PRIMARY RESEARCH PAPER

Zooplankton response to climate warming: a mesocosmexperiment at contrasting temperatures and nutrient levels

Michal Sorf • Thomas A. Davidson • Sandra Brucet • Rosemberg F. Menezes •

Martin Søndergaard • Torben L. Lauridsen • Frank Landkildehus •

Lone Liboriussen • Erik Jeppesen

Received: 13 February 2014 / Revised: 9 July 2014 / Accepted: 15 July 2014 / Published online: 6 August 2014

� Springer International Publishing Switzerland 2014

Abstract Zooplankton community response to the

combined effects of nutrients and fish (hereafter

N ? F) at contrasting temperatures was studied in a

long-term experiment conducted in 24 shallow lake

mesocosms with low and high nutrient levels. We

found a positive effect of N ? F on zooplankton

biomass, chlorophyll-a and turbidity. In contrast,

zooplankton species and size diversity decreased with

added N ? F, as did submerged macrophyte plant

volume inhabited (PVI). The community composition

of zooplankton in high N ? F mesocosms was related

to chlorophyll-a and turbidity and to macrophyte PVI

in the low N ? F mesocosms. Macrophytes can

protect zooplankton from fish predation. Compared

to N ? F effects, temperature appeared to have little

effect on the zooplankton community. Yet analysis of

community heterogeneity among treatments indicated

a significant temperature effect at high N ? F levels.

The results indicate an indirect temperature effect at

high N ? F levels that can be attributed to tempera-

ture-dependent variation in fish density and/or chlo-

rophyll-a concentration.

Keywords Zooplankton � Mesocosms � Nutrients �Climate change

Introduction

Shallow lakes, an important component of the land-

scape, are seriously threatened by climate change,

eutrophication and their combined effects (Jeppesen

et al., 2009; Moss et al., 2011). Being sandwiched in-

between fish and phytoplankton, zooplankton plays aHandling editor: Karl E. Havens

M. Sorf (&)

Faculty of Science, University of South Bohemia,

Ceske Budejovice, Czech Republic

e-mail: michal.sorf@prf.jcu.cz

T. A. Davidson � S. Brucet � R. F. Menezes �M. Søndergaard � T. L. Lauridsen � F. Landkildehus �L. Liboriussen � E. Jeppesen

Department of Bioscience, Aarhus University, Silkeborg,

Denmark

T. A. Davidson � T. L. Lauridsen � E. Jeppesen

Arctic Research Centre, Aarhus University, Aarhus,

Denmark

S. Brucet

Department of Environmental Sciences, University of

Vic, Vic, Spain

R. F. Menezes

Departamento de Ecologia, Centro de Biociencias,

Universidade Federal do Rio Grande do Norte, Natal,

Rio Grande do Norte, Brazil

T. L. Lauridsen � E. Jeppesen

Sino-Danish Centre for Education and Research (SDC),

Beijing, China

123

Hydrobiologia (2015) 742:185–203

DOI 10.1007/s10750-014-1985-3

key role in the pelagic food web of temperate shallow

lakes and contributes to maintaining clear water

conditions at low fish predation (Meijer et al., 1994;

Hansson et al., 1998). How zooplankton will be

affected by climate warming is widely debated.

Zooplankton may be directly impacted by the rising

temperature depending on their ontogeny and physi-

ology (Huntley & Lopez, 1992) and indirectly by

altered size and nutrient quality of their main food

source, phytoplankton (van de Waal et al., 2010).

Warming may also modify the zooplankton commu-

nities through increased top–down regulation (Gyll-

strom et al., 2005; Jeppesen et al., 2010; Shurin et al.,

2012) or by altering their competitive interactions

(Fey & Cottingham, 2012). Field studies using a

space-for-time approach indicate that the size of

zooplankton will decline; this has been attributed to

the metabolic benefits of small specimens in warm

lakes (Moore et al., 1996; Gillooly & Dodson, 2000),

but an alternative, though not mutually exclusive,

explanation is increased fish predation as demon-

strated in several recent studies (Jeppesen et al., 2010,

2011; Iglesias et al., 2011; Havens & Beaver, 2011).

Cross-comparisons of fish populations in comparable

systems in South America and Europe (Teixeira-de

Mello et al., 2009) and within Europe have shown that

lower-latitude fish species are typically not only indi-

vidually smaller (Griffiths, 1997; Jeppesen et al., 2010),

but also grow faster, mature earlier and have shorter life

spans than species at higher latitudes (Blanck &

Lammouroux, 2007). Thus, temperature-related

changes in fish population structure may be an important

factor, resulting in higher predation on large-bodied

zooplankton. Accordingly, zooplankton biomass is

often lower in warm lakes than would be expected from

the available food resources (low zooplankton to

phytoplankton biomass ratio) (Gyllstrom et al., 2005;

Havens & Beaver, 2011; Meerhoff et al., 2012).

The observed effects of experimental warming on

zooplankton have, however, been highly variable. In a

study conducted in heated mesocosms in Canada,

zooplankton biomass was found to be suppressed due

to a decline in large-sized cladocerans, even in the

absence of fish (Strecker et al., 2004). By contrast, an

experiment conducted in heated mesocosms in the UK

showed no clear effects of warming on cladoceran and

copepod abundance (McKee et al., 2002).

