Download Ingredients for protist coexistence: competition, endosymbiosis and

Journal of Animal Ecology 2012, 81, 222–232
doi: 10.1111/j.1365-2656.2011.01894.x
Ingredients for protist coexistence: competition, endosymbiosis and a pinch of biochemical interactions
Johann P. Müller1,2*, Céline Hauzy3,4,5 and Florence D. Hulot1,6
1
UFR Sciences de la Vie, UPMC Univ Paris 06, Paris, France; 2Laboratoire Bioemco, Ecole Normale Supérieure, Paris,
France; 3IFM Theory and Modelling, Linköping University, Linköping, Sweden; 4Université Pierre et Marie Curie, Sorbonne
Universités, UMR 7625 Ecologie et Evolution, Paris, France; 5INRA, USC 2031 Ecologie des Populations et Communautés,
Paris, France; and 6Laboratoire d’Ecologie, Systématique & Evolution, UMR 8079, Univ Paris-Sud, Orsay, France
Summary
1. The interaction between mutualism, facilitation or interference and exploitation competition is
of major interest as it may govern species coexistence. However, the interplay of these mechanisms
has received little attention. This issue dates back to Gause, who experimentally explored competition using protists as a model [Gause, G.F. (1935) Vérifications expe´rimentales de la théorie mathe´matique de la lutte pour la vie. Actualités Scientifiques et Industrielles, 277]. He showed the
coexistence of Paramecium caudatum with a potentially allelopathic species, Paramecium bursaria.
2. Paramecium bursaria hosts the green algae Chlorella vulgaris. Therefore, P. bursaria may benefit from carbohydrates synthesised by the algae. Studying endosymbiosis with P. bursaria is possible as it can be freed of its endosymbiont. In addition, C. vulgaris is known to produce
allelochemicals, and P. bursaria may benefit also from allelopathic compounds.
3. We designed an experiment to separate the effects of resource exploitation, endosymbiosis and
allelopathy and to assess their relative importance for the coexistence of P. bursaria with a competitor that exploits the same resource, bacteria. The experiment was repeated with two competitors,
Colpidium striatum or Tetrahymena pyriformis.
4. Results show that the presence of the endosymbiont enables the coexistence of competitors,
while its loss leads to competitive exclusion. These results are in agreement with predictions based
on resource equilibrium density of monocultures (R*) supporting the idea that P. bursaria’s endosymbiont is a resource provider for its host. When P. bursaria and T. pyriformis coexist, the density of the latter shows large variation that match the effects of culture medium of P. bursaria. Our
experiment suggests these effects are because of biochemicals produced in P. bursaria culture.
5. Our results expose the hidden diversity of mechanisms that underlie competitive interactions.
They thus support Gauses’s speculation (1935) that allelopathic effects might have been involved
in his competition experiments. We discuss how a species engaged both in competition for a
resource and in costly interference such as allelopathy may counterbalance these costs with a
resource-provider endosymbiont.
Key-words: allelopathy, biochemicals, Chlorella
competition, Gause, Paramecium bursaria, R*rule
Introduction
Understanding species coexistence is a seminal issue in ecology. It is well known that the interplay of interspecific interactions such as competition and predation may allow species
to coexist (Chase et al. 2002; Holt 1984; Paine 1966). But
there is also a growing theoretical and experimental body of
evidence for the structural roles played by mutualism, facilitation or interference in interaction with exploitation compe*Correspondence author. E-mail: [email protected]
vulgaris,
endosymbiosis,
exploitation
tition (Amarasekare 2002; Bascompte & Jordano 2007;
Brooker et al. 2008; Gross 2008; Lankau & Strauss 2007;
Roy 2009; San Emeterio, Damgaard & Canals 2007; Vance
1984). The underlying question of the interplay of interspecific interactions is the conception of communities (or
ecosystems if abiotic components are taken into account).
Historically, the emphasis has been put on trophic links leading to food web as sufficient to characterise community or
ecosystem functioning (Goudard & Loreau 2008). Other
types of ecological interactions such as interference and
mutualism have been often ignored (Arditi, Michalski &
2011 The Authors. Journal of Animal Ecology 2011 British Ecological Society
Ingredients for protist coexistence 223
Hirzel 2005; Goudard & Loreau 2008). Recent theoretical
analyses have shown that communities and ecosystems properties may be strongly modified when non-trophic interactions are included in models. Arditi, Michalski & Hirzel
(2005) have shown that communities become ‘super-efficient’
with a certain proportion of mutualistic interactions. In a
comparison between interaction and food webs, Goudard &
Loreau (2008) showed that the addition of non-trophic interactions change several ecosystem properties such as the shape
of the positive relationship between diversity and biomass.
However, the interplay of mutualism, facilitation or interference interactions with exploitation competition has
received little attention in view of their potential effects on
community dynamics. Coexistence requires negative mutual
feedbacks on species abundances to limit interspecific effects
with regard to intraspecifics effects. However, mutualism
produces positive feedbacks that may prevent coexistence
(Schmitt & Holbrook 2003). Few studies explored how mutualism affects coexistence between competitors (Schmitt &
Holbrook 2003; Bever et al. 2010). As for interference, several theoretical studies showed that two species competing
for the same resource may coexist if interference limits them
(Amarasekare 2002; Case & Gilpin 1974; Vance 1984). Interference is in these cases an alternative strategy to increasing
competitive abilities (Case & Gilpin 1974). Theoretical models predict that interference is beneficial if the trade-off
between its cost and its effect is negative and the resource
overlap with the competitor that suffers from interference is
high (Case & Gilpin 1974). To ensure coexistence, costly
interference such as territoriality or allelopathy may be balanced with beneficial interference such as predation or parasitism that increases the agressor’s growth rate (Amarasekare
2002).
