Hydrobiologia (2018) 816:231–241

https://doi.org/10.1007/s10750-018-3587-y

PRIMARY RESEARCH PAPER

Herbivory effects on the periphytic algal functional diversity

in lake ecosystems: an experimental approach
Bárbara Dunck . Diogo Castanho Amaral . Ubirajara Lima Fernandes .
Natália Fernanda Santana . Taise Miranda Lopes . Liliana Rodrigues

Received: 4 July 2017 / Revised: 6 March 2018 / Accepted: 9 March 2018 / Published online: 19 March 2018
Ó Springer International Publishing AG, part of Springer Nature 2018

Abstract Herbivory can determine the rates of
ecosystem functions and is key factor to periphytic
algae development. Here, we aimed to evaluate the
effects of herbivory of zooplankton and omnivorous
fish in periphytic algae of lake ecosystems. Based on
prediction of grazer:prey size ratio, we tested that
periphytic algal biomass and density will be lower in
the fish treatments. Based on prediction of the size-
selective predation, size-efficiency hypothesis, and
general allometric equations, and that both herbivores
Handling editor: Mariana Meerhoff
Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s10750-018-3587-y) con-
tains supplementary material, which is available to authorized
users.
will feed with distinct mode, we tested the hypothesis
that periphytic algal functional diversity will be higher
in the mixed treatment. There were four treatments
(Control, T1-zooplankton, T2-fish, T3-zooplankton?-
fish). We evaluated algae biomass, density, functional
traits, functional diversity, and the herbivory effect.
Our results show that the presence of both herbivores
were related to higher functional diversity, lower
density values, and prostrate periphytic algae, and in
T2 occurs higher periphytic algae biomass via nutrient
facilitation. Fish was responsible for the higher
consumer effect size, and was more important mod-
ifier than zooplankton of the periphytic algae com-
munity. The greater variation in producer species traits
was directly related to mixed foraging patterns of
herbivores together, which allow rare species to

B. Dunck B. Dunck (&)

Programa de Pós-graduação em Ecologia, Universidade Universidade Federal do Pará, Instituto de Ciências
Federal do Pará, Belém, PA, Brazil Biológicas, Rua Augusto Corrêa, 01, Guamá, Belém,
PA CEP 66075110, Brazil
D. C. Amaral  U. L. Fernandes  N. F. Santana  e-mail: dunck.barbara@gmail.com
T. M. Lopes  L. Rodrigues
Programa de Pós-graduação em Ecologia de Ambientes
Aquáticos Continentais, Maringá, PR, Brazil

L. Rodrigues
Laboratório de Algas Perifı́ticas, Departamento de
Biologia, Núcleo de Pesquisa em Limnologia, Ictiologia e
Aquicultura, Universidade Estadual de Maringá, Maringá,
Paraná, Brazil