A mesocosm warming experiment simulating shal-

low lakes with contrasting nutrient loadings has been

running in Lemming, Denmark, since 2003 (Libori-

ussen et al., 2005; Jeppesen et al., 2010) and is to the

best of our knowledge the longest running warming

experiment in the world. Earlier studies conducted in

these mesocosms have shown that bacterioplankton

and protists (heterotrophic nanoflagellates and cili-

ates) are mainly influenced by nutrients but also, to

varying degrees, by complex interactions between

nutrients and warming (Christoffersen et al., 2006;

Ozen et al., 2013).

We followed the zooplankton in this warming

experiment (3 temperature scenarios crossed with 2

nutrient levels) during summer 2010 (May–August),

7 years after the start of the experiment when the

mesocosms were assumed to have passed the initial

transient phase. Mesocosms differed not only in

temperature and nutrient level but also in fish abun-

dance with higher fish stocks in the high-nutrient

mesocosms. We focused on the effect of warming and

the combined effect of increased nutrient levels and

fish predation on zooplankton community composi-

tion and size structure in shallow water bodies. We

hypothesised that (a) higher nutrient level would

increase the biomass of zooplankton due to enhanced

phytoplankton growth despite higher fish abundance,

(b) the zooplankton body size would be lower at high

nutrient level due to a higher fish predation pressure,

(c) zooplankton body sizes would decrease at higher

temperature, particularly at the high nutrient levels

where fish predation is expected to be highest, and

(d) the rise of temperature would affect zooplankton

community less than variations in fish predation and

nutrient loading.

Materials and methods

Experimental set-up

An ongoing outdoor mesocosm experiment was

established in August 2003 in Lemming, Central

Jutland, Denmark. It consists of 24 artificially mixed

flow-through mesocosms (diameter 1.9 m, water level

1 m, retention time ca. 2.5 months due to addition of

groundwater six times daily). The experimental set-up

is described in detail by Liboriussen et al. (2005). This

fully factorial designed experiment combines three

temperature scenarios [unheated; A2 scenario (IPCC,

2007); A2 ? 50% scenario (50% increase relative to

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the A2 scenario)] crossed with two nutrient levels, all

in four replicates. Temperature levels of the treatments

were modelled using 1961–1990 as reference period

and down-scaled to local conditions. The temperature

differences vary over the season (adjusted monthly;

Liboriussen et al., 2005). A2 scenario was in average

Fig. 1 Temperature, total phosphorus and nitrogen concentra-

tions in experimental mesocosms. Upper panel represents daily

mean temperature in unheated mesocosms (solid line), the A2

scenario (hatched line) and the A2 ? 50% scenario (dotted

line). Nutrient panels: left low-nutrient treatments, right high-

nutrient treatments. Columns represent ambient temperature

(empty bars), the A2 scenario (hatched bars) and the A2 ? 50%

scenario (crosshatched bars). All bars express mean ± SE

Hydrobiologia (2015) 742:185–203 187

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2.7�C higher and A2 ? 50% scenario 4.1�C higher

than unheated mesocosms during the summer period

that we studied (Fig. 1). The highest temperature was

obtained at the beginning of July: 24.2�C in unheated

mesocosms, 27.0�C in the A2 scenario and 28.2�C in

the A2 ? 50% scenario (Fig. 1).

Nutrients were added weekly to half of the mesocosm,

while the other half received no extra addition apart from

the groundwater input varying from 2 to 20 lg TP l-1

and 51–70 lg TN l-1. The nutrient loading to the high-

nutrient mesocosms was 2.7 mg P m-2 day-1 and

27.1 mg N m-2 day-1, respectively.

A 20 cm sediment layer (10 cm washed sand and

10 cm nutrient rich sediment from a nearby pond)

sieved through a 1 9 2 cm mesh was added initially to

each mesocosms. An inoculum of both limnetic and

benthic communities was added before the start of the

experiment, 7 years before our study began.

Fish

Three-spined sticklebacks, Gasterosteus aculeatus,

were introduced to the mesocosms in August 2003. In

order to reach densities typical for Danish lakes, one

individual was added to the low-nutrient mesocosms

and 12 fish were stocked in the high-nutrient meso-

cosms. Originally, only males were added to prevent

breeding. Later (in 2006), females were added to the

high-nutrient mesocosms, allowing breeding. In the

low-nutrient mesocosms we kept one male stickleback

per mesocosm as breeding would lead to unnaturally

high densities. The mesocosms are too small to hold

piscivores which in natural lakes keep abundance of

planktivores low at low nutrient concentrations, but

not at high concentrations where the proportions of

piscivores are low (Jeppesen et al., 2000).

We estimated catch per unit effort (CPUE) of fish

once (in August) using circular hand nets with a fine

mesh size (0.5 mm) for young-of-the-year (YOY)

fish and traps for older year classes. To estimate

CPUE of YOY fish, we conducted three standard-

ised haulings with a hand net at two opposite sides

of the mesocosms. Adult stickleback density was

determined during a 4-day period (4 nights) using

traps. The traps, made of perforated plastic, were

provided with bait (fish eggs) and weighed down to

keep them just above the bottom. The traps were

checked every day for number of fish, and body size

was measured before the fish were returned to the

mesocosms.