The aim of our work is to experimentally explore interactions between competition, interference and mutualism using
protists as model organisms. Laboratory experiments using
protists played a key role in the development of population
ecology starting with Gause (1935) and continue to play a
valuable role in bringing together theory, observations and
experiments (Holyoak & Lawler 2005). Indeed, community
ecology was kick-started by a set of laboratory experiments
conducted by Gause (Gause 1934, 1935; Holyoak & Lawler
2005). As stated by Gause (1935), the models proposed by
Volterra (1926) and Lotka (1932) were ‘based on observations of nature’ generating ‘conjectures’. Therefore, the aim
of Gause (1935) was ‘to provide a basis with several generations data in order to understand the reciprocal numerical
action of animal species living together’ (our translations
from French). In agreement with theoretical predictions, Gause showed competitive exclusion with Paramecium aurelia
and Paramecium caudatum (Gause 1935). Later, Gill (1972),
who reanalysed Gause’s classical competition experiments
with P. aurelia, suggested that interference, thanks to noxious endosymbionts, may play an important role in protozoan communities. In a second set of experiments, Gause
(1935) showed that Paramecium bursaria can coexist with
P. aurelia or P. caudatum. He had chosen species with a
priori different ecological niches, P. bursaria feeding predominantly on yeast cells and P. aurelia or P. caudatum on bacterial cells. However, he also conducted experiments with
different initial conditions leading to different final equilibria
characterised by P. bursaria dominance (Arthur 1987; Gause
1935), which is typical of allelopathic effects (Chao & Levin
1981; Durrett & Levin 1997; Hulot & Huisman 2004). Gause
(1935) concluded that these particular equilibria were
because of allelochemicals (‘metabolism’s products’) produced by P. bursaria.
In this respect, it may be significant that P. bursaria hosts
hundreds of cells of the green algae Chlorella vulgaris and
may benefit from carbohydrates synthesised by its endosymbiont presumably in exchange of protection (Karakashian
1963; Pado 1965; Weis 1969). Although the interaction
between P. bursaria and C. vulgaris looks mutualistic, it has
been suggested that P. bursaria is actually enslaved by its
partner (McPhearson 2004). We, therefore, use the neutral
term endosymbiosis (Douglas & Smith 1989). Chlorella vulgaris is known to produce allelochemicals that affect the
growth of other algal species (Inderjit & Dakshini 1994) and
may affect the dynamics of small communities (Hulot, Morin
& Loreau 2001). Hence, C. vulgaris may affect P. bursaria’s
interaction with other ciliates both as a resource provider and
as a producer of allelochemicals.
These findings support the hypothesis formulated by
Gause (1935) on the existence of allelopathic effects in his
experiments with P. bursaria but suggest also that the endosymbiont could have other roles in ciliate coexistence. Moreover, because the symbiosis of P. bursaria and C. vulgaris is
facultative, P. bursaria can be freed of its endosymbiont,
which allow studying the effect of this endosymbiosis on the
coexistence with other ciliates. Thus, P. bursaria is an interesting model to study the interplay between endosymbiosis,
competition and allelopathy. In the following, we will refer to
allelopathy as the biochemical interaction, whether stimulatory or inhibitory, among organisms (e.g. Folt & Goldman
1981). This definition generalises Molisch’s (1937) definition
of allelopathy that is restricted to primary producers (cited
by Gross 2003).
To explore under what conditions endosymbiosis and allelopathy may affect ciliate coexistence, we designed an experiment to separate the effects of resource exploitation,
allelopathy and endosymbiosis and to assess their relative
importance for the coexistence of P. bursaria with two ciliate
species Colpidium striatum and Tetrahymena pyriformis that
compete for the same resource, bacteria.
Materials and methods
STRAINS AND CULTURE
The three ciliate species, P. bursaria, C. striatum and T. pyriformis,
that were purchased from Carolina Biological Supply company
(Burlington, North Carolina, USA) are bacterivorous. Of these,
P. bursaria is the only species to be engaged in a symbiosis: one single
P. bursaria individual harbours several hundred green algal cells of
2011 The Authors. Journal of Animal Ecology 2011 British Ecological Society, Journal of Animal Ecology, 81, 222–232
224 J. P. Müller, C. Hauzy & F. D. Hulot
C. vulgaris. This endosymbiosis is facultative, and P. bursaria can
persist without hosting C. vulgaris (Siegel & Karakashian 1959 cited
by Karakashian 1963, 1963). To obtain algae-free paramecia, the herbicide Paraquat (N,N’-dimethyl-4,4¢-bipyridinium dichloride) was
added to P. bursaria cultures at a concentration of 10 lg mL)1 (Tanaka et al. 2002) and they were cultured at 20 C under controlled
light ⁄ dark cycle of 12.12 h. After 2 weeks, there remained some
C. vulgaris cells within P. bursaria individuals, but we considered that
P. bursaria was effectively free of their endosymbionts (See Fig. S1).
We monitored the chlorophyll-a concentration in cultures of endosymbiont-free P. bursaria during pilot experiments. Results showed
that this concentration remained very low (data not shown). During
all our experiments, including the one in which C. vulgaris was
removed from P. bursaria, ciliates were cultured in 50 mL Erlenmeyer flasks containing 15 mL of initially sterile medium
(0Æ75 mg L)1 of powdered Protozoan Pellet in Volvic water). This
medium provides resources for bacteria Serratia marcescens which is
consumed by ciliates. Serratia marcescens was the only bacterial species used in the experiments. But despite dilution because of successive
culture transplantations, we cannot exclude the presence of other bacterial species coming from purchased protist cultures. Cultures were
maintained at 20 C under controlled light ⁄ dark cycle of 12.12 h.
EXPERIMENTAL DESIGN
To investigate the importance of endosymbiosis, allelopathy and
exploitation competition for the coexistence of P. bursaria with competitors, either C. striatum or T. pyriformis, we conducted two experiments (one for each competitor) with the same experimental design.
It comprises seven treatments that were replicated four times: the
three species (P. bursaria, endosymbiont-free P. bursaria and a competitor) in monoculture; two competition treatments (P. bursaria or
endosymbiont-free P. bursaria with a competitor) and two allelopathy treatments where P. bursaria and its competitor were grown with
conditioned medium of the other species. We refer to a monoculture
of a given species that is filtered at 0Æ2 lm to eliminate all individuals
and particles and keep only dissolved compounds as a conditioned
medium of that species (Fig. 1).These were obtained from monocultures of all species, run in parallel to experimental cultures under
the same environmental conditions and with the same initial conditions. These parallel cultures are only a week older than the experimental treatments. Conditioned medium contains organic
compounds produced by the protists and bacteria individuals and
products of medium degradation. Because conditioned medium does
not separate biochemicals produced by the protists from other molecules present in the medium, we refer in the following to conditionedmedium treatment instead of allelopathy treatment.