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persist, decreasing the dominance of stronger com-
petitive species.
Keywords Benthic algae  Fish  Moenkhausia 
Traits  Trophic web  Zooplankton
Introduction
Determining the variation in the magnitude and
direction of species interactions and its ecological
consequences through field experiments is critical to
understand the functioning of ecosystem. Herbivory is
species interactions generally defined as a consump-
tion by a heterotrophic organism (the herbivores or
grazers) of all or part of a living, autotrophic organism
(Begon et al., 1990). This interaction predator–prey
have potential to mediate effects that cascade up and
down trophic chains (Liess & Hillebrand, 2004;
Schmitz, 2008) and can determine the rates of
ecosystem functions such as production, decomposi-
tion, and nutrient cycling.
The individual-scale properties of herbivores as
feeding mode, predation risk avoidance, body size,
and feeding selectivity mediate herbivory (Duffy,
2002; Schmitz, 2008). In freshwater aquatic ecosys-
tem, fish and zooplankton (mainly scraper cladoceran)
are important herbivores of periphytic algal commu-
nity (Feminella & Halkins, 1995; Steinman, 1996).
Both have distinct behavior and feeding mode, and can
change the physiognomic and taxonomic structure of
periphytic algal community in freshwater ecosystem
(Feminella & Hawkins, 1995; Steinman, 1996).
Top-down and bottom-up are relevant controls on
periphytic algal community (Fox, 2004; Hillebrand &
Cardinale, 2004; Lang et al., 2011; Aguilera et al.,
2015; Rakowski & Cardinale, 2016). The most
important direct negative effect of grazers on peri-
phytic algae reveals that algal biomass almost always
declines in the presence of herbivores (Cattaneo &
Mousseau, 1995; Steinman, 1996; Liess & Hillebrand,
2004; Hillebrand, 2009; Siehoff et al., 2009). But there
are the indirect effects, such as keystone predation,
trophic cascades and habitat facilitation, which can in
some cases lead to an increase of algal biomass of
some or all periphytic algal species (Liess & Hille-
brand, 2004). This positive effect can be through
excretion of nutrients, removal of senescent cells, or
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Hydrobiologia (2018) 816:231–241
increased uptake of nutrients by the remaining cells
(McCormick & Stevenson, 1991; Kahlert & Baun-
sgaard, 1999). Some studies showed that the distinct
feeding behavior of herbivores and their size can alter
periphytic algal community structure and composition
(Cattaneo & Kalff, 1986; McCormick, 1991) through
the keystone predation, also at functional traits level,
as the algae growth strategies and size (DeNicola et al.,
1990; Steinman et al., 1992; Hillebrand et al., 2000).
Cattaneo & Kalff (1986) analyzed the effect of grazer
size manipulation on periphytic algae communities
and showed that when large grazers are not present (as
fish, macroinvertebrates, or snails) small grazers as
zooplankton can alter periphytic algae communities
more in composition and species size than biomass.
However, patterns of the effect of herbivory on
periphytic algal diversity and functional diversity are
still unclear.
Considering that fish and zooplankton have distinct
behavior and feeding mode, they consume the peri-
phytic algae in different ways. The size-selective
predation by fish (Brooks & Dodson, 1965) demon-
strated that, as large herbivores, they graze mainly
higher periphytic algae, as filamentous and large
diatom (Cattaneo & Kalff, 1986). So, the mechanism
underlined is, by consuming that, it will reduce
competition for space, allowing the slower growing
algae to grow. It is also expected that this pattern for
periphytic algae is based on the size-efficiency
hypothesis (Brooks & Dodson, 1965) and general
allometric equations (Peters, 1983). For these, as
demonstrated in limnetic plankton communities, when
large grazers are removed from a community, small
grazers increase leading to grazing pressure of smaller
algae and nutrient turnover (Cattaneo & Kalff, 1986).
So, in conditions that both herbivores are present, fish
would consume larger and superficial individuals with
similar traits in the periphytic matrix, and in counter-
part, zooplankton, would consume smaller and more
prostrate species in the matrix, and this could allow
weaker competitively species occupy new spaces and
niches, and thus making more diverse periphytic algae
in terms of traits (Cattaneo & Kalff, 1986; DeNicola
et al., 1990; Steinman et al., 1992; Hillebrand et al.,
2000).
In the context of functional diversity, understanding
the structure of species traits can provide greater
generalization in ecological research (McGill et al.,
2006) and greater predictive power in community-
Hydrobiologia (2018) 816:231–241
based studies (Webb et al., 2010). This is because
species have evolved over time in response to natural
environmental variation (Heino et al., 2013), and their
existence in habitats over the years is provided by their
functional traits. These functional traits are basic units
which can be either morphological, physiological, or
phenological measurable at the individual level (Vi-
olle et al., 2007) and used as proxies of adaptation
strategies that allow species competition in a given
habitat (Tapolczai et al., 2016).
The functional diversity of periphytic algae has
been evaluated under such factors, as water levels of
aquatic ecosystems in floodplain and limnological
characteristics of environments (Dunck et al.,
2013, 2016). Ecological guilds for this community
(Larson & Passy, 2012; Law et al., 2014; Lange et al.,
2016) and for diatoms (Passy, 2007; Berthon et al.,
2011; Rimet & Bouchez, 2011; Stenger-Kovács et al.,
2013; Leira et al., 2015; Algarte et al., 2016) have
gained more attention lately, in order to assist
biomonitoring studies. However, yet considering the
functional diversity, there are still no patterns regard-
ing the different pressures by herbivory (Tapolczai
et al., 2016). In this context, only Lange et al. (2011)
considered this factor in an experimental study to
assess ecological diatoms guilds.
Considering the unclear patterns of the herbivory
effect on periphytic algal community, mainly in the
functional diversity, and the robustness that the
functional approach can provide for ecological studies
(McGill et al., 2006; Webb et al., 2010; Heino et al.,
2013), this study aimed to evaluate the herbivory
effect in freshwater trophic webs composed by
periphytic algae, zooplankton, and omnivorous fish.
Specifically, we evaluated the structure and dynamics
of the periphytic algae and their functional traits in
microcosms with three distinct freshwater food webs.
In relation to control (treatment without herbi-
vores), we hypothesized first that periphytic algal
biomass and density will be lower in the presence of
only fish (Moenkhausia aff. sanctaefilomenae Stein-
dachner 1907; T2 treatment). Our prediction is based
on the grazer:prey size ratio which can result in large
patches of the periphyton removed or dislodged
(Hillebrand, 2009). We do not expect this pattern in
the combined presence of fish and zooplankton (mixed
treatment, T3), by enabling another food source (the
zooplankton), would decrease the direct consumption
on periphyton.
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The second hypothesis is that periphytic algal
functional diversity will be higher in the combined
presence of fish and zooplankton (mixed treatment,
T3). Our prediction is that in the mixed treatment (T3)
both herbivores groups will feed with distinct mode,
and increase the chance of non-edible species or
unpalatable to present high diversity (Duffy, 2002),
leading to high prey functional diversity.
Materials and methods
Experimental design
The experiment was developed in microcosms (aquar-
ium of 40 l) placed in an open area at the advanced
field study base of the Universidade Estadual de
Maringá (see Algarte et al., 2015 for a study area map).
The experimental design of the microcosms included
four treatments, each one with 3 replicates, resulting in
12 sampling units: Control (no herbivory), T1 (zoo-
plankton), T2 (fish), and T3 (zooplankton?fish).
Periphytic algae community First, inert glass slides
of microscopy were colonized by periphytic algae in a
lake, and then taken to the advanced field study base of
Estate University of Maringá, for organization of
microcosms. These glass slides (artificial substrates)
were placed in wooden support to colonize for 28 days
near to Eichhornia azurea K. banks present in the
coastal area of the lake Garças in the upper Paraná
River floodplain (228540 30.300 S, 538380 24.300 W;
228440 50.7600 S; 538150 11.1600 W). The mean depth of
the slides during the colonization ranged from 10 to
30 cm deep. After 28 days of colonization, sufficient
time for colonization and maturation of the periphyton
(Patrick et al., 1954; Vercellino & Bicudo, 2006), the
glass slides were removed from the lake and taken into
cooler with ice to be put posteriorly in the microcosms.