Macrophytes

Macrophytes (mainly Elodea canadensis and Pota-

mogeton crispus) emerged naturally in most of the low-

nutrient mesocosms from May to June 2003 (before the

start of the experiment), whereas sparse shoots of these

species appeared in only two of the high-nutrient

mesocosms. As the plant populations developed non-

uniformly among the low-nutrient mesocosms, we

manipulated their distribution and coverage before the

experiment to obtain similar starting conditions in all

mesocosms (Liboriussen et al., 2005).

Elodea canadensis and/or Potamogeton crispus

also dominated in all the low-nutrient mesocosms,

while filamentous algae inhabited mostly the high-

nutrient mesocosms. The abundance of macrophytes

and filamentous algae floating on the water surface

was determined monthly using plant volume inhabited

(PVI) based on measurements of percentage coverage

and mean macrophyte height (Canfield et al., 1984).

Sampling and laboratory analyses

Sampling was conducted five times from May to

August 2010. Zooplankton was sampled using a 1-m

long tube sampler with a diameter of 7 cm at seven

stratified randomly selected sites. The samples were

subsequently pooled and an 8 l subsample was filtered

through a 50 lm mesh and preserved in 4% Lugol’s

iodine (v/v). Sampling took place twice a day: from 11

to 13 and from 23 to 1 h(s). All statistics are based on

the mean values of the two samples.

Lugol’s solution was rinsed off the sample prior to

counting, which was performed on a 100 ml subsample

at 639 magnification (cladocerans and copepods) using a

stereomicroscope (Leica MZ12, Wetzlar, Germany) or at

4009 magnification (rotifers) using an inverted micro-

scope (Leitz Labovert). Rotifer biomass was calculated

using standard dry weights (Dumont et al., 1975; Bottrell

et al., 1976). Cladoceran and copepod biomasses were

calculated based on length–weight relationships

(Dumont et al., 1975; Bottrell et al., 1976; Culver et al.,

1985). We only recorded few adults and, therefore,

grouped copepodites and adults in the statistical analyses.

Nauplii were recorded and analysed separately.

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Chlorophyll-a was used as a surrogate of phyto-

plankton biomass. A 100–1,000 ml water sample was

filtered through Whatman GF/C filters (47 mm in

diameter) depending on concentration. Chlorophyll-

a was determined spectrophotometrically after ethanol

extraction (Jespersen & Christoffersen, 1987). Besides

chlorophyll-a, the coverage of filamentous algae was

also determined using PVI. Nutrients were determined

monthly, and the water was frozen prior to analysis of

total phosphorus (TP) and ortho-phosphate (PO4–P)

(Grasshoff et al., 1983), total nitrogen (TN) (Solorz-

ano & Sharp, 1980) and nitrate ? nitrite (NO3–N)

using a cadmium reduction method (Grasshoff et al.,

1983). Turbidity was measured in the field.

To assess the effects of temperature and nutrients

on zooplankton size structure, we used three size

measures: zooplankton size diversity, zooplankton

mean body size and mean body size of cladocerans.

We calculated Shannon–Weaner diversity for zoo-

plankton size classes with a programme that uses the

size of the individual organisms (Quintana et al.,

2008). The proposed size diversity (l) is computed

based on the Shannon diversity expression adapted for

a continuous variable, such as body size. This measure

takes the form of an integral involving the probability

density function of the size of the individuals

described by the following equation:

l ¼ �Z þ1

0

pXðxÞlog2pXðxÞdx;

where px(x) is the probability density function of size

x. The non-parametric kernel estimation was used as a

probability density function, which is applicable to any

type of size distribution. Before we calculated size

diversity, the data were automatically standardised by

division of each size value by the geometric mean of

the size distribution. The size diversity index integrates

the extension of the size range and the evenness in the

same way that Shannon species diversity integrates the

number of species and their relative abundance (Brucet

et al., 2006; Quintana et al., 2008). Thus, its meaning is

easy to interpret: high size diversity means a wide size

range and/or similar proportions of the different sizes

along the size distribution.

Statistical analyses

Repeated measures ANOVA was used to test the

effect of warming, nutrient loading and their

interactions on species richness and on the Shannon–

Wiener diversity index. The same procedure was also

used to evaluate all studied effects in the course of

time. Comparison of fish CPUE between treatments

was done using one-way ANOVA. The dataset (with

the exception of ratios) was transformed by log(x ? 1)

before analyses. Ratios were transformed by arcsin

transformation.

p0 ¼ arcsinffiffiffipp

:

The assumptions of normality and homogeneity of

variance were tested for each group. Repeated mea-

sures and one-way ANOVA were applied under the

full factorial design using Statistica 6.0 (StatSoft,

USA).

