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RESEARCH ARTICLE
What drives the distribution of the bloom-forming
cyanobacteria Planktothrix agardhii and Cylindrospermopsis
raciborskii?
Sylvia Bonilla
1
, Luis Aubriot
1
, Maria Carolina S. Soares
2
, Mauricio Gonza
´lez-Piana
1
, Amelia Fabre
1
,
Vera L.M. Huszar
3
, Miquel Lu
¨rling
4,5
, Dermot Antoniades
1
, Judit Padisa
´k
6
& Carla Kruk
1
1
Grupo de Ecologı
´a y Fisiologı
´a de Fitoplancton, Seccio
´n Limnologı
´a, Instituto de Ecologı
´a y Ciencias Ambientales, Facultad de Ciencias,
Universidad de la Repu
´blica, Montevideo, Uruguay;
2
Department of Sanitary and Environmental Engineering, Universidade Federal de Juiz de
Fora, Luis de Fora, Brazil;
3
Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil;
4
Aquatic Ecology and Water Quality
Management Group, Department of Environmental Sciences, Wageningen University, Wageningen, The Netherlands;
5
Department of Aquatic
Ecology, Netherlands Institute of Ecology, Royal Netherlands Academy of Arts and Sciences, Wageningen, The Netherlands; and
6
Department of
Limnology, Pannon University, Veszpre
´m, Hungary
Correspondence: Sylvia Bonilla, Seccio
´n
Limnologı
´a, Facultad de Ciencias, Igua
´4225,
11400-Montevideo, Uruguay. Tel.:
+598 25258618, ext. 7148; fax:
+598 25258617; e-mail: sbon@fcien.edu.uy
Received 11 July 2011; revised 21 October
2011; accepted 28 October 2011.
Final version published online 28 November
2011.
DOI: 10.1111/j.1574-6941.2011.01242.x
Editor: Riks Laanbroek
Keywords
eutrophication; shallow lakes; invasive
species; Nostocales; Oscillatoriales; climate
change.
Abstract
The cyanobacteria Planktothrix agardhii and Cylindrospermopsis raciborskii are
bloom-forming species common in eutrophic freshwaters. These filamentous
species share certain physiological traits which imply that they might flourish
under similar environmental conditions. We compared the distribution of the
two species in a large database (940 samples) covering different climatic regions
and the Northern and Southern hemispheres, and carried out laboratory exper-
iments to compare their morphological and physiological responses. The envi-
ronmental ranges of the two species overlapped with respect to temperature,
light and total phosphorus (TP); however, they responded differently to
environmental gradients; C. raciborskii biovolume changed gradually while
P. agardhii shifted sharply from being highly dominated to a rare component
of the phytoplankton. As expected, P. agardhii dominates the phytoplankton
with high TP and low light availability conditions. Contrary to predictions, C.
raciborskii succeeded in all climates and at temperatures as low as 11 °C. Cylin-
drospermopsis raciborskii had higher phenotypic plasticity than P. agardhii in
terms of pigments, individual size and growth rates. We conclude that the
phenotypic plasticity of C. raciborskii could explain its ongoing expansion to
temperate latitudes and suggest its future predominance under predicted
climate-change scenarios.
Introduction
The excessive growth of planktonic cyanobacteria is
among the main threats endangering the use of water
resources in shallow lakes. Temperature increases in the
range of 0.2 °C per decade, and their effects on water
mixing regimes, are expected to increase the occurrence,
frequency and duration of cyanobacterial blooms in
several regions of the planet (Doney, 2006; Falkowski &
Oliver, 2007; Markensten et al., 2010). These future
changes in climate are also predicted to cause shifts in
the species composition of cyanobacterial blooms in
favour of invasive species (Mehnert et al., 2010). The
modern global distributions and environmental prefer-
ences of cyanobacterial species result from differences in
evolutionary adaptations and phenotypic traits (Whitton
& Potts, 2000). Understanding the characteristics that
allow cyanobacterial taxa to succeed in disparate environ-
ments is crucial for predicting future bloom-forming
behaviour in warming climates.
Filamentous cyanobacteria such as Planktothrix and
Cylindrospermopsis, as well as colonial genera at Microcys-
tis, are the most successful bloom-forming organisms in
shallow lakes (Padisa
´k & Reynolds, 1998; Nixdorf et al.,
2003; Paerl et al., 2011; Tomioka et al., 2011). In particu-
lar, Planktothrix agardhii (Order Oscillatoriales) and
ª2011 Federation of European Microbiological Societies FEMS Microbiol Ecol 79 (2012) 594–607
Published by Blackwell Publishing Ltd. All rights reserved
MICROBIOLOGY ECOLOGY
Cylindrospermopsis raciborskii (Order Nostocales) can be
used as model species because of the extensive informa-
tion available about their distributions. Planktothrix agar-
dhii is a resilient, shade-tolerant species that can produce
microcystins and is one of the most common bloom-
forming species in temperate lakes (Scheffer et al., 1997).
Blooms of C. raciborskii are becoming more frequent in
tropical (Figueredo & Giani, 2009; Gemelgo et al., 2009),
subtropical (Vidal & Kruk, 2008; Everson et al., 2011)
and temperate lakes (Hamilton et al., 2005; Stu
¨ken et al.,
2006) because of the apparently invasive behaviour of the
species (Padisa
´k, 1997). The expansion of C. raciborskii
has generated widespread concern as a result of its poten-
tial for producing two toxin types, cylindrospermopsins
and saxitoxins (Chorus & Bartram, 1999). To date no
consensus exists regarding the main mechanisms that
have permitted the expansion of C. raciborskii into tem-
perate regions. Proposed hypotheses include climate
change–associated water temperature increases (Wiedner
et al., 2007), an exceptionally good tolerance of transport
(Padisa
´k, 1997), the ecophysiological plasticity of the spe-
cies (Briand et al., 2004) and the existence of ecotypes
with different environmental preferences and tolerances
(Chonudomkul et al., 2004; Piccini et al., 2011).