Two days before inoculation with ciliates, bacteria S. marcescens
were added to the sterile medium in each vial. On day 0, we added to
each vial c. 50 cells mL)1 of each ciliate. From day 2, we sampled
1Æ5 mL of the experimental cultures every 2–3 days of which we used
0Æ1 mL for protist counting. The remaining 1Æ4 mL was filtered at
0Æ2 lm on cellulose acetate filters and kept available for dilution of
conditioned medium (Fig. 1). In the seven treatments, replacement
of the sampled 1Æ5 mL follows this design: 0Æ15 mL of fresh medium
with bacteria was added in each vial in addition to a volume of conditioned medium from parallel cultures diluted with the filtered remaining of the sample of the day to reach 1Æ35 mL (Fig. 1, Table 1). In
conditioned-medium treatments, media that were added came from
either the competitor or the P. bursaria parallel cultures and were
added to reach, in the replacement volume, the ciliate density in the
experimental treatment ‘monoculture’ which simulated the effects of
CMi
Pi
0·1 mL for
counting
Input
1·5 mL
Output
1·5 mL
1·4 mL
available
for dilution
Tj
0·15 mL fresh
medium
1·35 mL:
∑CMi
Fig. 1. Diagram of the experimental design. Pi, parallel culture of
species i; CMi, conditioned medium of species i; Tj, Treatments. i:
Paramecium bursaria, endosymbiont-free P. bursaria, Colpidium
striatum or Tetrahymena pyriformis. j: monocultures, polycultures
and conditioned-medium treatments. Thick grey bar: filtration at
0Æ2 lm. See text and Table 1 for explanations on the treatments and
the CMs added.
a potentially allelopathic species. To control for this addition of conditioned medium, monoculture and competition treatments also
received conditioned medium. The volume was calculated to reach,
in the replacement volume, the ciliate density in the ‘monoculture’
and ‘competition’ treatments (Table 1). As withdrawals represented
10% of the experimental cultures, the conditioned mediums were
added in experimental cultures in proportion of 1 : 10 of species densities. This experimental design induces an impoverishment of culture
mediums during the experiment and, as a consequence, population
equilibria with lower densities than expected without impoverishment, but it allows to add, on a regular basis, conditioned medium
and to separate biochemical effects from competition for resources.
For each sample, we counted individuals of each species present in
10 wells of 10 lL. The density in 100 lL was then extrapolated to
density in individuals per millilitre. When cells of endosymbiont-free
P. bursaria were counted, the colour and the presence of C. vulgaris
cells were carefully checked by microscopy to verify that C. vulgaris
density in its host remained very low.
BACTERIAL DENSITY MEASUREMENT AT EQUILIBRIUM
AND THE OUTCOME OF EXPLOITATION COMPETITION
Theoretical models of species competing for one unique resource predict the outcome of exploitation competition in the absence of any
other type of interaction. The species that has the lowest resource
density at equilibrium when growing alone will exclude its competitors (Grover 1997; Tilman 1982). In the absence of other types of
interactions, the resource density at equilibrium, often denoted R*,
does only depend on the consumer’s life-history parameters (Grover
1997; Tilman 1982). In addition, it does not take the presence of hosts
(virus, endosymbionts) into account. In our system, the R* rule gives
a prediction for the outcome of competition between endosymbiontfree P. bursaria and competitors in the absence of any other type of
interaction.
Paramecium bursaria hosts an endosymbiont C. vulgaris, which
may provide resources (organic carbon) to P. bursaria (Karakashian
2011 The Authors. Journal of Animal Ecology 2011 British Ecological Society, Journal of Animal Ecology, 81, 222–232
Ingredients for protist coexistence 225
Table 1. Source of conditioned medium (CM) added in the different treatments and equivalent density in the input. Equivalent density di refers
to the density of individuals in treatments before filtration
Treatments
Source of CM added
Equivalent density di in the input
Monoculture of species i
Competition of 2 species
Conditioned medium of Paramecium bursaria on the competitor
Conditioned medium of the competitor on P. bursaria
CM of species i
CM of the 2 species
CM of P. bursaria
CM of the competitor
Density of species i in monoculture
Density of the 2 species in competition
Density of P. bursaria in monoculture
Density of the competitor in monoculture
1963; Pado 1965; Weis 1969). Here, we will only consider the endosymbiont as an additional resource for P. bursaria (the potential
positive effect of P. bursaria on the endosymbiont will not been integrated explicitly). Resource competition theory (Grover 1997;
Tilman 1982) summarises also conditions of coexistence of two
competitors competing for two resources in the absence of any other
type of interaction. Coexistence is ensured if the competitor that is
the better exploiter of one resource is the worse exploiter for the other
(Phillips 1973) and if each competitor exploits proportionally more
of the resource limiting its own growth (León & Tumpson 1975). In
our system, C. vulgaris provides resources to P. bursaria that are not
exploited by the competitor for bacteria. As a consequence, theoretically, P. bursaria should persist whatever the ability of its competitor
to exploit the common resource, bacteria. The outcomes of competition range from coexistence, if the competitor has sufficient abilities
to exploit bacteria, to competitive exclusion of the competitor. These
predictions describe the expected outcome of exploitation competition between endosymbiont-carrying P. bursaria and the competitors
in the absence of any other type of interactions.
