# Cooperation-mediated plasticity in dispersal and colonization

Staffan Jacob, Priscilla Wehi, Jean Clobert, Delphine Legrand, Nicolas

Schtickzelle, Michele Huet, Alexis Chaine

► To cite this version:

Staffan Jacob, Priscilla Wehi, Jean Clobert, Delphine Legrand, Nicolas Schtickzelle, et al. Cooperation-mediated plasticity in dispersal and colonization. Evolution Agricole, 2016, 70 (10), pp.2336-2345. 10.1111/evo.13028. hal-04967694

HAL Id: hal-04967694

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1 Cooperation-mediated plasticity in dispersal and colonization

2

3 Staffan Jacob*¹,³, Priscilla Wehi*¹,², Jean Clobert¹, Delphine Legrand¹,³, Nicolas

4 Schtickzelle³, Michele Huet¹ and Alexis Chaine¹,⁴

5 * Co-first authors

6

7 ¹Station d'Ecologie Expérimentale du CNRS à Moulis USR2936, Evolutionary Ecology

8 Group, 2 route du CNRS, 09200 Saint-Girons, France,

9 ² now at Landcare Research Manaaki Whenua, Private Bag 1930, Dunedin 9054, New Zealand

10
11 ³ Université Catholique de Louvain, Earth and Life Institute & Biodiversity Research Centre,
Croix du Sud 4, L7-07-04, 1348 Louvain-la-Neuve, Belgium

12
13 ⁴ Institute for Advanced Studies in Toulouse, Toulouse School of Economics, 21 allée de Brienne, 31015 Toulouse, France

15 Corresponding author: Staffan Jacob; Station d'Ecologie Expérimentale du CNRS à Moulis

16 USR2936, Evolutionary Ecology Group, 2 route du CNRS, 09200 Saint-Girons, France &

17 Université Catholique de Louvain, Earth and Life Institute & Biodiversity Research Centre,
Croix du Sud 4, L7-07-04, 1348 Louvain-la-Neuve, Belgium;

19 Tel : +33 5 61 04 03 73; jacobstaffan@gmail.com

20

21 Running title: Cooperation and the plasticity of dispersal

22 Keywords: Cooperation, mobility, altruism, kin selection, density-dependent dispersal, colonization, social aggregation, reaction norms

24 Type of article: Original Article
25 Abstract

26 Kin selection theory predicts that costly cooperative behaviors evolve most readily when
27 directed toward kin. Dispersal plays a controversial role in the evolution of cooperation:
28 dispersal decreases local population relatedness and thus opposes the evolution of
29 cooperation, but limited dispersal increases kin competition and can negate the benefits of
30 cooperation. Theoretical work has suggested that plasticity of dispersal, where individuals can
31 adjust their dispersal decisions according to the social context, might help resolve this
32 paradox and promote the evolution of cooperation. Here, we experimentally tested the
33 hypothesis that conditional dispersal decisions are mediated by a cooperative strategy: we
34 quantified the density-dependent dispersal decisions and subsequent colonization efficiency
35 from single cells or groups of cells among 6 genetic strains of the unicellular *Tetrahymena*
36 *thermophila* that differ in their aggregation level (high, medium and low), a behavior
37 associated with cooperation strategy. We found that the plastic reaction norms of dispersal
38 rate relative to density differed according to aggregation level: highly aggregative genotypes
39 showed negative density-dependent dispersal, whereas low-aggregation genotypes showed
40 maximum dispersal rates at intermediate density, and medium-aggregation genotypes showed
41 density-independent dispersal with intermediate dispersal rate. Dispersers from highly
42 aggregative genotypes had specialized long-distance dispersal phenotypes, contrary to low-
43 aggregation genotypes; medium-aggregation genotypes showing intermediate dispersal
44 phenotype. Moreover, highly aggregation genotypes showed evidence for beneficial kin-
45 cooperation during dispersal. Our experimental results should help to resolve the evolutionary
46 conflict between cooperation and dispersal: cooperative individuals are expected to avoid kin-
47 competition by dispersing long distances, but maintain the benefits of cooperation by
dispersing in small groups.
48
49
50 **Introduction**

Cooperation exists in most taxonomic groups (West et al. 2007a) and lies at the heart of major transitions from unicellular live forms to multi-cellularity and societies (Maynard Smith and Szathmary 1995b; Crespi 2001). Explaining why organisms carry out costly cooperation is thus fundamental to our understanding of the evolution of various biological functions (Maynard Smith and Szathmary 1995a; Crespi 2001). The main hypothesis to explain the emergence and maintenance of cooperation is kin selection, which states that for cooperation to evolve, costly cooperative behaviors should be directed toward kin, thus indirectly increasing gene transmission (Hamilton 1964; West et al. 2007a). Genetic relatedness and population genetic structure consequently play a major role in the evolution of cooperation (Hamilton 1964; Griffin and West 2002 p. 200; West et al. 2002), since the maintenance of kin-based groups allows cooperative behaviors to be directed toward kin, providing a simple mechanism to control invasion by cheaters (Hamilton 1964; West et al. 2007a).