We use mature periphytic algal community because it
contains enough food to support the herbivores. The
microcosms were installed in open area in the field
study base.
Zooplankton We prepared 2 l of zooplankton mix
sampled by trawling in the coastal area near the
Eichhornia azurea banks in the limnetic zone of the
lake Garças. This mix was use in the treatments with
the presence of zooplankton (Rotifera, Cladocera,
Copepoda). The species and respective feeding type
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present in this mix are given in the supplementary
material (Table 1).
Fish Individuals of Moenkhausia aff. sanctaefilom-
enae Steindachner 1907 (Red-eye tetra) were sampled
by passive traps (plexiglass-type minnow traps, Dib-
ble & Pelicice, 2010; Cunha et al., 2012), and
acclimated in tanks (for a week) with air compressors.
The fish were fed (commercial fish feed) during
acclimatization.
Microcosms
In each microcosm, we put 35 L of the Parana River
water filtered by plankton net with 45 lm of opening
mesh as follows: Control Treatment (C): no herbi-
vores; Treatment T1: we added 350 mL of the
zooplankton mix; Treatment T2: we added 7 individ-
uals of Moenkhausia aff. sanctaefilomenae; Treatment
T3: we added 7 individuals of Moenkhausia aff.
sanctaefilomenae and 350 mL of the zooplankton mix.
The zooplankton quantification from each micro-
cosm was made from the minimum of 50 adult
individuals from each group (Rotifera, Cladocera,
Copepoda) counted in Sedgwick-Rafter chambers on
optical microscope in three subsequent sub-samples of
the same sample (Bottrel et al., 1976). Young cope-
pods were estimated separately from adults counting
at least 50 individuals too. The initial zooplankton
abundance in each microcosm was ± 20 ind L-1 and
± 23,000 ind L-1 in the end. These zooplankton and
fish densities in the microcosms were similar to those
of the ecosystem (see Simões et al., (2012) for
zooplankton, and Dias et al. (2017) for Moenkhausia
sanctaefilomenae). In the treatments, 67% of the
Cladocera species were scraper (Table 1, supplemen-
tary material).
Possible dead fish along the experiment were
collected and preserved in formaldehyde solution
4%, and new fish were replenished immediately in
each sampling unit. At the end of the experiment, all
the fishes were sampled and preserved.
We put styrofoam supports containing 21 colonized
glass slides on the surface of water in each microcosm.
We also put air compressors for aeration of the water.
We cover each microcosm with transparent tulle to
prevent entry of insects that could serve as food for
fish, and we create a shadow to control the temperature
of the microcosms. The microcosms were organized
randomly according to the treatment in an area in open
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Hydrobiologia (2018) 816:231–241
air in the field study base. In this way, the conditions of
climate and light were natural and this short experi-
ment lasted 7 days.
Samplings
We performed sampling one day after the mounting of
the experiment (day 1—time 1) and on the 7th day of
the experiment (time 2). In each sample, we take five
glass slides per sampling unit (quantitative analysis of
periphyton (1), qualitative analysis of the periphyton
(1), and periphyton chlorophyll-a (3)).
Abiotic variables
Limnological variables were measured in all sam-
plings with multiparameter Horiba U-50 (temperature
(°C), pH, conductivity (mS cm-1), dissolved oxygen
(mg L-1), and total dissolved solids (mg L-1). Water
samples were filtered through Whatman GF/F filters,
under low pressure (\ 0.5 atm) and stored at - 20°C
for later determination of total phosphorus (lg L-1)
and total nitrogen (lg L-1, Bergamin et al., 1978).
Analyses of environment variables were done follow-
ing Roberto et al. (2009). The light radiation was
estimated by radiometer Light Meter Li-250.
Quantification of periphytic algae
The periphytic material was removed from the glass
slides by a stainless steel blade wrapped in aluminum
foil and by jets of distilled water (Algarte et al., 2014).
The area colonized and scraped from the glass slides
was equal and standardized (33 cm2). The periphytic
material was removed from the substrate, fixed, and
preserved in 0.5% acetic acid lugol’s iodine solution
for further quantification.
We use the Utermöhl method (1958) to quantify the
algae, and the counting was conducted in random
fields on an inverted OlympusÒ CK2 microscope
according to two criteria, until at least 100 individuals
of the most common species (cells, colonies and
filaments) of each sample and a species–accumulation
curve were reached (Ferragut & Bicudo, 2012).
Chlorophyll-a values were estimated according to
Golterman et al. (1978) with 90% acetone extraction
and with the results expressed in lg cm-2.
The density of periphytic species was estimated
according to Ros (1979) with the results expressed as
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the number of individuals per area unit (ind cm-2). We
adopted the species classification system proposed by
Round (1971).
Functional traits
We examined three functional response traits (Violle
et al., 2007) of the periphytic algae distributed in 13
categories: life forms (non-motile unicellular, fila-
mentous, flagellate, or colonial), intensity of adher-
ence to substrate (firmly or loosely attached;
Sládecková & Sládecek, 1977), form of adherence
(mobile, entangled, prostrate, stalked, mucilage tube,
or heterotrichous-differentiated basal cell; Biggs et al.,
1998). These are the traits composing the functional
matrix. These selected functional response traits allow
the establishment of the periphytic algae on substrates,
and they were analyzed directly through the individ-
uals of each species.
Data analysis
Abiotic variables
Initial abiotic conditions between all the treatments
were tested by Anova (One-Way). A principal com-
ponent analysis (PCA) was performed only consider-
ing final time, and was applied to summarize
environmental variables (Legendre & Legendre,
1998) with variables previously log-transformed (ex-
cept pH) and standardized. Axis retention was eval-
uated under the Broken-Stick criterion (Jackson,
1993).
Periphytic algae community
To analyze algae community structure (composition
and density) between treatments, we performed
exploratory NMDS (non-metric multidimensional
scaling) based on a similarity matrix using the Bray–
Curtis coefficient (Bray & Curtis, 1957; Clarke, 1993)
on algae data. Species occurring in only one treatment
(all replicates) were excluded from this analysis. Data
were log-transformed and this analysis included 95
species.
The functional traits matrix was standardized and
transformed in a distance matrix through the mixed-
variables coefficient of distance proposed by Pavoine
235
et al. (2009). This distance matrix was transformed
into dendrogram by the unweighted pair-group
method with arithmetic mean (UPGMA). It provided
the calculation of the functional diversity through
mean pairwise distance (MPD), which considers the
density of species in the estimate. This measure is
considered basal (i.e., more sensitive to distant taxa)
for calculating the pairwise distance between each
species of a community (Webb, 2000).
In our sampling design, time 2 depends on time 1,
so we use a One-Way Anova Repeated measures to
test the hypothesis. In this analysis, the response
variables (biomass, density, and functional diversity)
are the between-subjects factor; the treatments (C, T1,
T2, and T3) are the between-factors, and the time of
the experiment is the within-factor. This analysis
allows us to analyze the Treatments*Time like a Two-
Way Anova. Density and biomass data were log-
transformed to meet the assumptions of normality and
homoscedasticity. If univariate between-subjects
effects were significant, pairwise comparisons with
Fisher’s LSD post hoc tests were performed for the
factors. We emphasize the final time results in
figures (Fig. 3).
To investigate the periphytic algal traits that show
the differences between grazing effects, we performed
an RLQ analysis (Dolédec et al., 1996). With this
analysis, we assess the relationship between the
abiotic variables of the microcosms and periphytic
algae functional traits.
The relative effect of herbivory on periphytic algae
was characterized using the log response ratio (LRR),
which is the natural log-transformed ratio of mean
algal biomass in ungrazed control relative to grazed
treatments (Hedges et al., 1999). Increasingly negative
values correspond to a greater fraction of algal
biomass consumed.
All of the analyses were performed using R
software (R Development Core Team, 2014). Pack-
ages ade4 (Chessel et al., 2014) and picante (Kembel
et al., 2010) were used to construct the distance
functional matrix and dendrogram; picante was used
to calculate the functional diversity through mean
pairwise distance, ade4 was used for the RLQ analysis,
and vegan (Oksanen et al., 2013) was used for the PCA
and Anova.
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Results