To visualise heterogeneity within the data set

comprised of zooplankton biomass, both uncon-

strained and constrained methods using similarity

measures were assessed. Principal coordinates analysis

(PCoA) was used to show between-sample distances

based on Bray–Curtis dissimilarity. Although the

produced model was calculated using both the low-

and high-nutrient treatments, the final plot shows only

high nutrient centroids. More distant centroids repre-

sent higher level of dissimilarity between two samples

and hence more different mesocosms in term of

zooplankton biomass. For better orientation, samples

belonging to the same treatment were enclosed by

envelopes (Fig. 7). Distance-based redundancy ana-

lysis (db-RDA; Legendre & Anderson, 1999) as a

constrained method of PCoA was applied for testing

the effects of treatments (temperature, nutrients) and

explanatory variables (PVI, turbidity, chlorophyll-

a concentration) on the community composition of

each particular mesocosm. Bray–Curtis dissimilarity

and correction for negative eigenvalues were used

during the db-RDA procedure. In principle, db-RDA

represents a constrained principal coordinate analysis

(PCoA) capable of displaying the position of samples

in the coordinate system based on (dis)similarity

measures. Therefore, the combination of these two

methods (PCoA, db-RDA) allows us to interpret

differences between treatments. All ordinations were

done using CANOCO 5.03 (ter Braak & Smilauer,

2012). The mean values of dry weight biomass from

midday and midnight of all sampling dates were used

after transformation by log(x ? 1) and centred by

species. Significances of explanatory variables and

canonical axes were tested by Monte Carlo

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Fig. 2 Abundance of sticklebacks (catch per unit effort) and

seasonal patterns of chlorophyll-a concentrations, turbidity and

macrophyte coverage. All bars express mean ± SE. Left low-

nutrient treatments, right high-nutrient treatments. Columns

represent ambient temperature (empty bars), the A2 scenario

(hatched bars) and the A2 ? 50% scenario (crosshatched bars)

190 Hydrobiologia (2015) 742:185–203

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permutation (a = 0.05). All permutations were done

under the full model with 999 permutations and

considering the temporal structure of the dataset.

Patterns revealed by a visual assessment in PCoA

and db-RDA were statistically evaluated by two

approaches: multivariate dispersion measure and

ANOVA-like statistics adapted for multivariate design

of the performed experiment. Multivariate dispersion

based on Bray–Curtis dissimilarity was calculated

using the vegan package in R 2.15 (R Core Team,

2013). The procedure determined whether variability

in community structure differed among treatments.

Subsequently, permutational multivariate analysis of

variance (PERMANOVA) based on Bray–Curtis dis-

similarity was used to determine the simultaneous

response of zooplankton biomass data to nutrient

levels and warming (Anderson, 2001). Permutations

of residuals were run under a full model.

Results

Nutrients, phytoplankton and turbidity

The average total phosphorus concentration was 13.3

and 285 lg P l-1 in the low- and high-nutrient treat-

ments, respectively, while the same figures for nitrogen

were 0.23 and 3.8 mg N l-1 (Fig. 1). Total nitrogen

concentrations decreased with increasing temperature

in the high-nutrient treatments (Fig. 1; repeated mea-

sures ANOVA: P = 0.04; F = 3.8; df = 2).

Chlorophyll-a increased in the low-nutrient meso-

cosms during the experimental period, from an average

across all low-nutrient treatments of 3.0–18.4 lg l-1

(Fig. 2; repeated measures ANOVA: P = 0.01;

F = 5.22; df = 3). Nutrient addition enhanced the

chlorophyll-a concentration, especially in the ambient

temperature and the A2 scenario where concentrations

up to 1,608 lg l-1 were found. Turbidity mirrored

chlorophyll-a, varying from 2.6 to 4.1 FNU in the low-

nutrient mesocosms, while the maximum was as high

as 243 FNU in the high-nutrient mesocosms (Fig. 2).

Macrophytes and fish

The high-nutrient mesocosms hosted only a few

macrophyte stands, if any, and mainly in the unheated

mesocosms, while mean PVI in the low-nutrient

mesocosms from May to August was 19.9% in the

unheated treatment, 30% in the A2 scenario and 14.8%

in the A2 ? 50% scenario (Fig. 2). One adult male

stickleback was observed in all the low-nutrient

mesocosms. In the high-nutrient mesocosms, adult

stickleback CPUE ranged between 4.3 (unheated) and

10.6 (A2 scenario) individuals per mesocosm per night

in the August sampling (Fig. 2). Underyearling stick-

lebacks (YOY) were most abundant at ambient

temperatures (one-way ANOVA: F = 4.2; df = 2;

P = 0.03).

Zooplankton diversity

A total of 55 zooplankton taxa (33 rotifer, 16

cladoceran and 6 copepod taxa) were recorded. Rotifer

assemblages were dominated by Keratella quadrata

and K. valga in all treatments. The low-nutrient and

heated mesocosms were co-dominated by Polyarthra

spp. Trichotria pocillum and Brachionus calyciflorus

became abundant in the low-nutrient A2 scenario as

well as in all high-nutrient mesocosms regardless of

temperature. The high-nutrient mesocosms with ambi-

ent temperatures included other abundant species such

as Synchaeta sp., B. angularis, Filinia longiseta and

Euchlanis dilatata. The number of rotifer taxa

declined significantly in both the high- and low-

nutrient treatments (repeated measures ANOVA:

