chose lakes with different conditions in order to test the hypothesis that initial community composition may have an
effect on the specific outcome of successions, but that a
general dominance of osmotrophic species adapted to low
light conditions may occur as eutrophication advances.
Water samples were collected from each lake at 1.0 m
depth with a Van Dorn bottle sampler (5 L volume,
Limnotec, São Carlos, Brazil). We measured water temperature, pH, conductivity and dissolved oxygen (DO)
concentration and saturation in situ with an YSI 556 MPS
Table I: Environmental characteristics of the
five small tropical lakes, from which
phytoplankton assemblages were isolated for
succession experiments
pH
DO (mg L21)
O2 saturation (%)
Conductivity
(mS cm21)
Temperature (8C)
Photic zone (m)
Nitrate þ Nitrite-N
(mg L21)
Ammonium-N
(mg L21)
SRP (mg L21)
POM (g L21)
Casa de
Pedra
Lake
CTAN Lake
(macrophyte Azul
dominated) Lake
Boa
Vista
Lake
Ferros
Lake
8.4A
8.2A
115.0A
23A
6.4B
3.9B
45.5B
56B
8.6A
8.9A
116.8A
105C
5.6C
7.9A
85.5C
11D
5.4C
8.6A
96.2C
47B
31.9A
3.2A
19.9A
23.7B
0.18B
26.2B
29.2A
21.0C
52.5C
19.2B
4.8D
25.5B
21.5B
2.3A
574.7D
28.1A
37.4B
30.0A
52.6C
79.6D
7.1A
0.01A
2.1B
0.10C
7.1A
9.2C
0.005B 0.005B
14.2D
0.01A
Same letters indicate no statistical differences (one-way ANOVA followed
by Tukey HSD test, P . 0.05).
multiparameter probe (Yellow Springs Instruments, Yellow
Springs, OR, USA) equipped with automatic temperature
and barometric pressure compensation and calibrated directly before sampling. Photic zone depth was determined
with a 30 cm Secchi disc (Limnotec, São Carlos, Brazil).
For nutrient analysis, 100 mL samples were kept in acidwashed plastic bottles and transported to the laboratory on
ice. Samples for dissolved inorganic nutrients (nitrate, ammonium and soluble reactive phosphorus) were filtered
through GF-F glass fiber filters (45 mm diameter,
Whatman) and kept at 2188C until further analyses.
Filters containing seston samples were dried at 1108C for
1 h and then incinerated at 5508C for 4 h for particulate
organic and inorganic matter determination. Inorganic
nutrient concentrations were determined by flow injection
analysis (FIAlab 2500, USA) according to standard spectrophotometric methods (APHA, 2006). For taxonomic
characterization and quantitative determination of the
phytoplankton communities, 250 mL samples were fixed
with acetic lugol iodine solution and kept in the dark until
counting. Cells were counted using an inverted microscope
(Olympus, IX50, Japan) until 100 cells of the most abundant species had been counted (Utermöhl, 1958). Samples
for succession experiments (1 L volume) were initially filtered in the field through 45 mm mesh filters in order to
remove zooplankton and rapidly transferred to the laboratory. In the laboratory, samples were concentrated by lowpressure filtration onto 5 mm filters and cells were
re-suspended in 100 mL axenic WC medium. This step
considerably reduced the bacterial density in the inoculum, which then consisted mainly of phytoplankton, but
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also unicellular cyanobacteria species. The presence of
bacteria in the culture flasks, although reduced, may have
favored phagotrophic species. However, phagotrophy can
be expected to occur in natural phytoplankton assemblages, and thus does not interfere with the objectives of
this study. Three subsamples were taken from the concentrate for later determination of inoculated phytoplankton
composition and density.
Succession experiments
For succession experiments, 5.0 mL phytoplankton concentrate was inoculated into culture flasks containing
100 mL axenic WC medium (Guillard and Lorenzen,
1972) adjusted to the ambient pH of each lake in triplicate
for each experimental growth condition. A fourth culture
flask was inoculated, but no subsample was taken during
the experiments. This flask served as a nutrient and DO
control, in order to assure that nutrient and DO concentrations were still high at the end of the experiments
(.1 mg L21 PO4-P, .10 mg L21 NO3-N, and . 92% of
the initial DO concentration in all assays). The culture
flasks were kept at 25 + 18C in a 12:12 h light/dark
regime (L þ N condition, at 52 mmol m22 s21 photosynthetic photon flux density), under the same light regime
with additional glucose at 50 mg L21 (L þ N þ OC
condition), and in the dark with additional glucose at
50 mg L21 (N þ OC condition) for 22 days.