Abiotic variables

Initial abiotic conditions (temperature, pH, conduc-

tivity, dissolved oxygen, total dissolved solids, and
total nitrogen) did not have significant differences
among the treatments (p \ 0.05). Only total phospho-
rus were different between microcosms, with higher
values in T2 (F3;8 = 5.42, p = 0.02; Tukey MS =
0.00016, df = 8).
The PCA first two axes explained 90% of the total
variation in the limnological data. T2 and T3 were
characterized by higher values of nutrients, TN for T2, Fig. 2 Non-metrical multidimensional scaling plots (stress =
and TP and conductivity for T3 (Fig. 1). 0.17) showing different periphytic algae taxonomic composi-
tions between microcosms (A Time 1, B Time 2, C control
Periphytic algal community without herbivores, T1 only zooplankton as herbivore, T2 only
fish as herbivore, T3 mixed of zooplankton and fish)

We recorded a total of 195 species of periphytic algae.

The algae species present in the treatments are in the
supplementary material (Table 2). The result of the
NMDS (Fig. 2) ordination analysis (0.17 stress)
indicated similarity in the periphytic algae structure
in T2 and T3, but also indicated that T1 at final time
has some similarity to T2 and T3 (axis 2).
The mean densities differed between treatments
(F = 125.60, p \ 0.001), time (F = 150.25,
p \ 0.001), and with the interaction time*treatment
(F = 31.64, p \ 0.001). At time 1, the highest values
Fig. 1 Site scores derived from a principal component analysis
applied to the environmental variables in final time. Arrows
indicate the Pearson correlations between original variables and
ordination scores (C control without herbivores, T1 only
zooplankton as herbivore, T2 only fish as herbivore, T3 mixed
of zooplankton and fish)
of densities were found, respectively, in C, T1, T2, and
T3 (Fig. 3A). The lowest values occurred in T3 at all
the times (Fig. 3A). Biomass differs between times 1
and 2 (F = 7.64; p = 0.02) but they did not differ
between treatments (F = 2.67; p = 0.11) (Fig. 3B).
The averages were higher in T2 at time 2 (Fig. 3B).
The functional diversity (MPD) values differed
between times 1 and 2 (F = 29.72, p = 0.006) and
the interaction time*treatments was also significant
(F = 4.14; p = 0.001), but they did not differ between
treatments (F = 1.03, p = 0.42). All treatments at time
2 were higher than initial C, and the highest average
MPD occurred in T3 at time 2 (Fig. 3C).
The first two axes of the RLQ with eigenvalues of
0.05 and 0.02 for axes 1 and 2 accounted for 60% and
30% of the coinertia, respectively (Fig. 4). The first
axis was more correlated with total phosphorus and
conductivity (both negatively), and the second axis
was more correlated with temperature (negatively)
and total nitrogen (positively) (Fig. 4). For traits, the
first axis was correlated with prostrate form and
loosely attached species (both positively), and the
second axis was correlated with unicellular species
(negatively) (Fig. 4). Species firmly attached mainly
with prostrate form of adherence were correlated to
higher values of total phosphorus and conductivity;
stalked species were related to higher values of
temperature, and entangled species to higher values
of dissolved oxygen. Prostrate species prevailed in

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Fig. 3 Species density (A), species biomass (B), and functional bars indicate significant differences between treatments, C
diversity (C) of periphytic algae at final time in the treatments. control without herbivores, T1 only zooplankton as herbivore,
Bars are the means ± 1 SEM (SD) of all (Different letters above T2 only fish as herbivore, T3 mixed of zooplankton and fish)

Fig. 4 Ordination of RLQ scores of periphytic algae functional traits (A) and environmental variables (B) in the microcosms (TEM-
water temperature, pH, COND water conductivity, TP total phosphorus, TN total nitrogen, DO dissolved oxygen)

grazed communities, and stalked and entangled Discussion

species in ungrazed ones.
The higher consumer effect size occurred in T3 at Our results of short-term experiment show that the
time 1, and in T2 at time 2 (Table 1). herbivory effect was seen on all attributes of peri-
phytic algae. The direct effects of grazers reveal that
the mixed treatment was related to lower values of
Table 1 Consumer effect LRR periphytic algae density and higher values of func-
size (LRR: log response tional diversity, partially corroborating our hypothe-
ratio) in periphytic algae in Time 1
time 1 and time 2 in the sis. The higher values of biomass in the presence of
T1 - 0,0536 only the fish were considered as a positive indirect
microcosms (C: control, no
herbivores, T1: with T2 - 0,05554 effect of this grazer, and this result differed from the
zooplankton, T2: with fish, T3 - 0,11987 lower values expected for this treatment. On the other
T3: with fish and Time 2
zooplankton) hand, when no herbivores were present, we did not
T1 - 0,01258 found the classic pattern of algal biomass increased
T2 - 0,29562 and the temporal variation in biomass decreased
T3 - 0,03442 (Rakowski & Cardinale, 2016).
Bold indicates higher values