Dispersal, the ability of individuals to move from one place to another during their life, is expected to decrease relatedness in local populations (i.e. average relatedness of neighborhoods; Clobert et al. 2012). Since kin selection theory predicts that stable groups of kin should maintain cooperation, cooperation should favor low dispersal (Hamilton 1964; West et al. 2002). As a corollary, dispersal has long been seen as an opposing force to cooperation (Hamilton 1964; West et al. 2002; Lion and Baalen 2008). However, while group stability among kin provides benefits of kin cooperation, it also increases kin competition, which can negate these benefits (Hamilton and May 1977; Taylor 1992; Queller 1994). This contradiction is even more intriguing given ample empirical evidence of cooperative species performing dispersal movements (Bourke and Franks 1995; O'Riain et al. 1996; Sinervo and Clobert 2003), which has led to several theoretical studies showing that cooperation and dispersal can coevolve (Le Galliard et al. 2005; Hochberg et al. 2008; Parvinen 2013).
Substantial theoretical work has demonstrated that various factors could allow cooperation to evolve and be maintained despite kin competition, including overlapping generations (Irwin and Taylor 2000), population structure and environmental context [e.g. range expansion (Datta et al. 2013), empty patches (Alizon and Taylor 2008), patch quality (Rodrigues and Gardner 2012); see also (Lion and Baalen 2008)], budding dispersal (Gardner & West 2006; Kümmerli et al. 2009), and dispersal-dependent cooperation (El Mouden and Gardner 2008).

However, most theoretical work on cooperation and dispersal assumes that dispersal is independent of ecological or social context (Le Galliard et al. 2005; Hochberg et al. 2008; Patterson et al. 2008). By contrast, a large body of empirical work shows that dispersal is a plastic trait that depends on the internal state of organisms and their environmental context (Bowler and Benton 2005; Clobert et al. 2009), including in cooperative species (O'Riain et al. 1996; Sinervo and Clobert 2003). Individuals are expected to adjust dispersal decisions to stay in habitats that match their phenotype, or to leave poor quality patches in order to find a better place to live (Bowler and Benton 2005; Edelaar et al. 2008; Clobert et al. 2012; Jacob et al. 2015a). For cooperative species, habitat quality is dependent upon the balance between kin cooperation and kin competition, and we might thus expect dispersal decisions to vary accordingly. Dispersal decisions that vary according to the balance between the costs and benefits of cooperative opportunities in a given social environmental context (i.e. plastic dispersal behavior) could resolve the cooperation-dispersal paradox and promote the evolution of cooperation (Pepper and Smuts 2002; Aktipis 2004, 2011; Pepper 2007). Although these models have pointed out the potential importance of conditional movements for the evolution of cooperation, experimental evidence for the existence of such cooperation-mediated plasticity of dispersal is still lacking.

Here we experimentally tested the hypothesis that conditional dispersal decisions are mediated by the cooperative strategy in the ciliated protozoa *Tetrahymena thermophila*. As
for many microorganisms, the survival and growth of *Tetrahymena thermophila* depends on a
quorum sensing mechanism: extracellular emission of chemical signals drives population
growth, especially by preventing mortality and favoring growth at low density (Christensen
and Rasmussen 1992; Christensen et al. 1995, 1996, 2003; Rasmussen et al. 1996; Straarup et
al. 1997; Witzany and Nowacki 2016). According to the classical definition of cooperation
(i.e. a behavior which provides a benefit to another individual and which is selected because
of this beneficial effect; West et al. 2007b), cells thus cooperate through the emission of
different molecules e.g. insulin, endorphins, “growth factors” (TPAFs; reviewed in
Rasmussen and Wheatley 2007; Csaba 2012). Furthermore, cells can produce growth
inhibition factors reducing cell division at high densities (Christensen et al. 1998), as expected
for kin cooperation among clones (Hamilton 1964).

