The maintenance of cooperation and altruism in the face of manipulation by exploitative cheaters that reap the benefits of cooperative acts without paying the associated costs is a conundrum in evolutionary biology. Cheaters should spread through a population causing it to crash, yet cooperation is common. There are many models and theories that attempt to explain this apparent contradiction. The social amoeba Dictyostelium discoideum, like many microbial species has been used as a model organism to test these theories and to begin to understand the genetic mechanisms behind social behaviours. The aim of this PhD project is to quantify the interactions that occur between naturally-occurring genotypes during social competition in order to identify the types of cheating behaviours and to understand the evolutionary consequences of such behaviours. I first demonstrate that there is a social hierarchy of genotypes and that cheaters can increase their own fitness by increasing their own spore allocation or decreasing their partner's allocation the precise nature of which is dependent upon unique interactions between each competing pair. I also show that the outcome of social competition is dependent upon the physical environment where it can be significantly reduced, or even avoided by segregation of genotypes during development. Finally, it is demonstrated in a collaborative project that much of the observed social behaviour can be explained in terms of the production of and response to developmental signals.

Social amoebae are eukaryotic organisms that inhabit soil of almost every climate zone. They are remarkable for their switch from unicellularity to multicellularity as an adaptation to starvation. When starved, millions of single cells aggregate and form a multicellular fruiting body, which contains reproductive spore cells and dead stalk cells, which help in spore dispersion. This costly behavior made social amoebae a model system for addressing major questions of the evolution of cooperation and multicellularity. In this study we look at three different aspects of social amoebae behavior; aggregation, non-aggregation and competition, and ask how they contribute to our understanding of cooperation in social amoebae and microbial systems in general.We explored the known but neglected observation that, upon starvation, not all cells aggregate and engage in multicellular development. We describe phenotypically and genetically non-aggregating cell proportion in D. discoideum species. Both aggregating and non-aggregating strategy are costly or beneficial depending on duration of starvation. With our computational model we propose that partitioning the population into unicellular and multicellular states is adaptive in fluctuating environments with unpredicted duration of starvation periods. Social amoebae may therefore lie at the intersection of cooperation and bet-hedging. In the second part, we provide a new framework for addressing the contrasting observations of high genetic diversity in natural populations of social amoebae and experimentally suggested low diversity-high relatedness required for cooperation. We propose that complex life cycle of social amoebae provides multiple competition points that can possibly play an important role in maintaining diversity and cooperation. We explore this experimentally and computationally by looking at competition over the whole life cycle between 6 natural isolates of D. discoideum. Our simulation model indicates that competition at different stages of the life cycle can lead to exclusion of "social winners". Though we failed to explain strain coexistence. Although preliminary, our results emphasize the importance of integrating species ecology in cooperative studies.Finally, we focus on a new aggregation dynamics in P. pallidum species observed in our lab. Aggregation is a population level process during which population gets divided into numerous subpopulations/aggregates that face selection independently. Such population partitioning can have strong evolutionary consequences on cooperation that have not yet been explored experimentally. We describe the population dynamics qualitatively and propose several quantitative measurements of population partitioning into aggregates. Our preliminary results suggest that there is a preference for aggregates of certain size, but there is no spatial organization of aggregates.

By combining laboratory experiments with field work, I have looked at the following aspects of cellular slime mould (CSM) biology: (a) the genetic structure of social groups (fruiting bodies) in the wild and its relation to the role of large mammals as dispersal agents; (b) social behaviour in clonal, intra-species polyclonal and interspecies social groups and (c) fitness-related trade-offs with respect to life history traits as a possible mechanism for coexistence and cooperative behaviour in CSMs. The major findings of this study are as follows: (a) individuals belonging to different strains of a species, different species and genera occur in close proximity, even on a speck of soil (250µm–1mm) or the same dung pat; (b) social groups formed in the wild by Dictyostelium giganteum and D. purpureum are generally multiclonal; (c) genetically diverse strains can co-aggregate and form chimaeric social groups; (d) in chimaeric social groups, strains differ in their relative sporulation efficiencies; (e) the fact that strains co-exist in spite of this may be attributable in part to trade-offs between various fitness-related traits as can be demonstrated in the case of wild isolates of D. giganteum in pair wise mixes. The Dictyostelids or CSMs are haploid, eukaryotic, soil dwelling social amoebae with an unusual life cycle (Bonner, 1967; Raper, 1984). They exist as single cells in the presence of food (bacteria, yeast, fungal spores). Once the food is exhausted, they enter the social phase of their life cycle. Approximately 102 to 106 amoebae aggregate at a common collection point and form a starvation resistant structure called the fruiting body. In many species a fruiting body is made up of an aerial stalk of dead cells and a ball of viable spores on top. In other CSM species (not part of this study), all amoebae in a fruiting body differentiate into spores and the stalk is an extracellular secretion. The CSM life cycle raises fundamental questions related to the evolution of an extreme form of ‘altruism’ in the form of reproductive