Cylindrospermopsis raciborskii and P. agardhii have sim-
ilar phenotypic traits, including tolerance to continuous
mixing of the water column, high phosphorus storage
capacity, buoyancy regulation and shade tolerance (Rey-
nolds, 1993; Padisa
´k & Reynolds, 1998; Istva
´novics et al.,
2000; Padisa
´k, 2003). These similarities are also reflected
in their morphology, indicating that they may be func-
tionally equivalent and occupy a similar ecological niche
(Kruk et al., 2010). However, some studies show that
C. raciborskii has higher light requirements for growth
(I
k
) than P. agardhii, suggesting differences in some
dimensions of their niches (Briand et al., 2004; Koko-
cin
´ski et al., 2010; Mehnert et al., 2010). Moreover, these
two species differ in their capacities to incorporate nitro-
gen. Cylindrospermopsis raciborskii has the capacity to fix
atmospheric nitrogen (N
2
) through heterocytes, as do
other Nostocales, conferring a competitive advantage in
nitrogen-depleted environments relative to P. agardhii,
which cannot fix nitrogen (Whitton & Potts, 2000). The
advantages that explain the recent worldwide expansion
of Cylindrospermopsis, combined with predicted changes
because of global warming, may imply an impending shift
from P. agardhii to C. raciborskii at intermediate lati-
tudes.
Although information about P. agardhii is extensive,
and data about C. raciborskii are increasingly available,
current knowledge derives either from experiments or
field sampling. Very few studies simultaneously compare
both species (Dokulil & Teubner, 2000; Wiedner et al.,
2007; Kokocin
´ski et al., 2010). Functional traits, including
morphological and physiological features, govern individ-
ual ecological performance and summarize organism
responses to the environment (McGill et al., 2006; Violle
et al., 2007; Kruk et al., 2010). A comparative approach
to studying the morphological and physiological traits
and distributions of P. agardhii and C. raciborskii can
provide insight into the behaviour of these key cyanobac-
terial species. Moreover, this approach can contribute to
more general ecological questions such as microorganism
invasions (McGill et al., 2006; MacDougall et al., 2009).
Our aim was to evaluate the global distribution and
ecological preferences of C. raciborskii and P. agardhii,
and to determine the implications for the geographical
expansion of C. raciborskii. We assembled a large database
spanning wide latitudinal gradients and different climatic
regions and carried out laboratory experiments to charac-
terize the morphological and physiological traits of the
two species.
Materials and methods
Field database
We constructed a database of 940 samples taken from 28
mesotrophic to hypereutrophic lakes where P. agardhii
and/or C. raciborskii were present in at least one sample.
In 125 samples neither species was present. Species data
were obtained from published (Padisa
´k, 1994; Aubriot
et al., 2000, 2011; Kruk et al., 2002, 2010; Marinho &
Huszar, 2002; Soares et al., 2009) and unpublished mate-
rial (kindly provided by F. Bressan, A. Ferreira and S. de
Melo). Three climate regions were represented in the lake
database: tropical (08°02′–22°33′S), subtropical (34°33′–
34°55′S) and temperate (35°30′–38°80′S and 46°50′–52°
23′N). Studied lakes in the temperate zone were from
Hungary (Balaton Lake), The Netherlands (Deest and
Ochten floodplain lakes: D1, D2, D3, D4, D5, O2, O3,
O4, O5, O6) and the Argentinean Pampas lakes AR19,
AR20, AR29, AR30, AR31 and AR32; in the subtropical
zone from Uruguay (Laguna Blanca, Canteras, Chica,
Javier, Rodo
´, Sauce and Ton-Ton); and in the tropical
zone from Brazil (Funil, Imboassica, Juturnaı
´ba, Tabocas
and Tapacura
´). All samples were used to determine the
distribution of each species in relation to selected envi-
ronmental variables, excluding observations with zero
biovolume. The environmental variables were lake area
(area, ha), maximum depth (Z
max
, m), mixing depth
(Z
mix
, m), euphotic/mixing depth ratio (Z
eu
/Z
mix
), water
temperature (T, °C), pH, conductivity (K, lScm
1
),
alkalinity (Alk, mg CaCO
3
L
1
) and total phosphorus
(TP, lgL
1
). The Z
eu
/Z
mix
ratio was used as a proxy of
the light available in the environment for phytoplankton
FEMS Microbiol Ecol 79 (2012) 594–607 ª2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Distribution of P. agardhii and C. raciborskii 595
growth (Jensen et al., 1994). The largest number of obser-
vations (~61%) was from shallow lakes (Z
max
<4 m),
although several cases corresponded to deep lakes (93
data points, Z
max
>20 m, maximum: Funil Reservoir,
Z
max
: 50 m). A wide range of lake areas were included
(0.5–7200 ha, plus Balaton Lake which is 59300 ha) but
only 19 lakes were smaller than 100 ha. Our data set had
an extensive range of TP (12–1653 lgL
1
) where few
observations (n=5) indicated mesotrophic status
(<30 lgL
1
TP) and 100 were from hypereutrophic
conditions (>200 lgL
1
TP).
Despite the diversity of lakes and locations, phyto-
plankton sampling always followed routine protocols, and
thus, the samples were representative of lake conditions.
Samples were obtained at different depths within the
mixed, illuminated zone, or in permanently mixed shal-
low lakes, the whole water column was integrated using
sample bottles or tubes. Phytoplankton samples were
fixed with Lugol’s solution and settled in counting cham-
bers (Utermo
¨hl, 1958); at least 100 individuals of the
most frequent species or 400 individuals in total were
counted in random fields in an inverted microscope as
described in (Kruk et al., 2010). Individual volume (V,
lm
3
) was calculated for each taxon according to simple
volumetric formula, considering the organism as the unit,
and biovolume was expressed as mm
3
L
1
. The surface
are volume ratio (S/V, lm
1
) and filament maximum lin-
ear dimension (MLD, lm) were estimated as detailed in
Kruk et al. (2010). The community was analysed in terms
of species richness (expressed as the number of taxa per
sample, S),theabsolutebiovolumeofP. agardhii and
C. raciborskii, the relative contribution of each to total
biovolume, and their frequency of occurrence (number of
observations). Total biovolume was considered low when
<1mm
3
L
1
, and a species was considered dominant
when it represented at least 30% of the total biovolume
in a particular sample. The frequency of occurrence, the
median and the range of the two species in terms of bio-
volume were analysed with all samples in the data set
(including zero data).