We assessed the ability to compete for bacteria of our ciliate species
(P. bursaria, endosymbiont-free P. bursaria, C. striatum and
T. pyriformis) by measuring in the treatment ‘monoculture’ the bacterial density when ciliates have reached their carrying capacity. To
measure bacterial density, we used the Di Aminido Phenyl lndol
method (Porter & Feig 1980) on 1 mL of monocultures sampled at
equilibrium in each replicate. In the experiment with C. striatum,
samplings were carried out on day 46 for P. bursaria and on day 29
for endosymbiont-free P. bursaria and C. striatum. The sampling
days are day 32 for P. bursaria and day 28 for endosymbiont-free
P. bursaria and T. pyriformis in the experiment with T. pyriformis.
DATA ANALYSIS
We used R (version 2.8.0) for all statistical analysis. We analysed the
effects of treatments on the dynamics of the competitor (C. striatum
or T. pyriformis) and P. bursaria. For the experiment with C. striatum, we conducted statistical analysis on subsets of the data only
because one or two treatments were missing after day 38 (For data
structure, see Fig. 2, Tables S2 and S3). We analysed species densities
across time using repeated measure anova, except for C. striatum
density at day 40. To perform repeated measure anova, we fitted a linear mixed effect model on species densities using the restricted maximum likelihood method. We tested the effect of time, treatments and
time · treatments using a linear mixed effect model where the random effect was the code attributed to each microcosm. For C. striatum density at day 40, we performed an anova using a linear model
fitted with generalised least square method. To take into account heteroscedasticity among treatments and over time and improve residuals’ normality, we constructed several models with different variance
structure. We selected the best model using the Akaike Information
Criterion (See Table S1). We tested the effect of time, treatments and
time · treatments on species density using F-tests.
When treatment effects or time · treatment interactions were significant, we investigated the effects of species interaction and medium, i.e. endosymbiosis, competition and conditioned medium, on
the dynamics of the competitor and of P. bursaria using a priori contrast analyses. We compared a priori pooled treatments using orthogonal contrasts. For the analysis of competitor density, the first
contrast tested the effect of competition, i.e. the competitor in monoculture or with P. bursaria conditioned medium vs. the competitor
with P. bursaria or endosymbiont-free P. bursaria. The second tested
the effect of conditioned medium on the competitor, i.e. the competitor in monoculture vs. the competitor with P. bursaria conditioned
medium. The third tested the effect of endosymbiosis on competitor
density, i.e. the competitor with P. bursaria vs. the competitor with
endosymbiont-free P. bursaria. For the analysis of P. bursaria density, the first contrast tested the effect of endosymbiosis, i.e. P. bursaria in monoculture or with competitor conditioned medium or with
the competitor vs. endosymbiont-free P. bursaria in monoculture or
with the competitor. The second contrast tested the effect of competition for P. bursaria, i.e. P. bursaria in monoculture or with competitor conditioned medium vs. P. bursaria with the competitor. The
third contrast tested the effect of competition for endosymbiont-free
P. bursaria, i.e. endosymbiont-free P. bursaria in monoculture vs.
endosymbiont-free P. bursaria with the competitor. The fourth contrast tested the effect of conditioned medium on P. bursaria, i.e.
P. bursaria in monoculture vs. P. bursaria with the competitor. For
data after day 38 of the experiment with C. striatum, we modified
these contrasts to take into account the absence of one or two treatments (See details in Tables S4 and S6).
The bacterial densities at equilibrium followed normality and
homoscedasticity assumptions. For each experiment (with C. striatum or with T. pyriformis), we performed one-way anova followed by
Tukey’s post hoc tests to compare the equilibrium bacterial densities
of P. bursaria, endosymbiont-free P. bursaria and their competitor.
Results
EXPERIMENT WITH COLPIDIUM STRIATUM AS A
COMPETITOR
Bacterial densities when ciliate monocultures are at their carrying capacity (R*) were significantly different (anova:
F2,9 = 94Æ1, P < 0Æ0001). Tukey’s post hoc tests showed that
the R* is significantly lower for C. striatum than for P. bursaria either with or without its endosymbiont (Table 2). It
should be noted that R* is significantly lower for P. bursaria
with endosymbionts than it is for P. bursaria without
(Table 2). As predicted by R* values, C. striatum did indeed
outcompete symbiont-free P. bursaria from day 38 (Fig. 2b).
Although bacterial densities at C. striatum carrying capacity
are significantly lower than that of P. bursaria, results show
2011 The Authors. Journal of Animal Ecology 2011 British Ecological Society, Journal of Animal Ecology, 81, 222–232
226 J. P. Müller, C. Hauzy & F. D. Hulot
Log [density (cells per mL)+1]
Competition: P. bursaria (
) and C. striatum (
)
(a)
(b)
3.0
3.0
2.5
2.5
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
) and
0.0
0.0
0
10
20
30
50
40
C. striatum: monoculture (
) and conditioned-medium
treatment (
)
(c)
Log [density (cells per mL)+1]
Competition: endosymbiont-free P. bursaria (
C. striatum (
)
0
3.0
2.5
2.5
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
20
30
40
50
Monoculture of P. bursaria (
) and endosymbiontfree P. bursaria (
) and conditioned-medium
treatment on P. bursaria (
)
(d)
3.0
10
0.0
0.0
0
10
20
30
40
Time (days)
0
10
20
30
40
50
Time (days)
Fig. 2. Ciliate dynamics in the experiment with Colpidium striatum [Mean ± SE in log (cell mL)1) + 1]. Treatments are represented as follows:
monocultures (—), competition (– –), and conditioned medium (...). (a) Competition between C. striatum (m) and Paramecium bursaria (d); (b)
Competition between C. striatum (4) and endosymbiont-free P. bursaria (s); (c) C. striatum monoculture ( ) and conditioned medium on
C. striatum (m); (d) P. bursaria (d) and endosymbiont-free P. bursaria (s) monocultures and conditioned medium on P. bursaria (d). (Colour
plates with all dynamics are in Fig. S2.)
Table 2. Equilibrium value of bacterial density for Paramecium
bursaria (Rp ), endosymbiont-free P. bursaria (Rf ) and their
competitor (Rc ) Colpidium striatum or Tetrahymena pyriformis.
Mean density (SE) of bacteria (cells mL)1) and statistical results in
the two experiments. When Tukey’s post hoc tests indicate a
significant difference in bacterial densities at equilibrium, the table
gives the R* ordering
Rp
Rf
Rc
Tukey’s
post hoc test
Experiment with
C. striatum
Experiment with
T. pyriformis
1Æ10 · 106 (0Æ04 · 106)
2Æ40 · 106 (0Æ60 · 106)
0Æ62 · 106 (0Æ16 · 106)
Rc < Rp (P = 0Æ0134)
Rc < Rf (P < 0Æ0001)
Rp < Rf (P < 0Æ0001)
1Æ49 · 106 (0Æ20 · 106)
4Æ21 · 106 (0Æ98 · 106)
1Æ48 · 106 (0Æ32 · 106)
Rc = Rp (P = 0Æ9999)
Rc < Rf (P = 0Æ0001)
Rp < Rf (P = 0Æ0002)
coexistence, at least until the end of the experiment (day 49)
(Fig. 2a).