Experimental flasks were manually shaken twice a day to
avoid sedimentation of algal cells. The glucose concentration used resulted in the highest phytoplankton biomass in
previous experiments (data not shown). We assumed that
the experimental growth conditions selected represent different scenarios of the eutrophication process. Thus, the
L þ N treatment was assumed to represent the early stages
of eutrophication, with high concentrations of inorganic
nutrients and light availability favoring autotrophic opportunists, but still low organic carbon concentrations, due to
limited decomposition and exudation. The L þ N þ OC
treatment, offering optimal inorganic nutrient and light
conditions, but also additional organic carbon, was
expected to simulate the second stage of the eutrophication
process, favoring a mixotrophic nutritional mode. The
N þ OC treatment, offering high concentrations of inorganic nutrients and organic carbon, but no light, was performed to simulate stressed assemblages during the late
eutrophication stage, with severe light limitation and high
saproby, favoring osmotrophic phytoplankton species. We
recognize that the growth conditions used simulate the eutrophication process in a highly simplified manner and as
extreme endpoints, but such experimental conditions were
necessary to provoke rapid phytoplankton assemblage
responses to changes in environmental conditions.
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During the experiments, we quantified changes in
phytoplankton species abundance and assemblage composition by taking three 1.0 mL subsamples each third
day. Subsamples taken from the cultures were fixed with
Lugol’s iodine solution and counted and identified under
a Zeiss microscope using a Sedgwick-Rafter chamber.
Counting was carried out until 100 cells of the most
abundant species had been counted. The Shannon –
Wiener index (H0 , based on the log2 of cell densities;
Shannon and Weaver, 1949) was used to calculate species
diversity during the entire experiment for each replicate
and trophic condition, as well as for the original lake
communities. The Pielou evenness index (J0; Pielou,
1966) was used to calculate assemblage evenness at the
end of the experiments in each trophic growth condition
and for the lake original community as well.
Statistical treatment
Lake environmental conditions were analyzed for differences using one-way ANOVA followed by Tukey post hoc
tests (Zar, 1998). Phytoplankton diversity was compared
among trophic growth conditions within each succession
experiment using one-way ANOVA followed by Tukey
post hoc tests. The same procedure was carried out to
compare diversity values within the same growth condition (e.g. L þ N) among different lakes. By comparing diversity results for the same growth condition among
lakes, we aimed to understand the effect of natural lake
original community composition and initial lake environmental conditions on the succession outcome. Student’s
t-tests were applied to compare initial and final diversity
values for a same growth condition. Normality of data
distribution and homocedasticity was tested using the
Shapiro–Wilk test and Bartlett tests, respectively (Zar,
1998). All univariate statistical procedures were performed
in STATISTICA for Windows, version 7.0 (Statsoft, USA).
In order to test for effects of original lake community
composition/adaptation and growth condition on succession outcomes, we created a positive difference matrix
between the taxon-specific cell density at the beginning
and end of the succession experiments. Based on this
matrix, we executed analysis of similarity (ANOSIM)
using Bray–Curtis dissimilarities between experimental
assays and 100 000 iterations with ‘lake’ (the five sampled
lakes) and ‘growth condition’ (L þ N, L þ N þ OC, N þ
OC) as grouping variables. We also performed a nonmetric multi-dimensional scaling (NMDS) analysis on
Bray–Curtis dissimilarities between experimental assays to
visualize the effects on succession outcomes. We used a
scree plot and a stress cutoff level of 0.15 to find the
parsimonious axis number. Confidence ellipses (95% SD)
were drawn for different lakes and growth conditions.
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ANOSIM and NMDS were performed using the ‘vegan’,
‘ecodist’, ‘BiodiversityR’ and ‘ellipse’ packages in the ‘R’
software (RCoreTeam, 2012).