Experimental data
The physiological and morphological responses of the
two species were compared using Uruguayan isolates:
P. agardhii (MVCC11) and C. raciborskii (MVCC14). Iso-
late MVCC11 was collected from Lago Rodo
´(34°55′S,
56°10′W), a eutrophic to hypereutrophic shallow lake
used for recreation (Area: 1.5 ha, Z
max
: 2.5 m, TP: 70–
565 lgL
1
) (Scasso et al., 2001). Isolate MVCC14 was
collected in Laguna Blanca (34°53′S, 54°20′W), a eutro-
phic shallow lagoon (Area: 40.5 ha, Z
max
: 2.6 m, TP:
86 lgL
1
) used as a drinking water supply (Vidal &
Kruk, 2008). Static cultures of the isolates were kept in
BG11 medium at 26 °C(±1°C), as described in the
study carried out by Piccini et al. (2011), which is the
normal summer water temperature in Uruguayan lakes
where the species were isolated (Vidal & Kruk, 2008;
Aubriot et al., 2011).
Two set of experiments were performed: light intensity
gradient and temperature experiments, and the growth
rates (physiological trait) of the two species were deter-
mined and compared. In addition, for the light intensity
gradient experiments, we evaluated the physiological
response of pigment structure change and the morpho-
logical trait changes of V, S/V and MLD.
To determine the effect of light intensity on growth
rates of P. agardhii, four growth curves under six light
intensity levels (from 5 to 180 lmol photons m
2
s
1
)
were repeated in 5-day experiments at 26 °C(±1°C).
Data for growth curves of C. raciborskii were obtained
from Piccini et al. (2011), who performed the experiment
under the same conditions. Before beginning the experi-
ments, cultures were acclimated to each light level for 10–
15 days and replicated when the biomass was duplicated
(three replicates, except for 5 lmol photons m
2
s
1
:
one). The experiments were run in 100-ml bottles, filled
with 80 mL BG11 medium and inoculated with cyano-
bacterial culture in exponential growth phase. Optical
density (OD, absorbance at 750 nm) was used as an indi-
cator of biomass and the initial inoculum for all experi-
ments was 0.1 absorbance units. Absorbance at 440 nm
was used to determine the light extinction coefficient
(Kirk, 1996) in order to calculate the light intensity inside
the bottles. Absorbance measurements were taken in a
spectrophotometer (Thermo Evolution 60). The growth
rate (l,d
1
) of each isolate was calculated in 24-h inter-
vals during the exponential phase as:
l¼ðln ODfln ODiÞ
ðtftiÞ
where OD
i
and OD
f
are the estimated biomasses at initial
(t
i
) and final (t
f
) times, respectively. Maximum specific
growth rate, l
max
, the initial slope, a, and the irradiance
at the onset of light saturation, I
k
(I
k
=l
max
/a), were
derived from the fitted model of Jassby & Platt, (1976)
for photosynthesis.
Samples were taken at the end of two growth experi-
ments (i.e. 20 and 100 lmol photons m
2
s
1
) in order
to quantify several characteristics of the two taxa. To
compare morphological changes, V, S/V and MLD were
calculated for each isolate and replicate, based on micro-
scopic measurements of 60 organisms made under an
Olympus BX40 optical microscope at 10009magnification.
To compare changes in relative pigment concentration,
ª2011 Federation of European Microbiological Societies FEMS Microbiol Ecol 79 (2012) 594–607
Published by Blackwell Publishing Ltd. All rights reserved
596 S. Bonilla et al.
in vivo relative concentrations of phycocyanin and Chl a
fluorescence were measured in a fluorometer (Turner,
Aquafluor), with phycocyanin relative concentration stan-
dardized against Chl a in vivo fluorescence. Finally, the
pigment structure of each taxon was characterized using
high-performance liquid chromatography (HPLC). Sam-
ples from one replicate of each taxon were filtered onto
GF/C glass-fibre filters and kept frozen (80 °C) until
pigment extraction. HPLC methods and protocols
followed those described in Bonilla et al. (2005). Carote-
noids were detected by diode-array spectroscopy (350–
750 nm), chromatograms were obtained at 450 nm (for
carotenoids), and Chl awas detected by a fluorescence
detector (excitation k=440 nm; emission k=650 nm).
The identification and quantification of the pigments
(Chl a, aphanizophyll, b,b-carotene, echinenone and zea-
xanthin) was based on commercial standards as detailed
in Bonilla et al. (2005). Unknown carotenoids were quan-
tified by applying the calibration curves used for b,b-car-
otene. The final concentration of each pigment is
expressed in nmol L
1
, and changes in carotenoids were
analysed using ratios to Chl a.
In order to determine the influence of low temperature
on growth rate, 4-day experiments were run for both iso-
lates at three temperatures: 15, 20 and 25 °C(±1°C) at
both 60 and 135 lmol photons m
2
s
1
, with four repli-
cates for each condition. The experimental setup and
growth conditions were the same as described earlier,
with cultures allowed to acclimate to each temperature
for 15 days (three times, except at 15 °C: once). Biomass
and growth rate were calculated as previously described
for light intensity experiments. The parameter Q
10
(15–
25 °C) for each light intensity (60 and 135 lmol pho-
tons m
2
s
1
) was calculated using the maximum average
growth rate (n=4) obtained at each temperature for
each species.
Statistical analysis
The annual coefficient of variation was calculated to
determine the variability of the biomass of the two spe-
cies in nature, based on temporal data series for 17 lakes
in the database, for temperate (11 lakes), subtropical (1)
and tropical (5) regions. As the objective of this particular
analysis was to determine the amplitude of biomass
change, all data, including observations with zero values,
were used.