In addition to cooperative behavior, *Tetrahymena* cells can form aggregative groups
among kin, a behavior frequently associated with cooperative behavior in other taxa (e.g.
Keller and Ross 1998; Queller et al. 2003; Griffin et al. 2004; Sinervo et al. 2006; Cornwallis
et al. 2010) and which is likely to facilitate the exchange of growth factors in this species.
Importantly, there is evidence for genetic variation in this aggregative behavior and it is
associated with specific behavioral and life-history traits that allowed the classification of six
*Tetrahymena* genotypes into a gradient of cooperation levels (Schtickzelle et al. 2009; Chaine
et al. 2010; Jacob et al. 2016). First, the expression of this aggregative behavior is modulated
by population relatedness: decreasing relatedness in populations lead to decreased levels of
aggregation as expected in cooperative species (Chaine et al. 2010). Second, genotypes
defined as highly aggregative move towards habitats that contain traces of a kin population,
whereas those defined as low aggregative move away from kin, and medium aggregative ones
show no preference (i.e. kin recognition and orientation; Chaine et al. 2010). Third, highly
aggregative genotypes show reduced growth rate much like cooperative lines in other taxa,
but live longer on a finite resource compared to low aggregative genotypes (i.e. growth rate is 125 negatively correlated to carrying capacity in this species (Fjerdingstad et al. 2007) and time 126 to reach 1/2 of the carrying capacity increases with aggregation level (Chaine et al. 2010)), 127 which is a classic resolution of the “tragedy of the commons”. Globally, variation in 128 aggregative behavior is consistently linked to variation in traits or behaviors predicted by 129 cooperation such that aggregation level can be used as a proxy for variation in cooperation. 130 Future work should confirm this link, but our present analysis focuses on variation in 131 aggregation level as a metric for cooperation. *T. thermophila* therefore provides an excellent 132 opportunity to investigate how cooperation shapes plastic dispersal decisions since genetically 133 distinct clonal strains are known to differ in their cooperation strategy (from highly to low- 134 cooperative genotypes; Schtickzelle et al. 2009; Chaine et al. 2010; Jacob et al. 2016). 135

According to kin selection theory, although an increase of population density generally 136 intensifies competition between individuals, it can also increase the benefits of cooperation in 137 cooperative species (Darch et al. 2012). Compared to low densities where competition is 138 weak but cooperative interactions are limited, higher densities should increase efficiency in 139 the use of extracellular chemicals for cooperative organisms (positive density-dependent 140 benefits of quorum-sensing; Darch et al. 2012). Accordingly, the negative effects of 141 increasing density in *T. thermophila* should be weaker in highly cooperative genotypes than in 142 low-cooperative ones (Chaine et al. 2010). Consequently, we expect that organisms differing 143 in their tendency to engage in cooperative interactions (i.e. cooperation strategy) should react 144 differently to the balance between kin cooperation and competition. Under the conditional 145 movement hypothesis, we therefore expect that the cooperation strategy should interact with 146 the social environment, especially population density, to drive dispersal decisions. 147

We manipulated density in clonal populations of 6 genetic strains of *T. thermophila* that 148 have previously shown consistently contrasted aggregation levels and thus cooperation 149
strategies (2 highly, 2 medium and 2 low aggregation strains (Schtickzelle et al. 2009; Chaine et al. 2010), and tested the consequences of the interaction between aggregation strategy and social context (density) on three key aspects of dispersal: emigration rate, the phenotype of dispersers, and colonization success by immigrants. First, we expected the interplay between aggregation strategy and social environment to result in the modification of dispersal rate (emigration) as a result of changes in the balance between the costs and benefits of cooperation. Specifically, low-aggregation genotypes should experience increased competition when density rises, whereas highly aggregative ones should also benefit from kin interactions at high densities. In aggregative genotypes, low competition but limited cooperative interactions at lower densities and increased benefits of cooperation as density rises should lead to a quadratic relationship between density and dispersal rate, with dispersal rate first decreasing and then increasing when density reaches higher levels. Second, dispersers and residents are often found to show different phenotypes, with dispersers being phenotypically specialized for reduced dispersal costs during transience and/or colonization (phenotype-dependent dispersal; Clobert et al. 2009; Le Galliard et al. 2012). Whilst all dispersers should be selected for reduced dispersal costs during transience, dispersers from cooperative species should also be specifically selected to escape kin competition, and thus be specialized for long-distance dispersal (Rousset and Gandon 2002; Schtickzelle et al. 2009; Bitume et al. 2013). We therefore expect aggregative genotypes to show dispersers specialized in long-distance movement (i.e. more elongated cells in the case of *T. thermophila*; Fjerdingstad et al. 2007; Schtickzelle et al. 2009) more often than for low-aggregative genotypes. Finally, the key immigration step, influencing gene flow and changes in a species distribution, depends on an organism's ability to colonize new environments (Clobert et al. 2012). We thus tested whether aggregation strategy affects colonization efficiency, and compared colonization efficiency from single founder cells with colonization
175 efficiency of several founder cells (akin to group dispersal). Since dispersing in groups is
176 expected to favor the evolution of cooperation ("budding dispersal"; Gardner and West 2006;
177 Kümmerli et al. 2009), we expect aggregative genotypes to perform better in colonizing a new
178 environment when colonization occurred from a group of individuals rather than from a single
179 founder cell.