R E S U LT S
Environmental conditions and original
phytoplankton communities
The five lakes differed in environmental conditions, especially in nutrient concentrations, depths of the photic zone
and conductivity (one-way ANOVA, all P , 0.05, n ¼ 15;
Table I). Nutrients were highest in the Ferros Lake
(Table I), probably due to the untreated sewage discharge
in that lake. The Casa de Pedra Lake had the lowest concentration of inorganic nitrogen, whereas the CTAN Lake
had the lowest SRP concentrations. The narrowest photic
zone was measured in the CTAN Lake due to the presence
of the floating macrophyte Salvinia sp., which nearly completely covered the water surface. This lake also had
higher concentrations of suspended particulate organic
matter (POM) than the other lakes (Table I). The highest
conductivity was measured in the Azul Lake, whereas the
lowest value was found in the Boa Vista Lake.
The natural phytoplankton community composition
also differed among lakes, following differences in environmental conditions. The poor nutrient and light sufficient
Casa de Pedra Lake was dominated by Chlorophyceae
(Fig. 1), mostly represented by Elakatothrix sp. and
Monoraphidium arcuatum. The light-limited CTAN Lake
showed an even distribution among taxa, with high relative contributions of Zygnemaphyceae (only represented
by Desmidiaceae) and flagellated forms belonging to
Euglenophyceae, Chrysophyceae and Cryptophyceae
(Fig. 1). The Azul Lake, which had low nutrient
concentrations and the highest conductivity and pH
values, was dominated by Desmidiaceae of the Cosmarium
genus and Chlorophyceae (Fig. 1), mostly represented by
Monoraphidium contortum. The Boa Vista Lake, with low nutrient concentrations and the lowest conductivity values,
was dominated by Chlorophyceae (Fig. 1), represented by
Kirchneriella obesa, Chlamydomonas sp. and Chlorella vulgaris.
The high nutrient and light sufficient Ferros Lake was
dominated by Desmidiaceae (Fig. 1), mostly represented
by Spondylosium panduriforme. The highest Shannon diversity
value was found for the light limited CTAN Lake (H0 ¼
3.14), followed by the low nutrient and light sufficient
lakes Boa Vista (H0 ¼ 1.87), Azul (H0 ¼ 1.56) and Casa de
Pedra (H0 ¼ 1.35). The nutrient and light sufficient Ferros
Lake had the lowest diversity value (H0 ¼ 0.44) (Table II).
Species richness and community evenness followed the
tendency of diversity, with the exception of the Azul Lake,
in which the lowest species richness among all lakes was
observed. However, community evenness was high, resulting in a higher diversity value than that of the Ferros Lake,
which had both low species richness and community evenness (Table II).
Succession experiments
Although succession dynamics differed in terms of phytoplankton assemblage composition during the experiments
(Figs 2–6), L þ N and L þ N þ OC growth conditions
generally enabled higher diversity than N þ OC conditions during the experiments for all lake assemblages
studied (Fig. 7). During all experiments, L þ N and L þ
N þ OC conditions resulted in increases in cell abundance towards the end of the experiment, whereas N þ
OC condition caused an increase in cell abundance in the
first week, followed by a decrease towards the end of the
Fig. 1. Phytoplankton community composition and relative densities in the studied lakes.