To evaluate the success of the two species in relation to
key environmental variables, we examined the maximum
relative contribution of each species to total biovolume
distribution along gradients of temperature, Z
eu
/Z
mix
and
TP. For these analyses, data were segregated into groups
every one degree Celsius, 0.1 Z
eu
/Z
mix
unit, and 10 lgL
1
TP. We then performed linear and nonlinear regressions
between species biovolume and each environmental vari-
able. The simple functions with best fit were selected fol-
lowing parsimony criteria of maximum explained variance
with the minimum number of parameters and best signifi-
cance (F-test). Linear relationships with breakpoints, such
as those suggested for Planktothrix biovolume to Z
eu
/Z
mix
and to TP plots, can indicate ecological thresholds (Toms
& Lesperance, 2003). We therefore applied a simple piece-
wise linear regression to these data and breakpoints were
determined after 200 iterations. Data were tested for nor-
mality and homogeneity of variance prior to analyses and
log
10
transformed when necessary (P. agardhii biovolume
distribution on Zeu/Zmix gradient).
The three climatic regions were compared by examin-
ing data from winter and summer months for each lake
and year in the dataset (n=445) in terms of tempera-
ture, Z
eu
/Z
mix
, TP, and the biovolume of the two species.
Differences between environmental and biotic variables
were analysed with nonparametric Kruskal–Wallis (K–W)
tests, all pairwise multiple comparison tests (Dunn’s
Method) and Mann–Whitney tests (when P. agardhii was
present in only two regions).
Differences between physiological and morphological
experimental responses of the two species to light and
temperature gradients were compared using t-test analy-
sis and, when normality failed, with the nonparametric
Mann–Whitney (M–W) test. All analyses were per-
formed with the programs STATISTICA 6.0 and SIGMA PLOT
11.0.
Results
Species distributions and their relation to
environmental factors
Cylindrospermopsis raciborskii was observed in a higher
number of samples than P. agardhii (306 and 199 sam-
ples, respectively), with the two species co-occurring on
34 occasions (all in Lake Balaton) in a wide range of
environmental conditions. We analysed lakes where one
of the two species was present on at least one sample.
Cylindrospermopsis raciborskii was absent in all samples of
subtropical (Lago Rodo
´) and small Dutch temperate
lakes, while P. agardhii was absent in most of the sub-
tropical Uruguayan lakes (except Lago Rodo
´) and all
tropical Brazilian lakes. Each species reached high biovo-
lume and had a high contribution to total biovolume in
several samples (Table 1, Fig. 1a and b). Cylindrospermop-
sis raciborskii was dominant (at least 30% of total biovo-
lume) more frequently than P. agardhii, in samples of
both high (>1mm
3
L
1
) and low (<1mm
3
L
1
) total
biovolume. In most cases, P. agardhii was a minor
FEMS Microbiol Ecol 79 (2012) 594–607 ª2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Distribution of P. agardhii and C. raciborskii 597
component of the phytoplankton, representing <10% of
total biovolume, but in several cases, it was strongly dom-
inant (>50% of total biovolume) and its maximum abso-
lute biovolume was one order of magnitude higher
than C. raciborskii (Table 1). Biovolume variability over
time also differed between species. Temporal data series
of 17 lakes showed that the annual variation of P. agar-
dhii biovolume was significantly higher (P<0.05) than
C. raciborskii, shifting from low to high values (Fig. 1c
and d). Phytoplankton species richness also differed when
the dominance of one or the other species occurred; in
general, the number of species was higher when C. raci-
borskii was dominant (Fig. 2a and b).
In the field, C. raciborskii and P. agardhii occurrences
differed significantly relative to temperature, lake area,
maximum depth, mixing depth, conductivity, alkalinity
and TP (Table 1). However, no significant differences
were found for pH and light availability (Z
eu
/Z
mix
)
(Table 1). In general, C. raciborskii was dominant at
higher temperatures than P. agardhii. Several occurrences
of P. agardhii were reported at temperatures below 15 °C,
and below 4 °C its contribution varied between 0.2% and
13% of total biovolume (0.1–1.4 mm
3
L
1
). Almost all
data for C. raciborskii appeared at temperatures higher
than 20 °C. However, it is notable that we observed C.
raciborskii with high biovolume at 11 °C (2.1 mm
3
L
1
,
95% of total biovolume) in a subtropical lake (Lago Ja-
vier, Uruguay). Both species were dominant in eutrophic
to hypereutrophic lakes. Cylindrospermopsis raciborskii
attained higher biomass under lower TP, and no occur-
rences of P. agardhii were found in samples with
<50 lgL
1
TP (Table 1).
Table 1. Community and environment characteristics (median, minimum–maximum between brackets cursive numbers indicate the number of
cases) for Planktothrix agardhii and Cylindrospermopsis raciborskii, when present
Lakes with P. agardhii Lakes with C. raciborskii P
Community
P.agardhii (mm
3
L
1
) 0.79 (0.11–149)
199
0(0–10.4)
306
*
C.raciborskii (mm
3
L
1
)0(0–45.0)
199
2.74 (<0.01–71.79)
306
*
P.agardhii (% total BV) 6.56 (0.19–98.8)
199
0(0–45.6)
306
*
C.raciborskii (% total BV) 0 (0–77.80)
199
32.9 (0.05–97.4)
306
*
Total BV (mm
3
L
1
) 14.6 (2.22–302)
199
12.9 (0.07–697)
306
*
S27 (3–123)
199
18 (3–59)
160
*
Environment
Temperature (°C) 13.3 (1.70–27.6)
156
26.7 (11.2–31.6)
154
*
Area (ha) 1.30 (1.30–6910
4
)
86
4300 (0.24–6910
4
)
313
*
pH 8.18 (6.89–9.25)
120
8.20 (5.49–9.91)
132
ns
Z
max
(m) 2.50 (1.40–6.00)
194
6 (0.3–45)
314
*
Z
mix
(m) 2.00 (0.66–5.16)
200
3.2 (0.3–30)
222
*
Z
eu
/Z
mix
0.75 (0.17–3.42)
162
0.68 (0.09–3.78)
61
ns
K(lScm
1
) 550 (380–1141)
123
91.9 (25.6–3457)
129
*
Alk (mgL
1
) 160 (65.0–546)
130
46.5 (0.01–180)
37
*
TP (lgL
1
) 130 (50.0–5600)
163
74.9 (12.4–658)
73
*
Significant differences (Mann Whitney, *P<0.05) between the measured variables for each species are indicated.
ns, not significant; BV, biovolume; S, species number; Z
max
, maximum depth; Z
mix
, mixing zone; Z
eu
, euphotic zone; K, conductivity; Alk, alkalinity;
TP, total phosphorus.