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The effect of the presence of fish was more evident
than zooplankton in periphytic algae composition and
biomass. They provided higher consumer effect size,
and were an important modifier of the spatial structure
of the periphytic algae community. But, they also
provided indirect effects leading to higher values of
biomass. This case of indirect effect is the most
common case of habitat facilitation by grazers through
nutrient regeneration (Liess & Hillebrand, 2004). The
nutrient values can be increased in the presence of
grazers, for increasing excreta or via nutrients cycling,
as suggested by some studies (DeAngelis, 1992;
Kupferberg, 1997). In this way, increasing nutrient
supply can improve the abiotic environment for algae,
leading to incorporate nutrients into periphytic algal
biomass. Many studies of water quality and impacts of
fish farming demonstrated increase of phosphorus and
nitrogen and reduction of dissolved oxygen (Borges
et al., 2010; Kaggwa et al., 2011; Sipaúba-Tavares
et al., 2014), as occurred in our microcosms. Lange
et al. (2011) also related the indirect effect of nutrients
over herbivory on benthic diatom communities.
The structure of periphytic algal community
between treatments also demonstrated that the pres-
ence of fish drove the similarity between the micro-
cosms. The greater similarity occurred between C and
T1 (no fish), and between T2 and T3 (both with fish).
Moenkhausia sanctaefilomenae is an omnivorous fish,
and their diet consists of allochthonous and auto-
chthonous items and debris (Tófoli et al., 2010). So,
although Moenkhausia sanctaefilomenae can also use
the zooplankton as feed (Tófoli et al., 2010), their
presence influenced the structure of algae and the
consumption was high. On the other hand, the lowest
density values in T3 may have been provided by the
presence of various types of herbivores in both
communities (fish and zooplankton) with differenti-
ated feeding mode.
As we expected, the mixed treatment with two
types of herbivores provided greater functional diver-
sity of periphytic algae. A possible explanation is that
the greater variation in producer species traits can
increase the chance of non-edible species or unpalat-
able to present high diversity (Duffy, 2002). And, the
differences in herbivore size (size-efficiency hypoth-
esis and general allometric equations), the size-
selective predation promoted by both herbivores, and
the more generalist feeding when both feed together
lead to a consumption of a wide variety of producers.
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Hydrobiologia (2018) 816:231–241
And this can allow rare species to persist, decreasing
the dominance of stronger competitive species, and
also allow higher diversity of distinct producer traits.
So, it is possible to say that in this treatment (T3)
occurred better use of available resources (producers)
by consumers. Thus, when both herbivores are
present, the dominance of single or few species was
clearly counteracted by the grazer presence (Liess &
Hillebrand, 2009), and the evenness of functional
traits increased in this condition.
In summary of the trait-based approach, it was
possible to identify the periphytic algae traits respon-
sible for differences between treatments. When we
consider trait structure, the effect of herbivory in the
treatments indicated that grazed communities are
simplified, as indicated by some studies (Poff &
Ward, 1995; Steinman, 1996). The differences were
mainly between prostrate species which prevailed in
grazed communities, and stalked and entangled
species in ungrazed ones. Studies have indicated that
stalked and entangled species are more susceptible of
removing from the biofilm by physical factors and
herbivores (Steinman, 1996; Dunck et al., 2016). In
communities with these species, new colonizers,
especially epiphytes, will find less available structure
to settle (Liess & Hillebrand, 2009). On the other
hand, an adaptive advantage of prostrate species is, to
be strategically positioned within the periphytic algal
matrix, that they can resist to herbivory (Burkholder,
1996; Steinman, 1996) and also persist in enriched
treatments by nutrients (Ferragut & Bicudo, 2010).
The removal of shading and epiphytes in the higher
layer of the periphytic algal matrix (as stalked and
entangled species) by herbivores in T3 is also an
indirect effect that grazers may have on the periphytic
algae (Kupferberg, 1997), a place where new coloniz-
ers can settle contributing to a greater diversity of
traits in this community.
Unlike the effect of fish on biomass in T2 treatment,
where specialist species in nutrient-enriched environ-
ment can dominate in biomass, in the presence of
zooplankton and fish, lower values of density
occurred. So, these results can suggest grazing–action
relationship by zooplankton. Both herbivores pre-
vented the development of the producers. Small
microzooplankton could have a strong grazing impact
on the small algae, and can be important food resource
for higher macrozooplankton, as the copepods (Bundy
et al., 2005; Agasild et al., 2007; Mahdy et al., 2014).
Hydrobiologia (2018) 816:231–241
The biofilm removal by different groups of zooplank-
ton can change interactions within the periphytic
matrix, and favor rare species by admitting more light,
nutrient recycling, and consequently increase distinct
species traits. On the other hand, fish can eat more
superficially on the periphytic matrix, since there is an
abundance of resources. And, all these factors together