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Table II: Shannon-Wiener diversity index (H 0 ), species richness (R) and Pielou evenness index (J 0 ) for
lake original communities (first value) and experimental assemblages at the end of the succession experiments
(second value, mean and standard deviation in parentheses) under different growth conditions (L þ N ¼
under light with nutrient addition, L þ N þ OC ¼ under light with nutrient and organic carbon addition,
and N þ OC ¼ in the dark with nutrient and organic carbon addition)
Shannon diversity (H0 )
Pielou evenness (J0 )
Species richness (R)
Lake
LþN
L þ N þ OC
N þ OC
LþN
L þ N þ OC
N þ OC
LþN
L þ N þ OC
N þ OC
Casa de Pedra
1.35
2.33A* (0.25)
3.14
1.80A* (0.34)
1.56
1.99A (0.40)
1.87
1.34A (0.35)
0.44
1.92A* (0.20)
1.35
1.88A* (0.54)
3.14
1.82A* (0.22)
1.56
1.40A (1.02)
1.87
1.61A (0.25)
0.44
2.31A* (0.60)
1.35
0.32B* (0.11)
3.14
1.22B* (0.57)
1.56
0.37B* (0.27)
1.87
0.94B* (0.11)
0.44
1.19B* (0.18)
24
11.3 (3.5)
41
12.0 (4.6)
6
7.7 (0.6)
25
6.7 (1.5)
10
10.0 (3.6)
24
9.7 (4.6)
41
17.7 (1.5)
6
8.3 (1.5)
25
7.0 (2.0)
10
13.3 (1.5)
24
5.7 (2.3)
41
5.3 (1.5)
6
3.7 (0.6)
25
2.0 (0.0)
10
3.0 (0.0)
0.42
0.99 (0.24)
0.84
0.74 (0.03)
0.87
0.97 (0.07)
0.58
0.71 (0.13)
0.19
0.88 (0.24)
0.42
0.86 (0.23)
0.84
0.63 (0.06)
0.87
0.69 (0.55)
0.58
0.84 (0.02)
0.19
0.89 (0.23)
0.42
0.23 (0.05)
0.84
0.76 (0.39)
0.87
0.31 (0.27)
0.58
1.0 (0.16)
0.19
1.0 (0.17)
CTAN
Azul
Boa Vista
Ferros
One-way ANOVA followed by Tukey HSD test tested for diversity differences among trophic growth conditions at the end of the succession experiments
(different letters indicate significant differences, P , 0.05). Student’s t-test tested for differences between initial and final diversity values for each trophic
growth condition (statistically significant differences are marked with an asterisk, P , 0.05).
experiment (Figs 2–6). Densities of all taxa were generally
lower under N þ OC growth conditions than under L þ
N and L þ N þ OC conditions (Figs 2–6). However, in
experiments conducted with assemblages from the nutrient limited Casa de Pedra Lake, Chlamydomonas sp. 1
reached higher peak abundance under N þ OC conditions than under L þ N and L þ N þ OC conditions
(Fig. 2). In experiments with assemblages from the Casa de
Pedra and CTAN lakes, two Cryptomonas species reached
the highest densities of all species towards the end of the
experiment under both L þ N and L þ N þ OC conditions (Figs 2 and 3). In experiments with assemblages from
the CTAN Lake, Cryptomonas erosa dominated the L þ N
and L þ N þ OC treatments towards the end of the
experiments, while Chlamydomonas sp. 1 dominated the assemblage during the first week under N þ OC conditions,
still maintaining higher densities than the other species at
the end of the experiment (Fig. 3). In experiments with
assemblages from the Azul Lake, Chlamydomonas dominated under L þ N and L þ N þ OC conditions.
Experimental N þ OC conditions resulted in the dominance of the dinoflagellate Peridinium umbonatum, with especially high abundance during the first 2 weeks of the
experiment (Fig. 4). In the experiment with assemblages
from the Boa Vista Lake, Chlamydomonas sp. 1, Scenedesmus
acuminatus and Cryptomonas erosa increased towards the end
of the experiment under both L þ N and L þ N þ OC
conditions (Fig. 5). Carteria cf. intermedia initially dominated
in experiments with assemblages from the Ferros Lake
under L þ N and L þ N þ OC conditions, but a decrease
was observed towards the end of the experiment (Fig. 6).
In general, N þ OC growth conditions resulted in the
lowest species diversity during the succession experiments
(Fig. 7), accompanied by the dominance of Chlamydomonas
sp. 1 in four out of five lakes (Figs 2C and 6C). Except for
the experiment performed with assemblages from Ferros
Lake, diversity tendencies were similar for all experiments,
showing a decrease at the beginning of the experiments,
followed by an increase from the second week on.