ª2011 Federation of European Microbiological Societies FEMS Microbiol Ecol 79 (2012) 594–607
Published by Blackwell Publishing Ltd. All rights reserved
598 S. Bonilla et al.
Planktothrix agardhii biovolume shifted sharply from
high to low values across thresholds in the temperature,
Z
eu
/Z
mix
and TP gradients (as identified by parameter c
in the logistic function and breakpoints in piecewise lin-
ear regressions in Fig. 3a, c and e). Planktothrix agardhii
biovolume decreased abruptly below 11 °C, above 1.62
Z
eu
/Z
mix
and above 159 lgL
1
TP. Cylindrospermopsis
raciborskii biovolume was inversely related to TP, and no
significant relation was found with temperature or Z
eu
/
Z
mix
(Fig. 3b, d and f). However, maximum C. raciborskii
biovolume was observed with Z
eu
/Z
mix
values 1. Also,
this species had higher biovolumes than P. agardhii in
fully illuminated water columns (Z
eu
/Z
mix
:3–4).
Comparison of the species among climates:
temperate, subtropical and tropical
There were significant differences (P<0.05) between
geographical regions in terms of temperature, light and
phosphorus. Water temperature was higher in the tropics,
more transparent waters were found in tropical systems,
and higher trophic states (TP) were found in subtropical
lakes (Table 2). Planktothrix agardhii occurred only in
temperate and subtropical water bodies, where it had a
significantly higher average contribution to total biovo-
lume than C. raciborskii.Cylindrospermopsis raciborskii
occurred in the three regions and, notably, had no signifi-
cant differences in its contribution to total biovolume
between tropical and temperate regions (Fig. 4).
Experimental data
Morphology, pigment structure and growth rate of
P. agardhii and C. raciborskii were compared under dif-
ferent light intensities and temperatures (Tables 3 and 4,
Fig. 5). Increments of light intensity from 20 to
100 lmol photons m
2
s
1
induced adaptive morpholog-
ical responses in C. raciborskii. A significant increase in
Lakes
Bala-96
Bala-97
O2-99
Bala-94
O5-00
O4-00
Rodo-00
O6-00
O5-98
O3-00
D2-98
D4-00
D4-98
Rodo-99
Bala-01
D5-00
Bala-06
D1-00
D3-99
Bala-02
O4-98
Bala-05
D5-98
O3-98
Bala-03
Rodo-98
Annual biovolume
coeficient of variation
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Lakes
Bala-93
Bala-87
Tapa-98
Bala-03
Bala-02
Bala-96
Tabo-98
Bala-06
Bala-86
Bala-05
Bala-82
Bala-84
Tabo-97
Bala-83
Bala-85
Bala-94
Bala-92
Bala-97
Imbo-00
Bala-01
Bala-88
Bala-89
Tapa-99
Jutur-97
Imbo-99
Funil-02
Annual biovolume
coeficient of variation
0.0
0.5
1.0
1.5
2.0
2.5
3.0
(c) (d)
(a) (b)
0 102030405060708090100
P. agardhii relative contribution
to total biovolume (%)
0
20
40
60
80
100
120
Frequency
(number of observations)
0 102030405060708090100
C. raciborskii relative contribution
to total biovolume (%)
0
20
40
60
80
100
120
Frequency
(number of observations)
Fig. 1. Frequency of the distribution of the relative biovolume of each species (percentage of total biovolume) for the data set (a: Planktothrix
agardhii, black bars and b: Cylindrospermopsis raciborskii, grey bars). Annual coefficient of variation of biovolume for 17 lakes with temporal data
(c: P. agardhii, black bars and d: C. raciborskii, grey bars). For c and d, the following lakes names are abbreviated: Balaton (Bala), Imboassica
(Imbo), Juturnaiba (Jutur), Tabocas (Tabo) and Tapacura
´(Tapa). The number after each lake name indicates the last two digits of the year.
FEMS Microbiol Ecol 79 (2012) 594–607 ª2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Distribution of P. agardhii and C. raciborskii 599
C. raciborskii MLD and individual V (M–W, P<0.05)
occurred when cultures grew at high light intensity
(Table 3).
Lipid pigment composition and responses to light
intensity also differed between species. Typical cyanobac-
terial carotenoids were detected in both species. Plankto-
thrix agardhii had higher concentrations of zeaxanthin, b,
b-carotene, an undetermined glycosidic carotenoid similar
to 4-keto-myxol-2′-methylpentoside (myxol-like) and an
unknown carotenoid (car 1), while C. raciborskii had
high concentrations of aphanizophyll and echinenone
(Table 3). In both species, protective and accessory pig-
ment concentrations changed in response to light inten-
sity, with notable differences. The phycocyanin/Chl a
ratio decreased with higher light intensity in P. agardhii
and increased in C. raciborskii. The magnitude of change
in carotenoids/Chl awas also higher in C. raciborskii
than in P. agardhii (Table 3). Total carotenoids increased
sixfold in C. raciborskii, largely because of aphanizophyll,
but also because of echinenone and b,b-carotene. Plankto-
thrix agardhii total carotenoids increased 1.5 times with
higher light, mainly because of myxol-like and carotenoid
1, whereas b,b-carotene, echinenone and zeaxanthin
decreased.
Growth curve experiments performed along a light
intensity gradient (from 5 to 180 lmol photons m
2
s
1
)
indicated strong similarities between the two species
under light-limited conditions (indicated by aand I
k
),
although C. raciborskii reached significantly higher growth
rates (l
max
) than P. agardhii (Table 4, Fig. 5a and b).
Temperature growth experiments at two light intensities
demonstrated the different behaviour of the two species.