P = 0.004; F = 4.22; df = 4).Cladoceran and cope-

pod communities were less diverse in the high

(Cladocera: 2.7, Copepoda: 0.3 taxa in average) than

in the low-nutrient mesocosms (Cladocera: 3.8,

Copepoda: 1.2 taxa in average) (repeated measures

ANOVA: Cladocera: P \ 0.001; F = 16.56; df = 1;

Copepoda: P \ 0.001; F = 23.75; df = 1). The most

abundant microcrustacean taxa were the small cla-

docerans Bosmina longirostris and Chydorus sphae-

ricus. Alona rectangula mainly occurred in the high-

nutrient mesocosms irrespective of temperature sce-

nario. Simocephalus vetulus was the only large-bodied

cladoceran present and Macrocyclops albidus the only

large-bodied copepod, both occurring in the meso-

cosms with dense macrophyte vegetation. The Shan-

non–Wiener diversity index was generally lower in the

high-nutrient mesocosms, particularly by the end of

the experiment (repeated measures ANOVA:

P = 0.04; F = 4.49; df = 1; Fig. 3).

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Fig. 3 Shannon–Wiener diversity index of Rotifera, Clado-

cera, Copepoda and total zooplankton. All bars express

mean ± SE. Left low-nutrient treatments, right high-nutrient

treatments. Columns represent ambient temperature (empty

bars), the A2 scenario (hatched bars) and the A2 ? 50%

scenario (crosshatched bars)

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Zooplankton biomass

Zooplankton biomass varied among treatments and

time, but the pattern differed depending on taxonomic

group (Fig. 4). The biomasses of rotifers, cladocerans

and copepod nauplii were generally higher in the high-

nutrient mesocosms that also had higher fish densities.

Although an effect of temperature was not found for

any of the tested taxonomical groups, a significant

effect of time and nutrient interaction on total

zooplankton biomass was found as could be expected

due to seasonal changes (Table 1). Rotifer and cope-

pod biomass differed between the low- and high-

nutrient mesocosms and with temperature (Fig. 4;

Table 1). While rotifer biomass continued to decline

in both the unheated and the low-nutrient mesocosms,

from 20 lg l-1 in May to 1 lg l-1 in August, an

increase from May to the end of June (up to

251 lg l-1) was recorded in the unheated high-

nutrient mesocosms, subsequently decreasing to a

mean value of 22 lg l-1 in August (Fig. 4). The

biomass of rotifers in the heated high-nutrient meso-

cosms reached its maximum earlier in the season (at

the end of May). There was no significant treatment

effect on cladoceran biomass (Table 1). Cladoceran

maxima, composed mainly of B. longirostris and

C. sphaericus, occurred in July in the high-nutrient

treatments. S. vetulus contributed significantly to the

cladoceran biomass in the low-nutrient A2 scenario

treatment in July. In the low-nutrient mesocosms at

ambient temperature, much lower cladoceran maxima

were reached in June.

In the low-nutrient mesocosms at all temperature

levels, copepod nauplii abundance increased from May

to June, followed by a decrease to low biomasses in

August. In contrast, nauplii biomass gradually increased

throughout the season up to 48.9 lg l-1 in the heated

high-nutrient mesocosms. We found a highly significant

effect of time and nutrient interaction on copepodites

and adult copepod biomass (Table 1). With the excep-

tion of the A2 ? 50% scenario treatment, copepods

were more abundant in the low-nutrient mesocosms. All

advanced stages of copepods were missing in the

unheated high-nutrient treatment (Fig. 4).

The zooplankton to phytoplankton biomass ratio

was generally much lower in the high than in the low-

nutrient treatments (Table 1). The highest ratio in the

high-nutrient treatments (0.22) was recorded at the

beginning of the season (Fig. 4). The cladoceran to

phytoplankton and copepods to phytoplankton bio-

mass ratios decreased with nutrients, while the rotifer

to phytoplankton biomass ratio revealed no nutrient

effect but a significant but a temperature treatment

interaction with time (Table 1).

Zooplankton size structure

Total zooplankton and average cladoceran body size

were significantly lower in the high- than in the low-

nutrient mesocosms (Table 1, repeated measures

ANOVA: P = 0.01; F = 8.13; df = 1 and

P \ 0.001; F = 33.0; df = 1, respectively). Cladoc-

eran body size did not change during the study period in

the low-nutrient mesocosms, while a decrease was

observed in the high-nutrient mesocosms in June

(Fig. 5). Time-nutrient level interaction was revealed

for average cladoceran body size and B. longirostris

(repeated measures ANOVA: P = 0.001; F = 5.14;

df = 4 and P = 0.04; F = 2.6; df = 4; Fig. 5).

Zooplankton size diversity was positively related to

average zooplankton body size and was significantly

higher in the low- than in the high-nutrient treatments

(Table 1; Fig. 6). Size diversity also changed with

time, being highest in May and tended to decrease in

summer (Table 1; Fig. 6). However, no temperature

treatment effect was found.

Ordination

Principal coordinates analysis produced a model

where the distances of the high-nutrient treatments at

ambient temperature were smaller than for the A2 and

A2 ? 50% scenarios (total variation 40.3%; Fig. 7).

Although the temperature treatments at the high-

nutrient level overlapped, the highest variability

occurred in the A2 ? 50% scenario.