The analysis of P. bursaria dynamics using repeated measure anovas showed that before day 40, the effect of treatments on P. bursaria density has varied over time
(treatments · time: F56,209 = 29Æ81, P < 0Æ0001; Table S3)
whereas, after day 40, P. bursaria density was affected by
treatments only (treatments: F3,12 = 45Æ51, P < 0Æ0001;
time: F4,46 = 1Æ19, P < 0Æ3248; Table S3). Before day 40,
the effect of treatments on C. striatum density depends on
time
(repeated measure anova,
treatment · time:
F42,168 = 18Æ40, P < 0Æ0001; Table S2). On day 40, C. striatum density was affected by treatments (anova: F2,9 = 16Æ77,
P = 0Æ0009; Table S2). After day 40, repeated measure anova showed C. striatum density was affected neither by treatments (F1,6 = 0Æ01, P = 0Æ9042; Table S2) nor by time
(F3,18 = 0Æ49, P = 0Æ6902; Table S2). Orthogonal contrasts
on treatments indicate that endosymbiosis, competition and
conditioned medium had a significant effect on the dynamics
of the competitor and P. bursaria (contrast analyses are summarised in Table 3 and are detailed in Tables S4 and S6).
There was a significant effect of endosymbiosis on P. bursaria densities from the ninth day until the end of the experiment (Table 3, contrast ‘endosymbiosis’). Paramecium
bursaria density was significantly higher (Fig. 2a,d; Colour
plates with all dynamics are in Fig. S2) than endosymbiontfree P. bursaria density (Fig. 2b,d). Moreover, endosymbiosis has decreased significantly C. striatum density from day 2
to 30 and from day 37 until day 40 (Table 3, contrast ‘endo-
2011 The Authors. Journal of Animal Ecology 2011 British Ecological Society, Journal of Animal Ecology, 81, 222–232
Ingredients for protist coexistence 227
Table 3. Summary of competition, conditioned-medium and endosymbiosis effects on the dynamic of the competitor (Colpidium striatum and
Tetrahymena pyriformis) and on Paramecium bursaria (carrying or free of its endosymbiont) in the two experiments. The table summaries days
of significant differences (threshold of 5%, NS: non-significant). The upper case letters represent the seven treatments: (A) monoculture of the
competitor, (B) competitor with P. bursaria conditioned medium, (C) competitor and P. bursaria in competition, (D) competitor and
endosymbiont-free P. bursaria in competition, (E) monoculture of P. bursaria, (F) P. bursaria with competitor conditioned medium and
(G)monoculture of endosymbiont-free P. bursaria. Asterisk indicates treatment missing from day 40 (*) and after day 40 (**) in the experiment
with C. striatum
Effects on competitor dynamic of
Competition
Conditioned medium
Endosymbiosis
Effects on Paramecium bursaria dynamic of
Endosymbiosis
Competition for P. bursaria
Competition for symbiont-free P. bursaria
Conditioned medium
Contrasts
Experiment with C. striatum
Experiment with T. pyriformis
(A*, B**) vs. (C, D)
A* vs. B**
C vs. D
2–11, 16–18, 35–40
NS
2–30, 37–40
2–4, 11, 21-end
7–9, 25–42, 51–58
2–42, 49-end
(E, F, C) vs. (G*, D)
(E, F) vs. C
G* vs. D
E vs. F
9-end
21–24, 30, 37-end
21, 28, 32–37
11–16
9-end
26–42
21, 25–30
21, 28
symbiosis’; Fig. 2a,b). The contrast ‘competition’ indicated
that C. striatum and P. bursaria had significant reciprocal
effects (Table 3). Paramecium bursaria has a clear negative
effect on population density of C. striatum (Fig. 2a,c) and
similarly, competition has a negative effect on P. bursaria
dynamics (Fig. 2a). Paramecium bursaria densities are significantly lower in the presence of C. striatum (treatment ‘competition’, Fig. 2a) than in its absence (treatments
‘monoculture’ and ‘conditioned medium’, Fig. 2d) at least in
the last part of the experiment (from day 21) (Table 3,
Fig. S2d). Contrasts on conditioned medium have revealed
no significant effects of P. bursaria conditioned medium on
C. striatum (Table 3, contrast ‘Allelopathy’; Fig. 2c). But
when filtrates of C. striatum cultures were added to P. bursaria, its growth during the exponential growth phase (from
11th to 16th day) was significantly enhanced over P. bursaria
growth in monocultures (Table 3, contrast ‘conditioned medium’; Fig. 2d).
EXPERIMENT WITH TETRAHYMENA PYRIFORMIS AS A
COMPETITOR
anova showed significant differences in bacterial density at
equilibrium between the three ciliates (F2,9 = 38Æ2,
P < 0Æ0001). Tukey’s post hoc tests indicate that the R* of
endosymbiont-free P. bursaria was significantly higher than
the R* of P. bursaria and T. pyriformis (Table 2). No significant difference between the R* of T. pyriformis and of P. bursaria was found (Table 2). As predicted by the R* rule, we
observed a competitive exclusion at day 20 of endosymbiontfree P. bursaria by T. pyriformis (Fig. 3b). Because the R* of
P. bursaria and T. pyriformis are not significantly different,
the prediction of the outcome of exploitation competition is
indeterminate. Competition treatments showed that P. bursaria and T. pyriformis coexist during the time course of the
experiment (until day 60), but with oscillations of T. pyriformis densities whose oscillation amplitude decreases over time
(Fig. 3a).
Repeated measure anovas of species density across time
showed that the interaction treatment · time is significant,
for T. pyriformis (F75,299 = 52Æ02, P < 0Æ0001; Table S2)
and P. bursaria (F84,315 = 53Æ90, P < 0Æ0001; Table S3).