can modify the architecture of the matrix, and reduce
the dominance of competitively stronger species.
Based on scarcity of studies that compare the effect
of zooplankton in the periphytic algae and in the
prominent fish effect over zooplankton effect, in our
study, we suggest some points that should be consid-
ered in future studies. To support the results of grazing
by zooplankton on periphytic algae, and to be able to
separate it from indirect effects, we indicate that
studies should also consider to supplement with
stable isotope approaches or zooplankton gut content
to confirm evidence of direct consumption and to
confirm the causality of the mechanisms analyzed. The
direct effect of the entire community of zooplankton is
much more complex, and this could act as trammels
for studies of this nature. Perhaps because of this,
studies have evaluated the effect of single species of
zooplankton on periphytic algae (Siehoff et al., 2009;
Mahdy et al., 2014; Rakowski & Cardinale, 2016).
Conclusion
In summary, our results imply that multifactor exper-
iments are required to determine the main forces
driving the functional diversity of periphytic algae.
The indirect effects of grazers were not marginal in
this study, and might contribute to structuring func-
tional traits of this community in freshwater ecosys-
tem. The presence of both herbivores was related to
higher functional diversity, lower density values, and
prostrate algae species, and in the presence only of the
fish as herbivore higher periphytic algae biomass was
the pattern via nutrient facilitation. Our study thus
highlights the importance of considering functional
roles and identity of generalist consumers on spatial
structure of the entire landscape.
Acknowledgements We would like to thank two anonymous
reviewers for excellent comments that substantially improved
this paper. We thank the Coordination for the Improvement of
Higher Education Personnel (CAPES) for granting B.Dunck
with post-doctoral scholarship and D.C.Amaral, U.L.Fernandes,
239
T.M.Lopes and N.Santana with doctoral scholarships; the
National Council for Scientific and Technological
Development (CNPq) for granting L.Rodrigues with
productivity support; the Long-Term Ecological Research
(CNPq-PELD-Brazil) and the Research Center in Limnology,
Ichthyology and Aquaculture (Nupélia) for technical and
logistical support during the conduction of this study; and the
Laboratory of Zooplankton and Ecology of fish for scientific
support.
References
Agasild, H., P. Zingel, I. Tonno, J. Haberman & T. Noges, 2007.
Contribution of different zooplankton groups in grazing on
phytoplankton in shallow eutrophic Lake Vortsjaev (Es-
tonia). Hydrobiologia 584: 167–177.
Aguilera, M. A., N. Valdivia & B. R. Broitman, 2015. Herbi-
vore-alga interaction strength influences spatial hetero-
geneity in a kelp-dominated intertidal community.
PlosOne. https://doi.org/10.1371/journal.pone.0137287.
Algarte, V. M., B. Dunck, A. Bichoff & L. Rodrigues, 2015.
First record of Pithophora oedogonia (Montagne) Wittrock
(Pithophoraceae) in the Upper Paraná River floodplain,
Brazil. Check List 11: 1722–1725.
Algarte, V. M., B. Dunck, J. A. Leandrini & L. Rodrigues, 2016.
Periphytic diatom ecological guilds in floodplain: ten years
after dam. Ecological Indicators 69: 407–414.
Algarte, V. M., L. Rodrigues, V. L. Landeiro, T. Siqueira & L.
M. Bini, 2014. Variance partitioning of deconstructed
periphyton communities: does the use of biological traits
matter? Hydrobiologia 722: 279–290.
Begon, M., J. L. Harper & C. R. Townsend, 1990. Ecology:
individuals, populations and communities. Blackwell,
Oxford.
Bergamin, H., B. F. Reis & E. A. G. Zagato, 1978. A new device
for improving sensitivity and stabilization in flow injection
analysis. Analytica Chimica Acta 97: 427–431.
Berthon, V., A. Bouchez & F. Rimet, 2011. Using diatom life
forms and ecological guilds to assess organic pollution and
trophic level in rivers: a case study of rivers in southeastern
France. Hydrobiologia 673: 59–271.
Biggs, B. J. F., R. J. Stevenson & R. L. Lowe, 1998. A habitat
matrix conceptual model for stream periphyton. Archiv fur
Hydrobiologie 143: 21–56.
Borges, P. A. F., S. Train, J. D. Dias & C. C. Bonecker, 2010.
Effects of fish farming on plankton structure in a Brazilian
tropical reservoir. Hydrobiologia 649: 279–291.
Bottrell, H. H., A. Duncan, Z. M. Gliwicz, E. Grygierek, A.
Herzig, A. Hillbricht-Ilkowska, H. Kurasawa, P. Larsson &
T. A. Weglenska, 1976. Review of some problems in
zooplankton production studies. Norwegian Journal of
Zoology 24: 419–456.
Bray, J. R. & J. T. Curtis, 1957. An ordination of the Upland
Forest Communities of Sourthern Wisconsin. Ecological
Monographs 27: 325–349.
Brooks, J. L. & S. I. Dodson, 1965. Predation, body size and
composition of plankton. Science 150: 28–35.
Bundy, M. H., H. A. Vanderploeg, P. J. Lavrentyev & P.
A. Kovalcilk, 2005. The importance of microzooplankton
123
240
versus phytoplankton to copepod populations during late
winter and early spring in Lake Michigan. Canadian
Journal of Fish and Aquatic Sciences 62: 2371–2385.
Burkholder, J. M., 1996. Interaction of benthic algae with their
substrata. In Stevenson, R. J., M. L. Bothwell & R. L. Lowe
(eds), Algal Ecology. Academic Press, San Diego:
253–298.
Cattaneo, A. & J. Kalff, 1986. The effect of grazer size
manipulation on periphyton communities. Oecologia 69:
612–617.
Cattaneo, A. & B. Mousseau, 1995. Empirical analysis of the
removal rate of periphyton by grazers. Oecologia 103:
249–254.
Chessel, D., A. B. Dufour & J. Thioulouse, 2014. The ade4