Multivariate dispersion as a measure of variability

within and between treatments revealed a clear

temperature effect on zooplankton community struc-

ture. The low-nutrient mesocosms had similar disper-

sion at the different temperature levels, whereas

dispersion in the high-nutrient treatments differed

markedly. The higher the temperature, the greater

were the multivariate dispersion and heterogeneity of

the zooplankton communities (Fig. 8).

Distance-based redundancy analysis constrained by

treatment effects, chlorophyll-a concentration, macro-

phyte coverage (PVI) and turbidity revealed nutrient

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Fig. 4 Mean biomass

(±SE) of rotifers,

cladocerans and copepods,

the nauplii ratio and the

zooplankton:phytoplankton

biomass ratio from May to

August 2010. Left low-

nutrient treatments, right

high-nutrient treatments.

Columns represent ambient

temperature (empty bars),

the A2 scenario (hatched

bars) and the A2 ? 50%

scenario (crosshatched

bars)

194 Hydrobiologia (2015) 742:185–203

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treatment and turbidity to be the most important

explanatory variables (Fig. 9). The results of the

Monte Carlo permutation test of significance of all

canonical axes of the proposed model were highly

significant (total variation = 40.4%; F = 5.4;

P \ 0.01). The first canonical axis, explaining 16.1%

of the variability, correlated with nutrients. Chloro-

phyll-a concentration and turbidity were closely

correlated with the first axis. Macrophyte coverage

was negatively correlated with both chlorophyll-a and

turbidity. Although the temperature treatments in the

db-RDA following the second axis explained only

2.5% of the variance, the effect was still significant

(P = 0.035 for the A2 ? 50% scenario).

Permutational multivariate analysis of variance

(PERMANOVA) based on zooplankton biomass data

Table 1 Results of two-way repeated measures ANOVA testing the effect of treatment (temperature = W; nutrients = N) and time

(T) on zooplankton biomass, average body size, number of taxa (richness), the Shannon–Wiener diversity index (S–W) and evenness

Warming (W) Nutrients (N) Time (T) W 9 N W 9 T N 9 T W 9 N 9 T

Total phosphorus ***

Total nitrogen *** *

Chlorophyll-a *** **

Turbidity *** ** **

PVI *** *** **

Rotifera

Dry weight *** *

Richness **

S–W **

Evenness *

Cladocera

Dry weight

Average body size *** ** ***

Richness **

S–W ***

Evenness * **

Nauplii

Dry weight **

Copepodites and adult copepods

Dry weight ** **

Copepoda (all stages)

Dry weight **

Total zooplankton

Dry weight ** *

Average body size **

Richness ** **

S–W * *

Evenness *

Zooplankton size diversity *** **

Rotifera:Phytoplankton ** *

Cladocera:Phytoplankton *** * *

Copepoda:Phytoplankton *** ** ***

Zooplankton:Phytoplankton *** * **

‘9’ = interaction of two factors. Ratios are based on carbon biomass. Statistical significance is marked as follows: * P \ 0.05;

** P \ 0.01; *** P \ 0.001

Hydrobiologia (2015) 742:185–203 195

123

Fig. 5 Average body size (mean ± SE) of cladocerans, cope-

pods (copepodites and adults), Bosmina longirostris and

Chydorus sphaericus. Left low-nutrient treatments, right high-

nutrient treatments. Columns represent ambient temperature

(empty bars), the A2 scenario (hatched bars) and the A2 ? 50%

scenario (crosshatched bars)

196 Hydrobiologia (2015) 742:185–203

123

revealed a significant effect of both time (df = 4;

F = 2.59; P \ 0.001) and treatment (df = 5;

F = 3.61; P \ 0.001) when temperature and nutrient

input were considered as a single factor; no significant

multivariate interactions were traced (Table 2). In

contrast, at high-nutrient levels time and temperature

were the only significant factors, demonstrating the

importance of seasonal change and temperature treat-

ment for the heterogeneity of the community. Heter-

ogeneity in the zooplankton community did not differ

between temperature treatments for the low-nutrient

mesocosms where only time or seasonal variation was

important (df = 4; F = 1.44; P = 0.05; Table 2).

The lower degree of variability in the low-nutrient

treatments is also reflected in the distance-based RDA

analysis (Fig. 9). The temperature effect was more

pronounced in the high-nutrient treatments where

samples were arranged according to the temperature

level from reference to A2 ? 50% as well as to

centroids representing the treatment (cf. Table 2).

Discussion

Our results confirm a set of well-known effects of

increased nutrient levels in shallow lakes on zoo-

plankton biomass, size and community composition

(Jeppesen et al., 2000; Hansson et al., 2004). We found

a higher phytoplankton biomass and a concomitant

higher zooplankton biomass in the high-nutrient

mesocosms. Moreover, the zooplankton consisted of

fewer and smaller-sized taxa and, accordingly, the

mean zooplankton body length was significantly lower

in the high-nutrient mesocosms and decreased during

Fig. 6 Size diversity and average body size (mean ± SE) of

zooplankton across examined taxonomical groups. Left low-

nutrient treatments, right high-nutrient treatments. Columns

represent ambient temperature (empty bars), the A2 scenario

(hatched bars) and the A2 ? 50% scenario (crosshatched bars)

Hydrobiologia (2015) 742:185–203 197

123

summer. This is consistent with the higher fish

biomass in these mesocosms and appearance of

YOY fish in summer exerting a higher predation on

zooplankton. Furthermore, the distance-based redun-

dancy analysis clearly distinguished the low-nutrient

mesocosms with clear water and high macrophyte PVI

from the turbid high-nutrient mesocosms with high

chlorophyll-a concentrations (Fig. 9; Moss, 1990;

Scheffer et al., 1993).