180

181 Methods

182 We performed all experiments in microcosms using experimental populations of 6 genetically
183 distinct *T. thermophila* strains (i.e. isolated genotypes kept as clonal populations) that differ in
184 their aggregation behavior, indicative of differences in cooperation strategy (low = 4A, 7;
185 medium = D3, P; high = E, Q; 5 replicates per experimental design level, *see below*;
186 Schtickzelle et al. 2009; Chaine et al. 2010). In a first experiment, we measured the density
187 dependence of dispersal rate as well as disperser phenotype using standard connected
188 microcosms: two habitat patches consisting of 1.5ml microtubes, connected by a corridor
189 made of 4mm internal diameter, 2.5cm long silicone tube. Cells were maintained at 22°C and
190 fed with growth media (2% Difco proteose peptone, 0.2% yeast extract) as described in
191 previous studies (Fjerdingstad et al. 2007; Schtickzelle et al. 2009; Chaine et al. 2010;
192 Pennekamp et al. 2014). Cells were placed in the start patch at each of three different densities
193 (100000, 250000 and 400000 cells/ml) that correspond to the range of densities commonly
194 observed in our culture conditions, from the minimum density required to reliably quantify
195 dispersal through to near carrying capacity (Chaine et al. 2010; Pennekamp et al. 2014) and
196 allowed to disperse towards the target patch for 17 hours (about ½ to 1 generation time at
197 23°C; Chaine et al. 2010; Jacob et al. 2015b). There was no difference in carrying capacity
198 between aggregation strategies on the basis of previously published data (F<sub>2,3</sub>=1.63; p=0.33; 3
199 replicates per genotype; *data from* Fjerdingstad et al. 2007), meaning that the population
densities we tested reflect a similar environmental context for the 6 different genetic lines. Furthermore, cell condition in this experiment should be similar given that all six genetic lines were raised under standard conditions immediately prior to the experiment. We created 5 replicates of the two-patch systems for each density by genotype combination, for a total of 90 two-patch microcosms.

We measured 1) dispersal rate as the proportion of cells in the target patch compared to the start patch, and 2) phenotype of dispersers as cell size and shape (measured as cell area and cell major/minor axis ratio of a fitted ellipse (Fjerdingstad et al. 2007) using automated digital analysis of pictures taken under dark field microscopy (Pennekamp and Schtickzelle 2013) using ImageJ software (version 1.47, National Institutes of Health, USA, http://imagej.nih.gov/ij). The measure of dispersal rate, used in several previous studies (Fjerdingstad et al. 2007; Schtickzelle et al. 2009; Chaine et al. 2010; Pennekamp et al. 2014; Jacob et al. 2015b), has been found insensitive to density-dependent effects on growth (Pennekamp et al. 2014). We summarized disperser phenotype through the first axis of a principal component analysis on cell size and shape (loading factors: cell size = -0.833; cell shape = 0.833; variance explained = 69.4%). Disperser phenotype increases when cells became small and elongated, and decreases when dispersers became bigger and less elongated. In this species, elongated cells are specialized for long-distance movement and show greater swim speed and straighter movements (Nelsen 1978; Fjerdingstad et al. 2007; Schtickzelle et al. 2009; Pennekamp et al. 2014). The induction of this phenotype is especially common when escaping starvation conditions (Nelsen 1978).

In a second experiment, we tested whether cooperation strategy affects the efficiency of dispersing cells to colonize a new environment. To this end, we transferred disperser cells from the above experiment into new empty patches (single unconnected 1.5ml standard microtubes), either as a single disperser cell (individuals isolated using a 10µl pipetteman) or
as a group of disperser cells transferred as a fixed volume from the target patches of the dispersal systems [1.4µl resulting in 175.57 ± 8.39 cells; no significant difference of number of cells transferred between density treatments (F₁,₈₃ = 2.92; p = 0.09) and aggregation strategies (F₂,₃ = 1.62; p = 0.33)]. For each of the 90 two-patch dispersal microcosms, we created 10 single cell replicates and one group dispersal replicate. We defined colonization efficiency of a new patch as population growth after a standard period of time (i.e. four days for the single disperser case, 24 hours for the group dispersal case). For colonization from several cells, colonization efficiency was measured as the final population size (Nₜinal) relative to initial (Nᵢntial), and calculated as follows: (Nₜinal - Nᵢntial) / Nᵢntial. This measure is not confounded by colonization failure, which did not occur in our experiments.