Planktothrix agardhii growth rates were significantly
higher than those of C. raciborskii at 15 and 20 °C at low
light intensity (60 lmol photons m
2
s
1
) (Fig. 5c),
although no differences were found at 25 °C. However,
C. raciborskii grew significantly faster than P. agardhii
(Fig. 5c) at high light intensity (135 lmol pho-
tons m
2
s
1
)at25°C. Q
10
values also showed that
C. raciborskii growth rate had a higher response to a tem-
perature increase at 135 lmol photons m
2
s
1
than
P. agardhii (Table 4).
Discussion
Our extensive data set and laboratory experiments indi-
cated that although C. raciborskii and P. agardhii overlap
in their distribution relative to temperature, light and tro-
phic status, they differed in their biovolume distributions
along these gradients. Our results support the hypothesis
that Cylindrospermopsis is tolerant to a wide range of cli-
mates, from tropical to temperate (Briand et al., 2004).
Although many studies have suggested that the optimum
water temperature of the species is from 25 to 35 °C
(Saker & Eaglesham, 1999; Briand et al., 2004; Mehnert
et al., 2010), high biomass has been observed in
subtropical lakes at 19 °C (Everson et al., 2011), and
C. raciborskii was equally dominant throughout the year
in a tropical lake independent of water temperature varia-
tion (17–24 °C) (Figueredo & Giani, 2009). Still other
studies found some strains to be capable of sustaining
biomass or growing at temperatures as low as 14–17 °C
(Chonudomkul et al., 2004; Piccini et al., 2011). Fabre
et al. (2010) observed C. raciborskii occurrence during
winter in a subtropical lake (Lago Javier, Uruguay), and
in our database the biovolume of C. raciborskii in this
lake reached 95% of the total (i.e. 2.2 mm
3
L
1
) in win-
ter (water temperature: 11.2 °C). To our knowledge, this
is the lowest temperature at which C. raciborskii has been
observed to reach high biovolume and dominate the phy-
toplankton. The success of C. raciborskii in a wide range
1
10
20
30
40
50
60
70
80
90
100
110
120
130
S
0
20
40
60
80
100
P. agardhii percentage
of total biovolume (%)
1
10
20
30
40
50
60
70
80
90
100
110
120
130
S
0
20
40
60
80
100
C. raciborskii percentage
of total biovolume (%)
(a)
(b)
Fig. 2. Contribution of Planktothrix agardhii (a) and Cylindrospermopsis
raciborskii (b) to total biovolume, when each species was >0, in
relation to species number of the community (S). Median (square),
percentiles 25% and 75% (box) and range (vertical lines).
ª2011 Federation of European Microbiological Societies FEMS Microbiol Ecol 79 (2012) 594–607
Published by Blackwell Publishing Ltd. All rights reserved
600 S. Bonilla et al.
of temperatures observed in our data set and other recent
studies (Vidal & Kruk, 2008; Kokocin
´ski et al., 2010; Ev-
erson et al., 2011) suggests that current concepts of C.
raciborskii as a tropical species may be due more to a lack
of information than to any physiological restriction.
We observed P. agardhii only in temperate and sub-
tropical lakes, but in a wide range of temperature condi-
tions. This species can reach high biomass in a range of
temperatures from <2°C (Toporowska et al., 2010) to
29 °C in tropical ecosystems (Crossetti & Bicudo, 2008;
Gemelgo et al., 2009). In our database, P. agardhii domi-
nated the phytoplankton of Lago Rodo
´(subtropical) in
all seasons at temperatures ranging from 10 to 31 °C,
indicating substantial tolerance to temperature variation.
Our Q
10
data indicated that C. raciborskii grows faster
than P. agardhii when temperatures shift towards warmer
conditions and thus may be favoured by climate warming.
Experimental studies showed that P. agardhii maximum
0.0 1.0 2.0 3.0 4.0
P.agardhii % of total biovolume, Log
10
0.0
0.5
1.0
1.5
2.0
2.5
TP (μg L
–1
)TP (μg L
–1
)
0 100 200 300 400 500 600 1600
P. agardhii % of total biovolume
0
20
40
60
80
100
Temperature (°C)
0 5 10 15 20 25 30 35
P. agardhii % of total biovolume
0
20
40
60
80
100
r
2
= 0.57
P < 0.001
Temperature (°C)
0 5 10 15 20 25 30 35
C. raciborskii % of total biovolume
0
20
40
60
80
100
Z
eu
/Z
mix
Z
eu
/Z
mix
0.0 1.0 2.0 3.0 4.0
C. raciborskii % of total biovolume
0
20
40
60
80
100
(a) (b)
(c) (d)
(e) (f)
0 100 200 300 400 500 600
C. raciborskii % of total biovolume
0
20
40
60
80
100
r
2
= 0.49
P < 0.05
r
2
= 0.53
P < 0.05
r
2
= 0.63
P < 0.001
Fig. 3. Maximum values of the contribution to total biovolume of Planktothrix agardhii (left, black circles) and C. raciborskii (right, grey circles) in
relation to water temperature (a and b), Z
eu
/Z
mix
(c and d) and TP (e and f). Sigmoidal logistic regression (a, parameters, a: 18.2, b: 49.3, c: 11.1
and d: 99.2), linear piecewise regressions (c, breakpoint: 1.62 and e, breakpoint: 159.2 lgL
1
) and linear regression (f) were fitted. No
significant model was found for b and d. Coefficient (r
2
) and significance (P) is indicated in each plot.
FEMS Microbiol Ecol 79 (2012) 594–607 ª2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Distribution of P. agardhii and C. raciborskii 601
growth occurred between 20 and 25 °C (Post et al., 1985;
Sivonen, 1990), and its growth rate increased significantly
between 15 and 25 °C (Oberhaus et al., 2007). Cylindro-
spermopsis raciborskii has been shown to benefit more
than other cyanobacteria from high temperatures (Mehn-
ert et al., 2010) and also to have higher photosynthetic
activity and lower light requirements than other cyano-
bacterial species (Wu et al., 2009). Dominance of this
species, however, cannot be predicted from any single
factor. In this sense, our Q
10
values (at 60 and
135 lmol photons m
2
s
1
) suggest that C. raciborskii
could have a competitive advantage over P. agardhii at
conditions with both high light and high temperature.