The zooplankton to phytoplankton biomass ratio

was low (usually below 0.2) in all the high-nutrient

mesocosms (cf. Fig. 4), which can be attributed to

high fish predation (Hessen et al., 2003; Jeppesen

et al., 2012). The high proportions of nauplii recorded

in the high-nutrient mesocosms provide further evi-

dence of size selective predation (Lehtiniemi et al.,

2007). In contrast, the higher zooplankton to phyto-

plankton biomass ratio in the low-nutrient mesocosms

indicated a more efficient grazing pressure on phyto-

plankton and a lower predation pressure by fish, as

expected from the lower fish density.

We found only a modest effect of warming. In the

high-nutrient mesocosms, stickleback density differed

among the temperature treatments. The lowest relative

densities of adult fish and the highest density of YOY

were observed at ambient temperature (Fig. 2). The

lowest density of YOY fish occurred in the A2

scenario despite high abundance of adult fish. Moran

et al. (2010) experimentally demonstrated a negative

effect of warming on three-spined stickleback survival

and reproduction, with a 60% reduction of stickleback

biomass at a temperature increase of 4�C. Moreover,

in their experiment the combined effect of heating and

enhanced nutrient loading led to a further stickleback

population decline, attributed to indirect effects of

warming such as low oxygen concentrations. We also

observed aggregation of fish near the surface, which

suggests a negative effect of the periodically measured

low oxygen concentrations (authors’ unpublished

data) on sticklebacks. The facts that the lowest adult

sticklebacks CPUE and the highest YOY recruitment

occurred in the unheated mesocosms suggest that

stickleback recruitment declined at the higher tem-

peratures. Correspondingly, Mehlis & Bakker (2014)

found that although fewer sperms fertilised more eggs

at higher temperature (25�C), the overall percentage of

motile sperms significantly decreased.

Despite the differences in fish abundance and size

between the temperature treatments at high nutrient

concentrations, the univariate statistics revealed no

direct temperature effect on any of the zooplankton

variables studied (Table 1). By contrast, Strecker et al.

(2004) found that warming suppressed zooplankton

biomass, reflecting higher dominance of rotifers at the

expense of large-bodied cladocerans. A temperature

effect was, however, identified by multivariate

analyses in our study (Figs. 7, 8, 9). The high-nutrient

mesocosms showed an increase in zooplankton bio-

mass heterogeneity with rising temperatures as the

multivariate dispersion and heterogeneity in zoo-

plankton communities increased at higher tempera-

tures. Higher variability in community composition

with temperature was confirmed by distance-based

RDA (Fig. 9). The greater distances and hence higher

variability among temperature treatments in the high-

nutrient mesocosms could be attributable to variation

in fish density. Accordingly, multivariate dispersion

was more similar in the low-nutrient mesocosms that

had an equal number of sticklebacks (1 male in each

mesocosm). Alternatively, the change in seasonality,

especially a shift in the beginning of the season, could

also have contributed to the increase in heterogeneity

Fig. 7 Principal coordinate analysis plotting the distances

between the high-nutrient treatments using Bray–Curtis dissim-

ilarity. Although the produced model was calculated using both

low- and high-nutrient treatments, the final plot shows only high

nutrient centroids. For better orientation, samples belonging to

the same treatment are enclosed by envelopes. Empty triangles

(and full line) represent reference temperature, grey (and dotted

line) represents the A2 scenario and black (and dashed line) the

A2 ? 50% scenario

198 Hydrobiologia (2015) 742:185–203

123

of zooplankton assemblages with increases in temper-

ature. However, we found no evidence of seasonality

changes between nutrient and temperature treatments.

Copepods peaked earlier in the heated low-nutrient

mesocosms, but no differences were evident for

rotifers and cladocerans.

The metabolic theory hypothesis (MTH) predicts

that size should decrease with increasing temperature

(Brown et al., 2004). However, we found higher

stickleback fitness at ambient temperature at which

they produced more offspring, leading to lower

average size than in the warmer mesocosms. In our

experiment, cladoceran body size, moreover,

decreased in all high-nutrient mesocosms regardless

of temperature. Later in the season, slightly larger

individuals of B. longirostris, one of the most impor-

tant cladoceran species, occurred in the A2 scenario,

coinciding with a relatively lower abundance of YOY

fish. Higher zooplankton density and an overall smaller

average size of zooplankton, particularly that of

cladocerans, resulted in lower zooplankton size diver-

sity in the high-nutrient treatments. Zooplankton size

diversity in the high-nutrient mesocosms showed a

consistent, albeit insignificant, pattern during the

season, with the lowest size diversity occurring at

ambient temperature where fish predation was highest

(cf. Figs. 2, 6). Our results, therefore, indicate that

zooplankton body size structure responds more

strongly to indirect effects of warming (e.g. higher

fish predation) than to temperature per se; in our

experiment this meant that the smallest fish and the

lowest zooplankton size diversity were recorded at

ambient temperature and not in the heated mesocosms.