In order to investigate how cooperation strategy affects density-dependent dispersal rate, dispersal phenotype and colonization efficiency from a single cell or several cells, we tested for the interaction between aggregation strategy and density using linear mixed models (*lme*, *nlme* R-package (Pinheiro and Bates 1996). We used dispersal rate, disperser phenotype and colonization efficiency from a single cell or from several cells as dependent variables in four separate models. The population density treatment was used as a continuous factor (in linear and quadratic form), aggregation strategy as a fixed categorical factor (i.e. 3 levels of aggregation —low, medium, high— containing 2 strains each), and genotype as a random categorical factor to account for our nested design (2 genotypes per aggregation level). We computed linear models with a Gaussian distribution, and in all cases residuals of the models followed a normal distribution. We estimated the variance explained by the models using *r.squaredGLMM* (*MuMln* R-package (Nakagawa and Schielzeth 2013). Following a backward selection procedure, we removed cooperation, density and their interaction from the models when non-significant at the 0.05 level.

249
250 **Results**

251 First, we tested whether emigration decisions depend on the interplay between cooperation
252 strategy and social environment. We found that the effect of density on dispersal rate differed
253 depending on the aggregation strategy of genotypes (Table 1; Fig 1a). In highly aggregative
254 genotypes, the dispersal rate was the highest at low density (Density²: estimate ± SE = 0.10 ±
255 0.05; F₁,₂₆ = 4.33; p = 0.04; Density: -0.47 ± 0.20; F₁,₂₆ = 5.48; p = 0.03; Fig 1a), while the
256 dispersal rate in low-aggregation genotypes was the highest at intermediate densities
257 (Density²: -0.18 ± 0.06; F₁,₂₆ = 10.09; p = 0.004; Density: 0.63 ± 0.23; F₁,₂₆ = 7.59; p = 0.01;
258 Fig 1a). Finally, medium aggregation strains showed an intermediate level of dispersal rate
259 not significantly affected by density (Density²: 0.04 ± 0.04; F₁,₂₆ = 1.38; p = 0.25; Density: -
260 0.22 ± 0.15; F₁,₂₆ = 2.09; p = 0.16).

261 Second, we investigated whether disperser phenotype differed according to cooperation
262 strategy, and especially whether aggregative genotypes had dispersers that were
263 morphologically specialized for long-distance dispersal. We found a significant effect of the
264 aggregation * density interaction on disperser phenotype (Table 1; Fig 1b). First, dispersers
265 were small and elongated in aggregative genotypes and in contrast big and less elongated in
266 low-aggregation genotypes (Fig 1b), while dispersers from medium-aggregation strains
267 showed an intermediate dispersal phenotype between low- and high-aggregation strains.
268 Importantly, this difference is not simply a result of among genotype phenotypic variation.
269 Dispersers of highly aggregative genotypes were indeed more elongated than residents
270 (disperser *minus* resident phenotype: mean ± SE = 0.75 ± 0.15), whereas low-aggregation
271 dispersers were bigger and less elongated than residents (-0.57 ± 0.14), and medium-
272 aggregation dispersers were similar to residents (0.01 ± 0.09; aggregation level effect on
273 relative disperser phenotype: F₂,₃ = 11.57; p = 0.039). Second, apart from medium-
274 aggregation strains in which density has no significant effect on disperser phenotype
(Density²: -0.24 ± 0.12; F₁,₂₆ = 3.85; p = 0.06; Density: 0.82 ± 0.49; F₁,₂₆ = 2.88; p = 0.10), dispersers from low and high-aggregation strains were the biggest and least elongated at medium density (Low: Density²: 0.41 ± 0.17; F₁,₂₆ = 5.49; p = 0.03; Density: -1.72 ± 0.71; F₁,₂₆ = 5.90; p = 0.02; High: Density²: 0.61 ± 0.24; F₁,₂₆ = 6.50; p = 0.02; Density: -2.88 ± 0.49; F₁,₂₆ = 2.88; p = 0.10).

Finally, because colonization efficiency after dispersal is a key step if dispersal is to produce gene flow and changes in species distribution, we tested whether cooperation strategy affects colonization efficiency, both from single founder cells and from several cells. In the single founder cell experiments, the effect of density on colonization efficiency depended on the aggregation strategy (Table 1; Fig 2a). First, dispersers from highly aggregative genotypes showed significantly reduced colonization efficiency from a single cell compared to low and medium aggregation genotypes whatever initial population density (contrasts after Tukey adjustment: high vs low: -139.85 ± 16.87; t₁,₂₇ = 8.29; p = 0.008; high vs medium: -109.65 ± 16.87; t₁,₂₇ = 6.50; p = 0.01; low vs medium: 30.20 ± 16.87; t₁,₂₇ = 1.79; p = 0.31). In contrast to the specialized long distance dispersers of aggregative genotypes, dispersers from low and medium aggregation genotypes were bigger, a phenotype that we may hypothesize to be responsible for this increased colonization efficiency (Fig 3). Furthermore, pre-dispersal density had a significant positive effect on single cell colonization of low aggregation genotypes (13.84 ± 5.20; F₁,₂₇ = 7.08; p = 0.01), a negative effect in medium aggregation genotypes (-41.82 ± 9.79; F₁,₂₇ = 18.24; p < 0.001), and no significant effect in highly aggregative genotypes (-4.91 ± 5.17; F₁,₂₇ = 0.90; p = 0.35).