The contrast analysis showed significant effects of species
interactions, i.e. endosymbiosis, competition and conditioned medium, on the dynamics of the competitor and
P. bursaria (summarised in Table 3 and detailed in Tables S5
and S7). As with C. striatum, contrasts on endosymbiosis,
competition and conditioned-medium effects showed some
significant differences (Table 3). Endosymbiosis had a significant effect on P. bursaria dynamics from day 9 until the end
and on T. pyriformis from the day 9 to 42 and from day 49
until the end (Table 3, contrasts ‘Endosymbiosis’). Paramecium bursaria density was increased by the presence of endosymbionts (Fig. 3a,b,d), whereas they decreased T. pyriformis
density (Fig. 3a,b). Contrasts revealed significant competitive effects on T. pyriformis, P. bursaria and endosymbiontfree P. bursaria (Table 3, contrasts ‘Competition’). Tetrahymena pyriformis in competition with P. bursaria (Fig. 3a)
showed first a fast decline: at day 32, its density
(10 ± 12 cells mL)1) was much lower than when in monoculture (2465 ± 230 cells mL)1, Fig. 3c) or in competition
with endosymbiont-free P. bursaria (1225 ± 151 cells mL)1,
Fig. 3b). Then, by the end of the experiment, T. pyriformis
density had increased but remained low (30 ± 7 cells mL)1
on day 60, compared to 997 ± 132 cells mL)1 in monoculture) (Colour plates with all dynamics are in Fig. S3). Endosymbiont-free P. bursaria in competition with T. pyriformis
became extinct at day 32 whereas it persisted at low densities
until the end of the experiment when in monoculture
(Fig. 3b,d). In competition with T. pyriformis, P. bursaria
densities were significantly lower than in the absence of
T. pyriformis from day 16 to 42 (Fig. S3d). The exploration
of biochemical-mediated interactions shows that there are
no effects, except a negative one on days 21 and 28, of
T. pyriformis conditioned medium on P. bursaria dynamics
but significant effects of P. bursaria conditioned medium on
2011 The Authors. Journal of Animal Ecology 2011 British Ecological Society, Journal of Animal Ecology, 81, 222–232
228 J. P. Müller, C. Hauzy & F. D. Hulot
Log [density (cells per mL)+1]
(a)
Competition: P. bursaria (
) and T. pyriformis (
)
4
3
3
2
2
1
1
0
Competition: endosymbiont-free P. bursaria (
T. pyriformis (
)
) and
0
0
(c)
10
20
30
40
50
60
0
T. pyriformis: monoculture (
) and conditioned-medium (d)
treatment (
)
4
4
Log [density (cells per mL)+1]
(b)
4
3
3
2
2
1
1
0
0
0
10
20
30
40
50
60
Time (days)
10
20
30
40
50
60
Monoculture of P. bursaria (
) and endosymbiontfree P. bursaria (
) and conditioned-medium
treatment on P. bursaria (
)
0
10
20
30
40
50
Time (days)
Fig. 3. Ciliate dynamics in the experiment with Tetrahymena pyriformis [Mean ± SE in log (cell mL)1) + 1]. Treatments are represented as in
Fig. 2. (a) Competition between T. pyriformis (m) and Paramecium bursaria (d); (b) Competition between T. pyriformis (4) and endosymbiont-free P. bursaria (s); (c) T. pyriformis monoculture (m) and conditioned medium on T. pyriformis (m); (d) P. bursaria (d) and endosymbiont-free P. bursaria (s) monocultures and conditioned medium on P. bursaria (d).
T. pyriformis dynamics (Table 3, contrasts ‘conditioned
medium’). Interestingly, these allelochemical-mediated
effects are negative at days 7, 9, 25–42 but then become positive at days 51–58 (Fig. 3c) and match with a small delay the
variations of T. pyriformis density in competition treatment
(Fig. 3a).
Discussion
Comparing the dynamics of P. bursaria and C. striatum in
competition with their dynamics in monoculture allows us to
draw several conclusions. Firstly, according to predictions
based on resource competition alone, P. bursaria and C. striatum coexisted until the end of our experiment, while endosymbiont-free P. bursaria went extinct. Endosymbionts can
thus mediate the coexistence of the two competitors for the
same resource by providing a supplementary resource to its
host. Secondly, P. bursaria and C. striatum had reciprocal
competitive effects: densities of C. striatum were significantly
lower in the presence than in the absence of a competitor
either P. bursaria or endosymbiont-free P. bursaria. The
same conclusion is reached with the comparison of P. bursaria densities in monoculture and in competition with C. striatum. Thirdly, higher P. bursaria densities when C. striatum
conditioned medium was added than in monoculture suggest
a transitional facilitative effect of C. striatum medium on
P. bursaria because of biochemicals. Therefore, biochemicals
may modify the dynamic of species in resource competition.
However, we did not observe the decrease in equilibrium densities between the treatment monoculture and the conditioned medium that would indicate that coexistence involves
interference between species through allelochemicals (Vance
1984; Case & Gilpin 1974).
In the second experiment, the comparison of ciliate
dynamics in monoculture and in competition leads to similar
conclusions. In agreement with predictions based on resource
competition alone, T. pyriformis outcompeted endosymbiont-free P. bursaria. Tetrahymena pyriformis and P. bursaria
coexisted until the end of our experiment, and the low and
oscillating densities of T. pyriformis reveal a strong competitive effect of P. bursaria on this ciliate. Thus, competition
and monoculture treatments show again a positive effect of
the endosymbiont on P. bursaria, which allow the two species
to coexist. However, conversely to the experiment with
C. striatum, P. bursaria medium affected T. pyriformis until
the end of the experiment. These biochemical-mediated
effects were first negative to become positive later on. Thus,
biochemicals produced in P. bursaria medium elicit different
2011 The Authors. Journal of Animal Ecology 2011 British Ecological Society, Journal of Animal Ecology, 81, 222–232
Ingredients for protist coexistence 229
responses of T. pyriformis dynamics ranging from interference to facilitation. Biochemicals effects of P. bursaria medium might have been stronger in the treatment competition
than in the treatment allelopathy because biochemicals produced by P. bursaria medium are diluted 10 times in the treatment conditioned medium.
In conclusion, our experiments show that the presence of
the endosymbiont C. vulgaris allows P. bursaria to compensate a competitive disadvantage in the exploitation of bacteria. In both experiments, we observed conditioned-medium
effects on species dynamics in addition to endosymbiosis and
competitive effects, which suggests that biochemical may
interact with resource competition.