Latitude gradient studies have shown a decrease in

body size of cladocerans (e.g. Gillooly & Dodson,

2000), agreeing with the MTH. However, Iglesias et al.

(2011) found that large-bodied Daphnia became

dominant after fish kills in a subtropical Uruguayan

Fig. 8 Multivariate dispersion of the zooplankton community

based on biomass. Left low-nutrient treatments, right high-

nutrient treatments. Columns represent ambient temperature

(empty bars), the A2 scenario (hatched bars) and the A2 ? 50%

scenario (crosshatched bars)

Hydrobiologia (2015) 742:185–203 199

123

lake and in experiments ran at low fish abundances

despite high water temperatures in both cases, sug-

gesting that predation and not temperature is of key

importance for the size change of zooplankton along

the latitude (climate) gradient. Similarly, a cross-

comparison of zooplankton size structure in compara-

ble Mediterranean and temperate lakes (Brucet et al.,

2010) showed that temperature per se did not explain

the low abundances of large-sized zooplankton in the

warm lakes, since low abundances were also found in

the cold lakes with high fish densities. The observed

and marked decline in the zooplankton to phytoplank-

ton biomass ratio from high to low latitudes (Gyllstrom

et al., 2005; Havens & Beaver, 2011; Meerhoff et al.,

2012) is also considered a clear indicator of high fish

predation (Jeppesen et al., 2004).

Although a synergistic effect of temperatures and

nutrient concentrations is now widely accepted (Je-

ppesen et al., 2010; Moss et al., 2011; Kosten et al.,

2012), our study revealed no interactive effects of

temperature and nutrients on zooplankton with the

exception of the rotifer-to-phytoplankton biomass

ratio, which was higher in low- than in high-nutrient

mesocosms in the A2 ? 50% scenario. In contrast,

microbial communities studied by Ozen et al. (2013)

were significantly affected synergistically by

enhanced temperature and nutrient levels. The lack

of synergistic effects in our study may be a scale issue.

While mesocosm experiments are useful for studying

mechanisms, they suffer somehow from realism (e.g.

Jeppesen et al., 2014). Due to the small scale, we only

included one small-bodied fish species (three-spined

sticklebacks) and no piscivores. In a warmer world, we

can expect higher dominance of benthic fish species

such as bream (Abramis brama) and various species of

carp in North European lakes (Jeppesen et al., 2010;

Moss et al., 2011), which feed on invertebrates, like

sticklebacks. But they also disturb the sediment during

feeding, resulting in high release of nutrients to the

water and consequently higher eutrophication. Our

experiment did not include such an effect.

To conclude, a positive effect of nutrients and fish

over temperature was observed on zooplankton bio-

mass, chlorophyll-a and turbidity. Zooplankton spe-

cies and size diversity and macrophytes decreased

with increasing nutrient loading. Our results indicate

an indirect temperature effect at the high nutrient

level, which can be attributed to temperature-depen-

dent higher variation in fish density and/or chloro-

phyll-a concentration, while no clear temperature

effects were found at low nutrient concentrations.

Fig. 9 Distance-based RDA plotting the distances between

samples using Bray–Curtis dissimilarity constrained by explan-

atory variables. Treatment effects (temperature and nutrients)

are shown as nominal variables (black diamonds), explanatory

variables (PVI, chlorophyll-a concentration and turbidity level)

as arrows. Low-nutrient treatments are shown as circles, high-

nutrient treatments as triangles. Empty circles and triangles

represent reference temperature, grey represents the A2 scenario

and black the A2 ? 50% scenario

Table 2 The simultaneous response of the zooplankton com-

munity to nutrients and warming based on PERMANOVA

results

Design Factor df F P

All treatments Time 4 2.59 0.0002

Treatmenta 5 3.61 0.0001

Time 9 treatment 20 1.12 0.16

High-nutrient

mesocosms

Time 4 1.44 0.06

Temperature 2 0.30 0.52

Time 9 temperature 8 2.05 0.0002

Low-nutrient

mesocosms

Time 4 1.44 0.05

Temperature 2 0.88 0.39

Time 9 temperature 8 1.05 0.36

The analyses were run using Bray–Curtis dissimilaritya The combination of temperature and nutrient effects

200 Hydrobiologia (2015) 742:185–203

123

Acknowledgments This study was supported by the REFRESH

project (Adaptive strategies to Mitigate the Impacts of Climate

Change on European Freshwater Ecosystems) funded under the 7th

EU Framework Programme, Theme 6, Contract No. 244121, the

MARS project (Managing Aquatic ecosystems and water Resources

under multiple Stress) funded under the 7th EU Framework

Programme, Theme 6 (Environment including Climate Change),

Contract No. 603378 (http://www.mars-project.eu), CLEAR2 (a

Villum Kann Centre of Excellence project), CIRCE and CRES. We

thank Anne Mette Poulsen for language improvements. SB’s con-

tribution was supported by Marie Curie Intra European Fellowship

No. 330249 (CLIMBING).

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