Assemblages isolated from all lakes showed lower species
diversity at day 22 in experiments run under N þ OC
growth conditions than those under both L þ N and L þ
N þ OC conditions (one-way ANOVA, Tukey HSD test,
P , 0.05; Table II). Compared with the initial diversity at
day 1 of the experiment, experimental L þ N and L þ
N þ OC growth conditions supported similar final diversity in phytoplankton assemblages isolated from both Boa
Vista and Azul lakes (Student’s t-test, P . 0.05; Table II),
but a lower diversity occurred under N þ OC conditions
(Student’s t-test, P , 0.05; Table II) caused by a strong reduction in species richness (Table II). The absence of significant differences in species diversity between day 1 and
day 22 under L þ N and L þ N þ OC conditions,
observed for experiments with assemblages from the Boa
Vista Lake, was due to the fact that a significant reduction
in species richness was compensated for by an increase in
evenness (Student’s t-test, P , 0.05; Table II). For experiments with assemblages from the high nutrient Ferros
Lake, all three experimental treatments resulted in higher
diversity, due to the higher evenness observed for the
experimental communities, compared with that of the original lake community (Student’s t-test, P , 0.05; Table II).
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Fig. 2. Absolute cell densities during the succession experiments
performed with phytoplankton assemblages isolated from the Casa de
Pedra Lake under L þ N (A), L þ N þ OC (B) and N þ OC (C)
culture conditions.
Fig. 3. Absolute cell densities during the succession experiments
performed with phytoplankton assemblages isolated from the CTAN
Lake under L þ N (A), L þ N þ OC (B) and N þ OC (C) culture
conditions.
Despite the differences observed in diversity and composition patterns during the succession experiments,
there was no significant assemblage-wide difference in
succession outcomes between different trophic growth
conditions (ANOSIM, P . 0.1, R ¼ 20.02). However,
there was a significant difference in succession
outcomes between assays from different lakes (ANOSIM,
P , 0.01, R ¼ 0.39). Non-metric multidimensional
scaling extracted two parsimonious axes at a stress level
of 0.11 (Fig. 8). Overlapping confidence ellipses also indicated no differences in assemblage-wide succession outcomes between different growth conditions (Fig. 8A).
However, non-overlapping confidence ellipses pointed to
differences in succession outcomes between assays from
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Fig. 4. Absolute cell densities during the succession experiments
performed with phytoplankton assemblages isolated from the Azul Lake
under L þ N (A), L þ N þ OC (B) and N þ OC (C) culture conditions.
the eutrophic Ferros Lake on the one hand, and the more
oligotrophic lakes CTAN, Casa de Pedra and Boa Vista
on the other hand (Fig. 8B).
DISCUSSION
In our study, we tested for responses of natural phytoplankton assemblages to different non-nutrient-limiting
conditions, designed to select species more adapted to
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Fig. 5. Absolute cell densities during the succession experiments
performed with phytoplankton assemblages isolated from the Boa Vista
Lake under L þ N (A), L þ N þ OC (B) and N þ OC (C) culture
conditions.
autotrophic, mixotrophic and heterotrophic growth, in
order to simulate responses occurring at early, intermediate and late stages of eutrophication, respectively. The
assays performed, differing in light and organic carbon
availability, are certainly extreme simplifications of temporally and spatially dynamic eutrophication processes
occurring in lakes, and the N þ OC assay performed in
the dark represents an extreme stress situation. However,
studying the effects of extreme differences in growth
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Fig. 6. Absolute cell densities during the succession experiments
performed with phytoplankton assemblages isolated from the Ferros
Lake under L þ N (A), L þ N þ OC (B) and N þ OC (C) culture
conditions.
conditions on succession outcomes may give valuable
insights into potential eutrophication responses related to
species-specific metabolic abilities.
The nutritional strategy of plankton protists affects
their physiology and ability to compete for limited
resources (Sleigh, 2000). In aquatic ecosystems, phytoplankton communities can be simultaneously limited by
nutrients, light, organic carbon and temperature, besides
being affected by other factors, such as grazing, flushing,
pH and conductivity. This co-limitation may result in coexistence and high diversity but may also generate
non-equilibrium community composition, depending on
the physiological abilities of the competing species
(Brauer et al., 2012). Although the eutrophic state of lakes
does not necessarily implicate low diversity, the substitution of species with progressing eutrophication may result
in the dominance of species well adapted to low light,
leading to decreases in community diversity in both, temperate (Sommer, 1988, 1991) and tropical lakes
(Figueredo and Giani, 2001). Thus, the physiological
abilities of species may influence and modulate successions in such changing systems.