ENDOSYMBIOSIS AND COMPETITION
In the two experiments, comparison of P. bursaria and endosymbiont-free P. bursaria dynamics shows that the presence
of C. vulgaris allows higher densities of its hosts. This result
seems obvious as we expect an endosymbiont to have a positive effect on its host but deserves some discussion. The green
alga is a resource provider for P. bursaria as the ciliate may
benefit from products of the photosynthesis (Karakashian
1963; Pado 1965; Weis 1969). Paramecium bursaria also preys
upon bacteria. Comparison of bacteria density at equilibrium
in P. bursaria and endosymbiont-free P. bursaria monocultures shows that bacteria density is lower in the former than
in the latter. In other words, endosymbiont-carrying P. bursaria better exploits bacteria than endosymbiont-free P. bursaria. According to resource competition theory (Grover
1997; Tilman 1982), resource density at equilibrium depends
on life-history parameters of the consumer. For instance, if
consumption of the resource follows a Monod function and
if the consumer has a constant mortality rate, the resource
density at equilibrium depends upon the Monod function
parameters (uptake rate, affinity constant) and the predator
conversion efficiency and mortality rate. Therefore, the
decrease in bacteria density at equilibrium in the presence of
C. vulgaris suggests that the endosymbiont either increases
P. bursaria’s ability to exploit bacteria or decreases its host’s
mortality rate. Thus, the endosymbiont C. vulgaris can modify the impact of P. bursaria on bacteria.
Our results show that the presence of the endosymbiont
C. vulgaris promotes the coexistence of P. bursaria with its
competitors. Resource competition theory (Grover 1997;
Tilman 1982) predicts that two competitors may coexist with
two resources if, at equilibrium, each competitor is limited by
a different resource and if there is a trade-off between competitors’ requirements and their impacts on the resources. If
these conditions are not fulfilled, one of the competitors
excludes the other. In our system, C. vulgaris provides
resources to P. bursaria that are not exploited by the competitor. As a consequence, the endosymbiont provides the
opportunity to its host to persist whatever its ability to
exploit the common resource.
According to niche theory (see Chase & Leibold 2003 for a
history of the niche concept), two species can coexist if they
do not share the same niche. In our experiments, the presence
of a resource-providing endosymbiont can thus induce niche
differentiation and coexistence. When Gause (1935) designed
his experiments to explore conditions for coexistence, he
manipulated the prey – yeast or bacteria – and space use –
bottom or top of the test tub. It is difficult to explain the coexistence that he observed with these two resources as he did
not attempt to measure niches (Arthur 1987): neither space
use by competitors was not monitored nor prey choice. In
addition, there is no data on prey spatial distribution. Nevertheless, here, we show that the presence of C. vulgaris allows
P. bursaria’s to differentiate its ecological niche from those
of its competitors, which offers a key to understanding coexistence in Gause (1935) experiments. Thus, our results show
that endosymbiosis can promote coexistence of competing
species by allowing niche differentiation.
The importance of symbionts on the coexistence of species
in competition for resources has been observed in plant communities (e.g. Bever et al. 2010). Plants in competition can
vary in their dependence on a symbiont (mycorrhizal fungi or
symbiotic N-fixer) for a specific soil nutrient (phosphorous
or nitrogen, respectively). The coexistence of plants depending on the presence of symbionts may also be interpreted in
the light of resource competition theory (Bever et al. 2010;
Van der Heijden 2002). A symbiont that increases plant productivity and allows its host to persist at lower levels of a limiting nutrient could contribute to competitive exclusion of
other plant species (Hartnett & Wilson 1999; Marler, Zabinski & Callaway 1999) and even reverse the outcome of competition (Hetrick, Wilson & Hartnett 1989). However, these
non-shared symbionts can also promote coexistence of plant
species. For instance, some plant species are only able to
coexist with other plants if mycorrhizal fungi are present,
indicating that the symbiont enhances their competitive ability (Grime et al. 1987; Van der Heijden et al. 1998). Similarly,
there is strong indirect evidence of positive effect of symbiotic
N-fixers on plant coexistence between legumes and nonlegumes (Schwinning & Parsons 1996; Cramer, Van Cauter
& Bond 2010). Thus, the importance of symbiosis for the outcome of competition is not restricted to our experimental
model and seems relevant to other ecological systems. Our
results provide a straightforward demonstration of the coexistence mechanism.
BIOCHEMICAL-MEDIATED INTERACTIONS
In our experiments, we did observe effects mediated by biochemicals during the transient dynamics. These effects interacted with endosymbiosis differently in the two experiments.
In the first experiment, the medium conditioned by C. striatum had a transient positive effect on P. bursaria and may
have made the coexistence easier. Indeed, P. bursaria reached
its carrying capacity earlier in the treatment competition than
in the treatment monoculture. Therefore, this biochemicalmediated facilitation and endosymbiosis had a positive synergetic effect on P. bursaria densities in monoculture. In the
second experiment, the comparison between the dynamics of
2011 The Authors. Journal of Animal Ecology 2011 British Ecological Society, Journal of Animal Ecology, 81, 222–232
230 J. P. Müller, C. Hauzy & F. D. Hulot
T. pyriformis in monoculture and with P. bursaria conditioned medium reveals direct effects until the end of the
experiment of biochemicals present in P. bursaria medium.
P. bursaria medium contained biochemicals that first inhibited T. pyriformis growth and then stimulated it. These
effects of biochemicals are significant but their strengths (different by an order of magnitude between treatments) are
modest. However, in the treatment conditioned medium, biochemicals are diluted up to 1 ⁄ 10 by comparison to biochemicals produced in P. bursaria medium and present in the
treatment competition. Thus, significant effects of biochemicals produced in P. bursaria medium suggest that biochemical-mediated interaction may have played a role in
coexistence in this experiment. In the treatment competition,
T. pyriformis displayed large density changes, which
increases its extinction risk, while P. bursaria dynamics was
stable. Therefore, we conclude that, in this experiment, endosymbiosis and biochemical-mediated interaction contributed
to P. bursaria dominance.