Furthermore, we found that aggregative genotypes were as efficient as low and medium aggregation genotypes in colonization from a group of cells (Table 1; Fig 2b), and we found no significant effects of density * aggregation interaction on colonization from several cells (p>0.05).
300

301

Discussion

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311

Plasticity in dispersal decisions that balance the costs and benefits of cooperative behaviors among kin might resolve the cooperation-dispersal paradox, hence potentially promoting the evolution of cooperation (conditional movement; (Pepper and Smuts 2002; Aktipis 2004, 2011; Pepper 2007). Here, we demonstrated that the density-dependent reaction norms of dispersal rate and the phenotype of dispersers depended on the aggregation level, a behavior linked to cooperation strategy in the ciliated protozoa *Tetrahymena thermophila*. Furthermore, we found that the colonization efficiency of single founder cells differed depending on their aggregation strategy and its associated disperser phenotype. Interestingly, when colonization occurred from a group of cells, colonization success was equivalent among all three aggregation levels.

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Cooperation mediates density-dependent dispersal reaction norms

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We found that *T. thermophila* genotypes of different aggregation strategies show contrasted reaction norms of density-dependent dispersal decisions (Fig 1a). The benefits of cooperation between unicellular organisms have been predicted to increase with density, as seen in the bacteria *Pseudomonas aeruginosa* (Darch et al. 2012). However, when density reaches high levels, competition between kin is expected to negate the benefits of cooperative acts, leading to negative effects of high densities (Hamilton and May 1977; Queller 1992; Taylor 1992). In our study, highly aggregative genotypes showed a quadratic relationship between density and dispersal rate, dispersal rate being the highest at low density, as found in other studies (Kim et al. 2009; Matthysen 2012; Fronhofer et al. 2015). When density increased, dispersal rate decreased as expected if the benefits of cooperation show positive density-dependence (Darch et al. 2012). Highly aggregative genotypes have previously been
found to live longer at high density than genotypes with lower levels of aggregation, thereby showing positive density dependence of cooperation (Chaine et al. 2010). This suggests that highly cooperative genotypes may try to avoid habitats where density is either not high enough to generate benefits of kin cooperation or so high that negative effects of kin competition negates the benefits of cooperation, and instead will disperse to habitats with medium congener densities. Interestingly, the balance between kin cooperation and competition occurring in this clonally reproducing species led to a U-shape density-dependent dispersal comparable to that found in sexually reproducing colonial species, such as the Blue-footed Booby (*Sula nebouxii*) where benefits of population density result from opportunities for pair formation (Kim et al. 2009; see also Fronhofer et al. 2015).

Contrary to highly aggregative genotypes, non-aggregative genotypes should not benefit from cooperative behaviors when density increases, but rather will experience higher competition. While dispersal rate in medium- aggregative genotypes was not significantly affected by density, dispersal of low- aggregative genotypes was higher at intermediate density. Then when density reached high levels, these low- aggregative genotypes sharply decreased their dispersal rate. This decrease in dispersal at high density is unexpected since these genotypes show very low levels of cooperative behaviors even at high density and preferentially orient toward non-kin during dispersal (Chaine et al. 2010). One possible explanation for this decrease in dispersal rate could be that crowded habitats might hinder cell movements or lead to poorer cell condition, consequently reducing dispersal. For example, a recent study on a similar species (*Tetrahymena pyriformis*) found that increasing density in populations while keeping resources and chemical compounds exchanged among cells constant resulted in a decreased distance travelled by cells (Fronhofer et al. 2015). However, except for medium aggregation genotypes from a single cell, pre-dispersal density has no significant effect on colonization efficiency in our study (Fig 2), making density effects on
350 cell condition unlikely. Alternatively, high population density could be a good predictor of
351 high habitat quality: negative density-dependent dispersal is expected to occur when congener
352 density provides individuals with more reliable information about habitat quality than direct
353 estimation of the quantity of resources available (Clobert et al. 2009; Baguette et al. 2011;
354 Rodrigues and Johnstone 2014). While *T. thermophila* does use social information in
355 dispersal decisions (Jacob et al. 2015b), further experiments that decouple the effects of
356 crowding on movement ability and cell condition from kin cooperation and competition, for
357 instance by manipulating cooperative molecules in the culture media, are required to fully
358 understand these patterns.