These differences are attributable to the ability of C. raci-
borskii to increase light-harvesting capacity through
changes in shape and pigment composition and their
proportions as we demonstrated in our experiments.
Different photoprotective responses were also suggested
by pigment changes after light increases. In this sense, the
acclimation capacity of C. raciborskii is illustrative of its
phenotypic plasticity.
Planktothrix agardhii showed a higher competitive
capacity than C. raciborskii under low light and lower
temperatures, which agrees with its broad distribution in
turbid temperate lakes (Dokulil & Teubner, 2000; Nixdorf
et al., 2003). Based on the biovolume distribution and
growth of P. agardhii in our study, we suggest that this
species has limited plasticity, as its physiological response
to temperature increase under high light intensity was less
pronounced than that of C. raciborskii.
According to our I
k
values, both species are shade-tol-
erant, implying that they can succeed in turbid, eutrophic
lakes (Padisa
´k & Reynolds, 1998), as originally proposed
for Oscillatoriales (Scheffer et al., 1997). The I
k
values
that we obtained for both species were lower than those
reported in the literature (~20 lmol photons m
2
s
1
)
Table 2. Water temperature, light availability (Z
eu
/Z
mix
) and total phosphorus (TP) of studied lakes grouped by regions (temperate, subtropical
and tropical) and based on winter and summer data (median and minimum and maximum between brackets and number of samples in cursive)
Temperate (35°30′–38°80′S, 46°50′–52°23′N) Subtropical (34°33′–34°55′S) Tropical (08°02′–22°33′S)
Temperature (°C) 19.1 (0.50–27.6)a
207
22.8 (10.0–26.4)b
40
26.9 (20.0–31.6)c
107
Z
eu
/Z
mix
0.58 (0.17–4.15)a
257
0.84 (0.17–2.7)a
75
0.64 (0.09–3.0)b
56
TP (lgL
1
) 105 (50–1652)a
210
158 (46–422)b
56
91.7 (12.4–794)a
4
Significant differences (Mann–Whitney, P<0.05) between regions are indicated with different letters in the table.
ns, not significant; Z
eu
, euphotic zone; Z
mix
, mixing zone.
Table 3. Morphology (average ±standard deviation, n=60) and pigment structure (molar pigment ratios to Chl a)ofPlanktothrix agardhii and
Cylindrospermopsis raciborskii isolates grown under 20 and 100 lmol photons m
2
s
1
P. agardhii
MVC11
C. raciborskii
MVCC14
Light intensity (lmol photons m
2
s
1
) 20 100 20 100
Size and shape
Volume (lm
3
) 3573 ±1771 6169 ±3968 414 ±150 1406 ±523
MLD (lm) 258 ±111 318 ±200 132 ±48 200 ±75
S/V (lm
1
) 0.98 ±0.09 0.82 ±0.04 2.02 ±0.01 1.35 ±0.03
Carotenoid ratios to Chl a
Myxo-like (497/522) 0.07 0.14 nd nd
Aphanizophyll nd 0.18 0.56 3.24
Car 1 (477/505) 0.45 0.67 nd nd
Zeaxanthin 0.33 0.29 nd 0.05
Lutein nd 0.24 nd 0.03
Echinenone 0.13 0.08 0.32 0.77
b,b-carotene 0.40 0.25 0.15 0.46
T
CAR
/Chl a1.53 2.28 1.09 6.74
Phy/Chl a48.9 11.4 77.7 49.4
The maximum absorbance peaks are indicated between brackets for the unknown carotenoids (Myxol-like and Car 1).
S / V, surface/volume ratio; Myxol-like, 4-keto-myxol-2′-methylpentoside-like; T
CAR
, total carotenoids; Phy, phycocyanin; nd, not detected.
ª2011 Federation of European Microbiological Societies FEMS Microbiol Ecol 79 (2012) 594–607
Published by Blackwell Publishing Ltd. All rights reserved
602 S. Bonilla et al.
(Talbot et al., 1991; Shafik et al., 2001; Briand et al.,
2004) which may support the existence of ecotypes sug-
gested by Piccini et al. (2011). The presence of ecotypes
with different environmental preferences confers a wide
intra-specific variability to C. raciborskii; ecotypes are
among the hypotheses advanced to explain the species’
expansion. The pigments we identified in both species
were typical for cyanobacteria (Millie et al., 1990),
although they were present in markedly different propor-
tions.
Planktothrix agardhii is favoured in continuously
mixed, shallow lakes (Scasso et al., 2001; Kruk et al.,
2002; Stu
¨ken et al., 2006). Similarly, blooms of C. raci-
borskii are commonly reported in mixed conditions (Bou-
vy et al., 1999; Huszar et al., 2000; Briand et al., 2002;
Figueredo & Giani, 2009) and rarely in stratified deep
reservoirs (Padisa
´ket al., 2003). This clearly indicates that
both species have a wide tolerance for mixing. While
in our study there was no clear relationship between
C. raciborskii biovolume and light availability (Z
eu
/Z
mix
),
P. agardhii biovolume was higher in turbid conditions
below the threshold value of 1.62 Z
eu
/Z
mix
ratio, suggest-
ing its dependence on turbidity.
The cyanobacterial contribution to total phytoplankton
biomass increases markedly above 30 lgL
1
TP in tem-
perate lakes (Watson et al., 1997; Dokulil & Teubner,
2000), and P. agardhii biovolume distribution in our data
set reflect this general pattern. However, the sudden
changes we observed in biovolume distribution in hype-
reutrophic conditions suggested a threshold near
160 lgL
1
TP, above which other factors affected biovo-
lume accumulation. In contrast, there was a negative rela-
tionship between trophic status and C. raciborskii
contribution to total biovolume, with increasing domi-
nance of the phytoplankton below 200 lgL
1
TP. Higher
ranges of phosphorus cell quota in C. raciborskii relative
to P. agardhii may permit C. raciborskii to better exploit
low P environments (Ducobu et al., 1998; Istva
´novics
et al., 2000). Some studies suggest that P. agardhii growth
is greatly dependent on high-frequency phosphate avail-
ability (Catherine et al., 2008; Crossetti & Bicudo, 2008;
Kokocin
´ski et al.,2010;Aubriotet al., 2011), while
C. raciborskii is able to dominate with small, low-
frequency phosphate inputs (Posselt & Burford, 2009). Phy-
toplankton from oligotrophic and mesotrophic ecosystems
may thus be sensitive to a replacement by C. raciborskii
as a dominant species under small nutrient enrichments.