package-I- One-table methods. R News 4: 5–10 [available
on internet at https://www.r-project.org/doc/Rnews/bib/
Rnewsbib.html].
Clarke, K. R. & M. A. Ainsworth, 1993. Method of linking
multivariate community structure to environmental vari-
ables. Marine Ecology-Progress Series 92: 205–219.
Cunha, E. R., S. M. Thomaz, R. P. Mormul, E. G. Cafofo & A.
B. Bonaldo, 2012. Macrophyte structural complexity
influences spider assemblage attributes in wetlands. Wet-
lands 32: 369–377.
DeAngelis, D. L. 1992. Dynamics of nutrient cycling and food
webs. Chapman and Hall, London.
DeNicola, D. M., C. D. McIntire, G. A. Lamberti, S. V. Gregory
& L. R. Ashkenas, 1990. Temporal patterns of grazer-pe-
riphyton interactions in laboratory streams. –. Freshwater
Biology 23: 475–489.
Dias, R. M., J. C. G. Ortega, L. C. Gomes & A. A. Agostinho,
2017. Trophic relationships in fish assemblages of
Neotropical floodplain lakes: selectivity and feeding
overlap mediated by food availability. Iheringia Série
Zoologia 107(e2017035): 1–11.
Dibble, E. D. & F. M. Pelicice, 2010. Influence of aquatic plant-
specific habitat on an assemblage of small neotropical
floodplain fishes. Ecology of freshwater fish 19(3):
381–389.
Doledec, S., D. Chessel, C. F. J. Ter Braak & S. Champley,
1996. Matching species traits to environmental variables: a
new three-table ordination method. Environmental and
Ecological Statistics 3: 143–166.
Duffy, J. E., 2002. Biodiversity and ecosystem function: the
consumer connection. Oikos 99: 201–219.
Dunck, B., J. C. Bortolini, L. C. Rodrigues, S. Jati, S. Train & L.
Rodrigues, 2013. Flood pulse drives functional diversity
and adaptative strategies of planktonic and periphytic algae
in isolated tropical floodplain lake (Brazil). Brazilian
Journal of Botany 36: 257–266.
Dunck, B., V. M. Algarte, M. V. Cianciaruso & L. Rodrigues,
2016. Functional diversity and trait–environment rela-
tionships of periphytic algae in subtropical floodplain
lakes. Ecological Indicators 67: 257–266.
Feminella, J. W. & C. P. Hawkins, 1995. Interactions between
stream herbivores and periphyton: a quantitative analysis
of past experiments. Journal of the North American Ben-
thological Society 14: 465–509.
Ferragut, C. & D. C. Bicudo, 2010. Periphytic algal community
adaptive strategies in N and P enriched experiments in a
123
Hydrobiologia (2018) 816:231–241
tropical oligotrophic reservoir. Hydrobiologia 646:
295–309.
Ferragut, C. & D. C. Bicudo, 2012. Effect of N and P enrichment
on periphytic algal community succession in a tropical
oligotrophic reservoir. Limnology 13: 131–141.
Fox, J. W., 2004. Effects of algal and herbivore diversity on the
partitioning of biomass within and among trophic levels.
Ecology 85: 549–559.
Golterman, H. L., R. S. Clymo & M. A. M. Ohmstad, 1978.
Methods for Physical and Chemical Analysis of Fresh
Waters. Blackwell, Oxford.
Hedges, L. V., J. Gurevitch & P. S. Curtis, 1999. The meta-
analysis of response ratios in experimental ecology. Ecol-
ogy 80: 1150–1156.
Heino, J., D. Schmera & T. Erós, 2013. Macroecological per-
spective of trait patterns in stream communities. Fresh-
water Biology 58: 1539–1555.
Hillebrand, H., 2009. Meta-analysis of grazer control of peri-
phyton biomass across aquatic ecosystems. Journal of
Phycology 45: 798–806.
Hillebrand, H. & B. Cardinale, 2004. Consumer effects decline
with prey diversity. Ecology Letters 7: 192–201.
Hillebrand, H., B. Worm & H. K. Lotze, 2000. Marine
microbenthic community structure regulated by nitrogen
loading and grazing pressure. Marine Ecology Progress
Series 204: 27–38.
Jackson, D. A., 1993. Stoping rules in principal components
analysis: a comparison of heuristical and statistical
approaches. Ecology 74: 2204–2214.
Kaggwa, M. N., D. M. Liti & M. Schagerl, 2011. Small tropical
reservoirs and fish cage culture: a pilot study conducted in
Machakos district, Kenya. Aquaculture International 18:
839–853.
Kahlert, M. & M. T. Baunsgaard, 1999. Nutrient recycling-a
strategy of a grazer community to overcome nutrient lim-
itation. Journal of the North American Benthological
Society 18: 363–369.
Kembel, S. W., P. D. Cowan, M. R. Helmus, W. K. Cornwell, H.
Morlon, D. D. Ackerly, S. P. Blomberg & C. O. Webb,
2010. Picante: r tools for integrating phylogenies and
ecology. Bioinformatics 26: 1463–1464.
Kupferberg, S., 1997. Facilitation of periphyton production by
tadpole grazing: functional differences between species.
Freshwater Biology 37: 427–439.
Lange, K., C. R. Townsend & C. D. Matthaei, 2016. A trait-
based framework for stream algal communities. Ecology
and Evolution 6: 23–36.
Lange, K., A. Liess, J. J. Piggott, C. R. Townsend & C.
D. Matthaei, 2011. Light, nutrients and grazing interact to
determine stream diatom community composition and
functional group structure. Freshwater Biology 56:
264–278.
Larson, C. A. & S. I. Passy, 2012. Taxonomic and functional
composition of the algal benthos exhibits similar succes-
sional trends in response to nutrient supply and current
velocity. FEMS Microbiology Ecology 80: 352–362.
Law, R. J., J. A. Elliott & S. J. Thackeray, 2014. Do functional or
morphological classifications explain stream phytobenthic
community assemblages? Diatom Research. https://doi.
org/10.1080/0269249X.2014.889037.
Hydrobiologia (2018) 816:231–241
Legendre, P. & L. Legendre, 1998. Numerical Ecology, 2nd ed.
Elsevier, Amsterdam.
Leira, M., M. L. Filippi & M. Cantonati, 2015. Diatom com-
munity response to extreme water-level fluctuations in two
Alpine lakes: a core case study. Journal of Paleolimnology
3: 289–307.
Liess, A. & H. Hillebrand, 2004. Direct and indirect effects in