Communities may respond to severe eutrophication
with decreases in community diversity (i) due to lower richness and/or (ii) due to lower evenness, caused by the dominance of opportunistic species (Huszar et al., 1998;
Figueredo and Giani, 2001). In the Shannon–Wiener
index, species richness can indeed exert an effect on diversity in communities with a richness of less than 15 species
(Sager and Hasler, 1969). Interestingly, lower species diversity observed at the end of our experiments with phytoplankton assemblages isolated from four out of five lakes
was generally caused by a much stronger reduction in
species richness than in assemblage evenness. This result
suggests that species exclusion can occur as a response to
eutrophication, rather than co-existence and dominance
of strong competitors. This pattern was especially obvious
in experiments conducted under N þ OC conditions,
which were assumed to represent a more advanced eutrophication scenario. However, the N þ OC treatment
was the most extreme treatment in our experimental setup,
and advanced eutrophication stages would be better represented by low light conditions, rather than no light at all.
Such a low light treatment could result in different assemblages, however, probably with a co-dominance of osmotrophic flagellated and cyanobacteria species (Sommer,
1988; Calijuri et al., 2002).
Chlamydomonas sp. 1 was the dominant taxon under experimental N þ OC conditions in all experiments, except
for those with isolates from Azul Lake, in which only
Chlamydomonas sp. 2 occurred in very low densities in the
natural phytoplankton community. This success of
Chlamydomonas sp. 1 in N þ OC treatments suggests a high
adaptation of this taxon under severe light limitation and
high dissolved organic matter conditions, characteristic of
shallow lake systems in advanced eutrophication stages.
This result corroborates previous findings that osmotrophy,
i.e. the use of dissolved organic carbon, is the most successful strategy adopted by different Chlamydomonas species
under light limitation (Lalibertè and Noüie, 1993; Tittel
et al., 2005). Interestingly, the Chlamydomonas species investigated grew well on glucose, which is not common for this
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Fig. 7. Shannon– Wiener diversity index (H0 , based on the log2 of cell densities) during the succession experiments performed with phytoplankton
assemblages isolated from the Casa de Pedra Lake (A), Azul Lake (B), CTAN Lake (C), Boa Vista Lake (D) and Ferros Lake (E), under L þ N
(triangles), L þ N þ OC (asterisks) and N þ OC (open circles) culture conditions.
genus (Droop, 1974). Moreover, Chlamydomonas, as well as
other osmotrophic flagellates belonging to Cryptophytes
and Euglenophytes, were abundant in the most light
limited and POM rich lake in our study (CTAN Lake), as
well as in other hypereutrophic systems in the region investigated (Figueredo and Giani, 2001).
In contrast to all other experiments in our study, diversity increased during the experiments carried out for the
Ferros Lake, the most eutrophic of the lakes studied, compared with the original lake community. As predators had
been removed from the assays, the lower diversity observed
in the natural community could suggest a strong top-down
control by herbivores in this lake. Top-down control is
known as an important mechanism of phytoplankton succession in shallow eutrophic lakes (Sommer et al., 1986;
Sarnelle, 1993) and could be an important regulator of the
natural phytoplankton community in Ferros Lake, in
which high densities of Copepoda, Cladocera and
Rotifera are present (personal observation). Moreover,
predation can also modulate phytoplankton competition
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Fig. 8. Non-linear multi-dimensional scaling results of succession
outcomes and 95% SD confidence ellipses for different growth
conditions (A) and lakes (B).
outcomes in controlled laboratory studies (Sommer, 1989).
In order to test the hypothesis that physiological abilities of
species can affect succession outcomes of phytoplankton
assemblages in experimental eutrophication scenarios, we
had to remove the influence of predators. Otherwise, we
would not have been able to separate both effects.
However, the removal of predators from experimental
treatments limits the extrapolation of results in terms of
final community composition in the lakes studied.