According to our data, C. raciborskii can dominate the
phytoplankton at lower overall biovolume than P. agar-
dhii, giving insight into the ability of C. raciborskii to colo-
nize and rapidly succeed in new habitats. While variations
of C. raciborskii biovolume during the year were gradual,
Table 4. Growth parameters under different light intensities
(maximum growth rate: l, slope of the light-limited portion of the
curve: a, and subsaturating light: I
k
); Q
10
based on the maximum
growth average (n=4) at 15 and 25 °C for both isolates
P. agardhii
MVCC11
C. raciborskii
MVCC14
Light growth response
lmax (d
1
) 0.54 ±0.03*0.60 ±0.02*
†
a(d
1
lmol photons
1
m
2
s) 0.08 ±0.03 0.08 ±0.03
†
I
k
(lmol photons m
2
s
1
) 7.27 ±3.15 8.49 ±3.43
†
Q
10
(15–25 °C)
At 60 lmol photons m
2
s
1
2.50 3.80
At 135 lmol photons m
2
s
1
3.43 6.46
Mean ±standard deviation for light growth response.
*Significant differences between species (t-test, P<0.05).
†Data from Piccini et al. (2011).
ST
TTR
Climate
ST
TTR
Climate
0
20
40
60
80
100
P. agardhii relative contribution
tototal biovolume(%)
0
20
40
60
80
100
C. raciborskii relative contribution
to total biovolume (%)
a
b
aa
b
(a)
(b)
Fig. 4. Contribution of Planktothrix agardhii (a) and Cylindrospermopsis
raciborskii (b) to total biovolume, in relation to the three geographical
regions when each species was >0. Median (square), percentiles
25% and 75% (box) and range (vertical lines). T, temperate; ST,
subtropical and TR, tropical regions. Different letters indicate
significant differences (K–W, P<0.001) between regions.
FEMS Microbiol Ecol 79 (2012) 594–607 ª2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Distribution of P. agardhii and C. raciborskii 603
P. agardhii was either dominant or scarce in phytoplank-
ton. Moreover, the dominance of P. agardhii in the
phytoplankton appears to occur in more eutrophic condi-
tions (i.e. higher phytoplankton biovolume). Scheffer
et al. (1997) observed similar behaviour in eutrophic tem-
perate shallow lakes and proposed hysteretic mechanisms
to explain the distribution and resilience of P. agardhii.
Our data indicated that higher diversity (as taxonomic
richness) is supported under dominance of C. raciborskii
than that of P. agardhii. This suggests a higher capacity
of C. raciborskii for co-existence with other species
(Kokocin
´ski et al., 2010) and may support the hypothesis
of its greater plasticity. Sperfeld et al. (2010) demon-
strated experimentally that the invasion and success of
C. raciborskii was not affected by the diversity of the host
phytoplankton community. The relatively higher diversity
associated with C. raciborskii dominance also has implica-
tions for food webs, as some studies also found positive
correlations between the biomass of C. raciborskii and
zooplankton (Bouvy et al., 2001; Soares et al., 2009).
Conversely, the lower diversity of communities domi-
nated by P. agardhii may result from a capacity of this
species to generate limiting conditions (i.e. high turbidity)
for potential phytoplankton competitors.
The greater plasticity of C. raciborskii in response to
key environmental factors (temperature and light inten-
sity) may explain its gradual response to changing envi-
ronments. Conversely, the lower plasticity of P. agardhii
fits with its narrower distribution in nature. Aquatic envi-
ronments are highly variable habitats in terms of light
and nutrient resources at the time scale of phytoplankton
life spans. Reversible plastic phenotypes represent an
advantage for organisms in highly variable environments
(Piersma & Drent, 2003), allowing the adjustment of their
functional responses and increasing their invasive poten-
tial (Litchman, 2010). Ecotypes with differing environ-
mental tolerances such as those shown in C. raciborskii
(Piccini et al., 2011) further strengthen its aptitude for
invasive behaviour and success in different climates.
In summary, C. raciborskii and P. agardhii behaved dif-
ferently as a result of contrasting strategies for responding
to environmental constraints. Further research is required
to determine whether this pattern may represent differing
strategies in bloom-forming filamentous cyanobacteria of
the orders Oscillatoriales and Nostocales. Differences
between P. agardhii and C. raciborskii, as well as between
other organisms with comparable strategies, will likely
affect the future distribution of these species in projected
warming climates where blooms will be enhanced. The
high phenotypic plasticity of C. raciborskii, and its wide
tolerance ranges to key environmental factors, explains its
current expansion to temperate latitudes and forecasts its
further increase in the future.
Acknowledgements
We thank Marie-Jose
´e Martineau for technical assistance
and Warwick F. Vincent for kindly providing access to
laboratory facilities. This work was financed by ANII
FCE2007_353 and PEDECIBA. We also thank two
anonymous reviewers for their comments.
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0.0
0.2
0.4
0.6
0.8
P. agardhii
C. raciborskii
P. agardhii
μ (d
–1
)μ (d
–1
)μ (d
–1
)
0.0
0.2
0.4
0.6
0.8
C. raciborskii
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–2
s
–1
)
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0.0
0.2
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0.8
**
*
(a)
(b)
(c)
15 °C
60
20 °C
60
25 °C
60
15 °C
135
20 °C
135
25 °C
135
Fig. 5. Experimental growth curves of Planktothrix agardhii (a) and
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and two light intensities (60 and 135 lmol photons m
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s
1
)
experiments. Bars are averages, and vertical lines are standard
deviations. *Significant differences (t-test, P<0.05) between species
at the corresponding experimental level of light and temperature.
ª2011 Federation of European Microbiological Societies FEMS Microbiol Ecol 79 (2012) 594–607
Published by Blackwell Publishing Ltd. All rights reserved
604 S. Bonilla et al.
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