359 Overall, our results demonstrate the existence of conditional dispersal decisions that
360 depend on the interaction between cooperation strategy and social context, a process which
361 has been theoretically shown to resolve the cooperation-dispersal paradox and favor the
362 evolution of cooperation (Pepper and Smuts 2002; Pepper 2007; Aktipis 2011).
363

364
**Cooperation-dependent disperser phenotype**
---

365 Specialized dispersal behaviors and morphs have been found in cooperative species
366 (Bourke and Franks 1995; O'Riain et al. 1996; Sinervo and Clobert 2003) which could help to
367 resolve the cooperation-dispersal paradox among kin (Schtickzelle et al. 2009). Here, we
368 found that the phenotypes of dispersing cells depend on the aggregation strategy of the
369 genetic strains (Fig 1b). Dispersers from highly aggregative genotypes were small and
370 elongated, dispersers from low-aggregation genotypes were big and round, and medium-
371 aggregation genotypes showed an intermediate phenotype. In *T. thermophila*, small and
372 elongated cells show greater swim speed and straighter movements, and are thought to be
373 more efficient dispersers because of reduced resistance during movement (Stein and Bronner
374 1989; Fjerdingstad et al. 2007; Schtickzelle et al. 2009; Pennekamp et al. 2014). Our results
suggest that highly aggregative genotypes develop specialized dispersal morphs and thus would be able to disperse longer distance, as expected for cooperators in order to escape regionally structured kin-based populations (Rousset and Gandon 2002; Lion and Baalen 2008; Hatchwell 2009; Schtickzelle et al. 2009; Bitume et al. 2013). In contrast, low-aggregation genotypes produced dispersers that were bigger and less elongated, with likely reduced long distance dispersal ability but probably greater energetic reserves and competitive ability for establishment in a new population (Fjerdingstad et al. 2007). Indeed, selection should favor competitive ability during colonization in low-cooperative individuals, with no real advantage of longer distance dispersal. Furthermore, we found that in both low and high-aggregative genotypes, dispersers were the biggest and least elongated at medium density (Fig 1b), showing that disperser phenotype is also a density-dependent plastic trait in this species.

## Cooperation and colonization efficiency

While emigration is the most frequent focus of empirical work, the fitness consequences of dispersal critically depend on the ability of dispersers to settle and reproduce in the colonized habitat (Clobert et al. 2012). Since dispersing in groups of relatives has been suggested to favor the evolution of cooperation (“budding dispersal”; Gardner and West 2006; Kümmerli et al. 2009), we measured colonization efficiency both from single founder cells and from a group of disperser cells. We found that highly aggregative disperser cells showed significantly reduced colonization efficiency compared to low- and medium-aggregation genotypes when colonization occurred from a single cell (Fig 2a). The larger cell size of dispersers from low-aggregation genotypes relative to highly aggregative ones may explain this difference in single cell colonization success (Fig 3), by providing advantages during colonization because of higher nutrient reserves or improved competitive abilities (Stein and
400 Bronner 1989; Fjerdingstad et al. 2007). The low colonization efficiency of a single cell in
401 highly aggregative genotypes might also result from a stronger need for cooperation during
402 colonization (Christensen et al. 1996, 2001; Gardner and West 2006; Kümmerli et al. 2009;
403 Schtickzelle et al. 2009).

404 When colonization occurred from a group of cells, highly aggregative genotypes were
405 indeed as efficient as low-aggregation genotypes (Fig 2b), as would be expected if group
406 dispersal evolved with cooperation, allowing an escape from kin competition while
407 maintaining the benefits of kin cooperation during dispersal (Gardner and West 2006;
408 Kümmerli et al. 2009; Schtickzelle et al. 2009). Our results suggest that cooperation between
409 kin might be required during transience and/or settlement for cooperative individuals to
410 efficiently colonize a new environment. The maintenance of cooperation in this species could
411 result from an increase in the colonization efficiency of highly cooperative genotypes through
412 group dispersal to levels similar to genotypes with lower cooperation levels.

413 Kin competition has been found to positively affect colonization success in lizards (Cote et
414 al. 2007). Here we found that, as in lizards, higher pre-dispersal density increased
415 colonization efficiency from a single founder cell in low-aggregation genotypes, while it
416 negatively affects colonization efficiency in medium-aggregation genotypes. Furthermore, we
417 found no significant effect of pre-dispersal density on colonization efficiency from a group of
418 cells, suggesting that the potential costs and benefits of pre-dispersal density on colonization
419 from one cell did not affect colonization efficiency when several cells arrived simultaneously
420 in a new patch.