Despite clear differences in succession outcomes
between different experimental conditions in terms of absolute phytoplankton abundance, diversity and species richness, multivariate ANOSIM and NMDS analyses did not
indicate that this was a consistent assemblage-wide difference across lakes. Nonetheless, these analyses showed significant assemblage-wide differences in succession
outcomes between the eutrophic Ferros Lake and several
oligotrophic lakes across different conditions. This points to
the importance of initial assemblage composition and
perhaps also the species’ physiological adaptation in determining successional responses to eutrophication. The fact
that Chlamydomonas sp. 1 dominated four out of five assemblages under N þ OC growth conditions, regardless of original lake conditions or initial assemblage composition,
suggests a strong effect of phytoplankton physiological abilities in determining assemblage composition and diversity
during advanced stages of eutrophication. These results
correspond well with findings from eutrophic Sicilian lakes,
in which Chlamydomonas species were strong bloom-forming
competitors (Naselli-Flores and Barone, 2000). The
absence of light, as simulated by N þ OC growth conditions, resulted in lower diversity in phytoplankton assemblages from all lakes, except for those from eutrophic Ferros
Lake. This finding may support the view that light limitation drives changes in community composition towards
later stages of eutrophication. However, the experimental
dark condition in our study was extreme and does not necessarily represent the reality of later eutrophication stages.
Interestingly, the most light-limited lake in our study, CTAN
Lake, had the highest diversity in situ, which suggests that
physiological abilities associated with osmotrophy can compensate for light limitation and maintain high natural
species diversity in less nutrient polluted shallow lakes.
Additionally, our study investigated how non-limiting
conditions may change taxonomic composition. Resourcebased competition theory predicts that mixotrophy only
supports higher fitness under limitation of the primary
organism’s resource (light, nutrient or an external carbon
source) (Rothhaupt, 1996; Boëchat et al., 2007). However,
consequences of ‘mixotrophy’ under non-limiting conditions, i.e. the community’s use of both organic carbon and
light as resources with the possible coexistence of protists
occurring at both ends of the light-organic carbon resource use spectrum, have rarely been studied. Except for
the Azul Lake, final diversity supported by L þ N þ OC
conditions (designed to favor mixotrophic species) did not
differ from that observed for L þ N conditions, although
assemblage composition and species richness tended to
differ between both experimental conditions. This result
suggests that changes in environmental conditions could
lead to changes in community composition and species
richness, without concomitant changes in community diversity. Consequently, the role of each phytoplankton
species and the entire phytoplankton community in
whole-lake matter cycling may be a more important question than predicting the final diversity of eutrophic tropical
lakes. Analyzing changes in phytoplankton functional
groups may be an interesting approach to investigate eutrophication effects in tropical lake phytoplankton communities (Reynolds et al., 2002).
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In conclusion, the extreme conditions applied in the experimental setup limit direct extrapolation of our results to
natural community processes during eutrophication and
should be interpreted with caution. Nonetheless, our
results are consistent with previous studies and suggest significant declines in diversity and species richness during
eutrophication in tropical lakes (Figueredo and Giani,
2001), a response already described for temperate lakes
(Sommer, 1988, 1991; Ptacnik et al., 2008). The comparison of succession outcomes among different growth conditions in our study is a new approach that analyses the role
of different physiological abilities of phytoplankton species
in population dynamics as succession advances.
Population dynamics resulted in species substitution as a
response to experimental eutrophication, rather than in
co-existence and dominance of better-adapted species.
Our results suggest that diversity and richness declines are
especially severe at later light-limited eutrophication
stages. In simulated late, i.e. heterotrophic, eutrophication
stages, the chlorophyte Chlamydomonas sp. 1 dominated the
phytoplankton assemblages of four of the five tropical
lakes investigated. Earlier experimental eutrophication
stages, simulated by conditions favoring autotrophy and
mixotrophy, did not differ in species diversity, but assemblage composition and species richness differed between
both conditions, pointing to a changing role of the phytoplankton community in whole-lake ecosystem processes
already in early eutrophication stages.
AC K N OW L E D G M E N T S
We would like to thank A. Contin, F.L. Nascimento,
A.T.B. Santos, R.C. Chaves, A.B.M. Paiva, A.P.C.
Carvalho, G.C. Silva and R.C.S. Silva for their help with
field sampling and maintenance of protist cultures during
the experiments. Three anonymous reviewers provided
helpful and constructive comments on an earlier version
of this manuscript. A. Giani facilitated phytoplankton
counting by granting access to her lab and microscopes.
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FUNDING
This work was supported by a fellowship to E.M.S. by
the Conselho Nacional de Desenvolvimento Cientı́fico e
Tecnológico (CNPq).
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