herbivore - periphyton interactions. Archiv fur Hydrobi-
ologie 159: 433–453.
Mahdy, A., U. Scharfenberger, R. Andrian & S. Hilt, 2014.
Experimental comparison of periphyton removal by chi-
ronomid larvae and Daphnia magna. Inland Waters 5:
81–88.
McCormick, P. V., 1991. Lotic protistan herbivore selectivity
and its potential impact on benthic algal assemblages.
Journal of North America Benthological Society 10:
238–250.
McCormick, P. V. & R. J. Stevenson, 1991. Grazer control of
nutrient availability in the periphyton. Oecologia 86:
287–291.
Mcgill, B. J., B. J. Enquist, E. Weiher & M. Westoby, 2006.
Rebuilding community ecology from functional traits.
Trends in Ecology and Evolution 21: 178–185.
Oksanen, J., F. G. Blanchet, R. Kindt, P. Legendre, P.
R. Minchin, R. B. O’hara, G. L. Simpson, M. P Solymos, H.
H. Stevens, & H. Wagner, 2013. Vegan: community
ecology package. R package version 20-9 2013. [available
on internet at https://cran.r-project.org/package=vegan].
Passy, S. I., 2007. Diatom ecological guilds display distinct and
predictable behavior along nutrient and disturbance gra-
dients in running waters. Aquatic Botany 86:171–178.
Patrick, R., M. H. Hohn & J. H. Wallace, 1954. A new method
for determining the pattern of the diatom flora. Notulae
Naturae 259: 1–9.
Pavoine, S., J. Vallet, A. B. Dufour, S. Gachet & H. Daniel,
2009. On the challenge of treating various types of vari-
ables: application for improving the measurement of
functional diversity. Oikos 118: 391–402.
Peters, R. H., 1983. The ecological implications of body size.
Cambridge University Press, Cambridge.
Poff, N. L. & J. V. Ward, 1995. Herbivory under different flow
regimes: a field experiment and test of a model with a
benthic stream insect. Oikos 72: 179–188.
R Development Core Team, (2014). R: A language and envi-
ronment for statistical computing. R Foundation for Sta-
tistical Computing, Vienna, [available on internet at https://
www.R-project.org].
Rakowski, C. & B. J. Cardinale, 2016. Herbivores control
effects of algal species richness on community biomass and
stability in a laboratory microcosm experiment. Oikos.
https://doi.org/10.1111/oik.03105.
Rimet, F. & A. Bouchez, 2011. Use of diatom life-forms and
ecological guilds to assess pesticide contamination in riv-
ers: lotic mesocosm approaches. Ecological Indicators 11:
489–499.
Roberto, M. C., N. F. Santana & S. M. Thomaz, 2009. Lim-
nology in the upper Paraná river floodplain: large-scale
spatial and temporal patterns, and the influence of reser-
voirs. Brazilian Journal of Biology 69: 717–725.
Ros, J., 1979. Práctica de Ecologia. Omega, Barcelona.
241
Round, F. E., 1971. The taxonomy of the Chlorophyta, 2.
Journal of the British Phycological Society 6: 235–264.
Schmitz, O. J., 2008. Herbivory from individuals to ecosystems.
Annual Review of Ecology, Evolution and Systematics 39:
133–152.
Siehoff, S., M. Hammers-Wirtz, T. Strauss & H. T. Ratte, 2009.
Periphyton as alternative food source for the filter-feeding
cladoceran Daphnia magna. Freshwater Biology 54:
15–23.
Simões, N. R., F. A. Lansac-Tôha, L. F. M. Velho & C. C. Bo-
necker, 2012. Intra and inter-annual structure of zoo-
plankton communities in floodplain lakes: a long-term
ecological research study. International Journal of Tropical
Biology 60: 1819–1836.
Sipaúba-Tavares, L. H., L. M. Seto & R. N. Millan, 2014.
Seasonal variation of biotic and abiotic parameters in
parallel neotropical fishponds. Brazilian Journal of Biology
74: 166–174.
Sládecková, A. & V. Sládecek, 1977. Periphyton as indicator of
the reservoir water quality II - pseudo-periphyton. Archiv
fur Hydrobiologie 19: 176–191.
Steinman, A. D., 1996. Effects of grazers on freshwater benthic
algae. In Stevenson, R. J., M. L. Bothwell & R. L. Lowe
(eds), Algal Ecology. Academic Press, San Diego:
341–373.
Steinman, A. D., P. J. Mulholland & W. R. Hill, 1992. Func-
tional responses associated with growth forms in stream
algae. Journal of the North American Benthological Soci-
ety 11: 229–243.
Stenger-Kovács, C., E. Lengyel, L. O. Crossetti, V. Vuveges &
J. Padisák, 2013. Diatom ecological guilds as indicators of
temporally changing stressors and disturbances in the small
Torna-stream, Hungary. Ecological Indicators 24:
138–147.
Tapolczai, K., A. Bouchez, C. Stenger-Kovács, J. Padisák & F.
Rimet, 2016. Trait-based ecological classifications for
benthic algae: review and perspectives. Hydrobiologia
776: 1–17.
Tófoli, R. M., N. S. Hahn, G. H. Z. Alves & G. C. Novakowski,
2010. Uso do alimento por duas espécies simpátricas de
Moenkhausia (Characiformes, Characidae) em um riacho
da Região Centro-Oeste do Brasil. Iheringia Série Zoo-
logica 100: 201–206.
Utermöhl, H., 1958. Zur Vervollkommnung der quantitativen
phytoplankton-methodic. Mitteilungen Internationale
Vereinigung für Theoretische und Angewandte Limnolo-
gie 9: 1–39.
Vercellino, I. S. & D. C. Bicudo, 2006. Sucessão da comunidade
de algas perifı́ticas em reservatório oligotrófico tropical
(São Paulo, Brasil): comparação entre perı́odo seco e
chuvoso. Revista Brasileira de Botânica 29: 363–377.
Violle, C., M. L. Navas, D. Vile, E. Kazakou, C. Fortunel, I.
Hummel & E. Garnier, 2007. Let the concept of trait be
functional. Oikos 116: 882–892.
Webb, C. O., 2000. Exploring the phylogenetic structure of
ecological communities: an example for rain forest trees.
American Naturalist 156: 145–155.
Webb, C. T., J. A. Hoeting, G. M. Ames, M. I. Pyne & N.
L. Poff, 2010. A structured and dynamic framework to
advance traits-based theory and prediction in ecology.
Ecology Letters 13: 267–283.
123