421

422 **Conclusion**

423 The interactions and potential coevolution between cooperation and dispersal have been
424 the focus of considerable theoretical work since dispersal should reduce the value of
425 cooperation among kin, yet alleviate kin competition (Hamilton 1964; West et al. 2002; Lion
426 and Baalen 2008; Aktipis 2011; Parvinen 2013). Our results provide experimental support for
427 the hypothesis that dispersal decisions vary according to an interaction between cooperation
428 strategy and social environment, a process which may help resolve the conflicting pressures
429 of kin-cooperation and kin-competition (Pepper and Smuts 2002; Aktipis 2004, 2011; Pepper
430 2007). Cooperation strategy governed how population density affected emigration and
431 subsequently influenced the efficiency of dispersers to colonize new environments.
432 Furthermore, our results suggest that cooperation between kin during transience and/or
433 settlement might be required for cooperative genotypes to be able to efficiently colonize new
434 habitats after dispersal. Overall, the results from these highly controlled experiments expand
435 our knowledge about the relationship between cooperation and dispersal, highlighting the role
436 of cooperation in shaping non-random movements of organisms across the landscape.

437
438

**Author contributions:** ASC, PW, SJ and JC defined the research theme; ASC, PW and MH
439 designed and performed the experiments. SJ, PW, and NS performed picture analyses, and SJ,
440 PW and DL analyzed the data. SJ wrote the manuscript, and PW, ASC, DL, NS and JC
441 contributed substantially to revisions.

442
443

**Acknowledgments:** This study was supported by the ANR EVO-INF-ECOL and INDHET to
444 SJ and JC, Royal Society of New Zealand ISAT grant to PW, ANR Netselect to ASC, and
445 ARC 10-15/031, F.R.S.-FNRS and UCL-FSR to NS, DL and SJ. NS is Research Associate
446 and DL is Postdoctoral Researcher of the F.R.S.-FNRS. This work is part of the Laboratoire
447 d'Excellence (LABEX) entitled TULIP (ANR-10-LABX-41) for SJ, ASC, MH and JC, and is
448
449 contribution BRCXXX of the Biodiversity Research Centre to which NS, DL and SJ are

450 affiliated.
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616 **Figure 1:** Cooperation governs dispersal decisions in *Tetrahymena thermophila*.

617 **a)** Cooperation strategy mediates reaction norms of dispersal rate to congener density
618 (aggregation * density² interaction: F<sub>2,78</sub> = 9.53; p < 0.001).

619 **b)** The phenotype of dispersers differed depending on their cooperation strategy and
620 population density (aggregation * density² interaction: F<sub>2,78</sub> = 5.70; p = 0.005). Highly
621 aggregative genotypes produced specialized disperser phenotypes that were small and
622 elongated, while low-aggregative genotypes produced dispersers that were bigger but less
623 elongated.

624 Each curve, fitted using individual replicates as data points, corresponds to one aggregation
625 strategy and contains 2 genetic strains with five replicates each [low aggregation strategy (7,
626 4A); medium aggregation strategy (D3, P); high aggregation strategy (E, Q)]. Mean ± SE for
627 the two genotypes of each density per aggregation combination are shown. Points are
628 staggered on the x-axis for clarity.

629
630 **Figure 2:** Cooperation strategy **a)** governed colonization efficiency from one single cell, but
631 **b)** did not significant affect colonization efficiency from several cells. Each curve, fitted using
632 individual replicates as data points, corresponds to one aggregation strategy and contains 2
633 genetic strains [low aggregation strategy (7, 4A); medium aggregation strategy (D3, P); high
634 aggregation strategy (E, Q)]. Mean ± SE for the two genotypes of each density per
635 aggregation combination are shown. Points are staggered on the x-axis for clarity.
636
637

638
639 **Figure 3:** Cell phenotype, cooperation strategy and colonization efficiency from single
638 founder cells. Each curve, fitted using individual replicates as data points, corresponds to one
639 aggregation strategy and contains 2 genetic strains [low aggregation strategy (7, 4A); medium
640 aggregation strategy (D3, P); high aggregation strategy (E, Q)]. Mean ± SE for the two

641 genotypes of each density per aggregation combination are shown.

642
**Table 1:** Factors affecting dispersal decisions and colonization efficiency. Final models after backward elimination are shown (non-significant fixed effects are shown for colonization from a group for clarity). Marginal (*R*²*m*, fixed effects alone) and conditional [*R*²*c*, combined fixed and random effects (i.e. genotypes)] estimates of model fit are shown.

647