Gause (1935) already suggested that allelopathic interactions occurred in his experiments with P. bursaria. We
observed biochemical-mediated interactions, in the experiment with T. pyriformis, where biochemicals produced in
P. bursaria medium affected T. pyriformis dynamics from
day 25. The absence of conditioned- medium effects at the
beginning of the experiment is consistent with the idea that
biochemicals have effects when their concentration become
higher than a threshold concentration (Chao & Levin 1981;
Durrett & Levin 1997; Hulot & Huisman 2004). However,
after day 25, the impact of organic compounds produced in
P. bursaria medium changed during the time course of the
experiment, from negative to positive and matched T. pyriformis density changes in competition treatment. This qualitative change might be the consequence of three hypotheses
that are not mutually exclusive.
First, biochemicals could modify demographic rates of
T. pyriformis and hence modify the timing of T. pyriformis
dynamics. Indeed, the transitory fluctuations displayed by
T. pyriformis in the treatment conditioned medium seem to
appear earlier than in the treatment monoculture. This timing difference then could correspond to negative followed by
positive conditioned-medium effects. Second, the production
of biochemicals by P. bursaria could have varied over time.
Similar changes in allelochemical-mediated effects had been
documented for instance with rye (Reberg-Horton et al.
2005) leading the authors to speak about allelopathy phenology. In our experiment involving animals, the phenology
terminology is difficult to use as individuals do not mature as
plants. However, the synthesis of biochemicals may have
changed directly or indirectly with P. bursaria population
dynamics allowing the recovery of T. pyriformis populations
in competition and conditioned-medium treatment. Third,
the sensitivity of T. pyriformis populations could vary over
time because of a selection of T. pyriformis individuals that
are less sensitive to biochemicals. Tetrahymena pyriformis
population could have been initially mainly sensitive to biochemicals, explaining the observed negative effect. The
increase in T. pyriformis densities after reaching very low values could be the result of the selection of individuals insensitive to biochemicals produced in P. bursaria medium. This
selection process could occur during a period of 23 days
(from day 16 to 39), which amounts to c. 61 generations with
a generation time of c. 9 h. Our experiments do not permit
more definitive conclusions, but we hope that this discussion
about these three hypotheses will stimulate further experimental and theoretical investigations into the mechanisms
involved in population response to biochemicals, in particular in the context of competition by exploitation.
INTERACTION BETWEEN COMPETITION,
ENDOSYMBIOSIS AND BIOCHEMICAL-MEDIATED
INTERACTIONS
Theory suggests that interference through allelochemicals
can promote coexistence (Vance 1984; Case & Gilpin 1974).
A weaker competitor for a shared resource can coexist with a
stronger competitor if the weaker competitor interferes sufficiently with the other (Vance 1984; Case & Gilpin 1974). In
contrast, our results suggest that the coexistence observed in
the treatment competition is not caused solely by biochemical-mediated interference. However, in these pioneer models,
the resource was not explicit. With the analysis of a model of
exploitative and interference competition, Amarasekare
(2002) predicted that a species engaged in costly interference
such as allelopathy should not be able to coexist with a superior competitor unless it is also engaged in beneficial interference mechanisms such as predation or parasitism. In the
experiment with T. pyriformis, where the outcome of competition could not be predicted, P. bursaria coexisted with its
competitor, while effects of P. bursaria medium on T. pyriformis did occur. To our knowledge, P. bursaria was not
engaged in beneficial interference mechanisms but benefited
from the presence of an endosymbiont. Our results suggest
that this positive effect was a condition for P. bursaria coexistence with T. pyriformis. In this case, endosymbiosis provided resources analogous to the predation or parasitism
hypothesised by Amarasekare (2002) and offered conditions
for coexistence. This result may be generalised to plants that
cannot develop beneficial interference based on predation or
parasitism but are nevertheless engaged in a costly interference competition mediated by allelochemicals. Endosymbiosis is then an additional axis for resource partitioning
allowing coexistence.
Conclusion
The results of our two experiments show that endosymbiosis
and biochemical-mediated interactions interact and may
change the dynamics of populations that are engaged in competition for the same resource. In particular, the presence of
an endosymbiont that is a resource-provider ensures the persistence of its host in competition. This might be the key
explanation of coexistence in Gause’s (1935) classical experiment. We speculate that a species engaged in competition for
2011 The Authors. Journal of Animal Ecology 2011 British Ecological Society, Journal of Animal Ecology, 81, 222–232
Ingredients for protist coexistence 231
a resource and in costly interference such as allelopathy may
counterbalance these costs by enlisting a resource-provider
endosymbiont instead of embarking on predation or parasitism to harm its competitors.
Acknowledgements
We thank Minus van Baalen, Sébastien Barot, Jeremy Fox, Michel Loreau
and the two anonymous reviewers for helpful comments on the manuscript
and Toshinobu Suzaki for his help to obtain chlorella-free P. bursaria. This
work was supported by ANR IFB Biodiversité 2005 to F.D. Hulot.
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Received 12 August 2010; accepted 1 July 2011
Handling Editor: Phil Warren
Supporting Information
Additional Supporting Information may be found in the online
version of this article.
Fig. S1. Picture of Paramecium bursaria and endosymbiont-free
P. bursaria obtained thanks to paraquat.
Fig. S2. Ciliate dynamics in the experiment with Colpidium striatum.
Fig. S3. Ciliate dynamics in the experiment with Tetrahymena
pyriformis.
Table S1. Selection of variance structure for the analysis of ciliate
densities across time.
Table S2. Analysis of the density of the competitors (Colpidium striatum or Tetrahymena pyriformis) across time.
Table S3. Analysis of Paramecium bursaria density across time in
Colpidium striatum experiment or Tetrahymena pyriformis experiment.
Table S4. Summary of competition, conditioned-medium and endosymbiosis effects (P-values) on Colpidium striatum density across
time.
Table S5. Summary of competition, conditioned-medium and endosymbiosis effects (P-values) on Tetrahymena pyriformis density
across time.
Table S6. Summary of endosymbiosis, competition and conditionedmedium effects (P-values) on Paramecium bursaria density across
time in Colpidium striatum experiment.
Table S7. Summary of endosymbiosis, competition and conditionedmedium effects (P-values) on Paramecium bursaria density across
time in Tetrahymena pyriformis experiment.
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2011 The Authors. Journal of Animal Ecology 2011 British Ecological Society, Journal of Animal Ecology, 81, 222–232