division of labour in social groups. The spore–stalk distinction in the CSMs is analogous to the germ–soma distinction in metazoans, although, the CSMs achieve multicellularity not by repeated divisions of a zygote but via the aggregation of many cells which may or may not be clonally related (Bonner, 1982; Kaushik and Nanjundiah, 2003). Social behaviour in the CSMs offers interesting parallels to what is seen in the social insects (Gadagkar and Bonner, 1994). The origin and maintenance of ‘altruism’ has been a long-standing issue in sociobiology. Because of their simple life cycle and experimental tractability, the CSMs are ideal for studying the evolutionary origin and maintenance of social behaviour, in particular of ‘altruistic’ behaviour. By elevating spores above soil level, stalk cells, protect them from noxious compounds and predators present in soil and also facilitate their passive dispersal. In the course of doing so they die. The death of stalk cells appears to be an extreme form of altruism. Knowledge of the genetic structure of social groups and populations including patterns of kinship is essential for modelling the evolution of ‘altruism’. Thus, it is important to understand the genetic structure of CSM social groups in the wild. For this, social groups (fruiting bodies) of CSMs were isolated from undisturbed forest soil of the Mudumalai forest reserve in South India. Soil and animal dung samples were brought to the laboratory and quasi-natural social groups were generated by inoculating the samples on non-nutrient agar. The fruiting bodies from various CSM species were formed by these isolates. Since soil and dung samples were not perturbed in any way, the fruiting bodies were formed as they would have in nature. When compared to soil, dung samples contained a higher CSM diversity and more CSM propagules. The presence of CSMs in fresh animal dung makes it likely that they were transported and dispersed over long distances through the gut of these animals. Such dispersal is likely to be preceded by a thorough mixing of spores in the gut. That increases the probability of co-occurrence of different genotypes in a social group. This possibility was confirmed by genetically characterizing spores in social groups of Dictyostelium giganteum and D. purpureum collected from the wild. Random amplification of polymorphic DNA (RAPD), a simple and reliable molecular technique, was used for genotyping spores within a fruiting body. 17 fruiting bodies (8 from animal dung and 9 from soil) were studied. 15 out of 17 (9 out of 11 of D. giganteum and 6 out of 6 D. purpureum) were polyclonal; the minimum number of distinct clones in a single fruiting body was 3 to 7 (animal dung) and 1 to 9 (soil). Therefore in D.giganteum and D. purpureum, chimaeric social groups seem to be the norm. This suggests that other species of CSMs form intra-species chimaeric social groups in wild, though clonal fruiting bodies occur too. The next objective of this thesis was to test whether genetic heterogeneity had functional consequences. That is, when different strains come together in an aggregate, do they contribute equally to the reproductive (spore) and non-reproductive (stalk) pathways? Amoebae of different clones (strains) of D. giganteum or D. purpureum were mixed and developed together and the number of spores formed by each strain was counted. These experiments confirmed that strains of D. giganteum or D. purpureum can aggregate together and form chimaeric fruiting bodies. The ability to mix (measured as the frequency of chimaerism) depended on the strains used and varied from one mix to another. One strain was often found to ‘exploit’ the other during sporulation, that is, it formed more spores than its expected share. Despite this, strains are found in very close proximity in the soil, which raises an important question: when one strain is more efficient at sporulating than other, how can the two co-exist stably? To investigate what might lie behind the stable co-existence of strains, I studied various fitness-related traits in the life cycle of D. giganteum. They included the rate of cell division, the time taken to go through multicellular development, the efficiency of slug migration through various depths of soil and the probability of differentiation into a spore. Measurements were carried out on strains taken separately and on their pair wise mixes. Five different D. giganteum wild strains (46a3, 46d2, 48.1a1, F5 and F16) were used. All were isolated from the Mudumalai forest (India). 46a3 and 46d2 came from soil within 10 cm of each other, 48.1a1 from soil about 200m away from 46a3; and F5 and F16 from the same fruiting body (Kaushik et al., 2006; Sathe et al., 2010). Members of a pair differed significantly in the measured fitness-related traits. For example, in the case of 48.1a1 and 46d2, 48.a1 grew faster than 46d2 both individually and in a mix. After starvation, 48.1a1 formed fruiting bodies faster than 46d2; a mix of the two developed at the rate of the faster member, implying that the slower one (46d2) gained from the association with 48.1a1. During slug migration, slugs formed by 48.1a1 came up through a higher depth of soil than 46d2 slugs and did so earlier. Chimaeric slugs were like the more efficient member, 48.1a1, in terms of the maximum depth of soil that was covered, but like the less efficient member, 46d2, in terms of the time taken for slugs to be seen on the soil surface. 48.1a1 seems to have an advantage over 46d2 in all these respects. However, during sporulation in chimaeras, 48.1a1 formed relatively fewer spores than 46d2. Similar trade-offs were seen in all mixes. F5 and F16 displayed an unexpected feature during sporulation; the spore-forming efficiency of either strain depended on its proportion in the initial mix in a frequency-dependent manner that was consistent with a stable equilibrium. Thus, trade-offs between different fitness-related traits contribute to the co-existence of strains. Next, I studied interactions between members of different CSM species. Several species of CSMs were isolated from the same environment (Sathe et al., 2010); a question of interest was to see if amoebae of different species came together to form a chimaeric multicellular body. Five strains (two D. purpureum and three D. giganteum) were used in this study. Amoebae of D. giganteum and D. purpureum co-aggregated. However, there were factors that caused amoebae of the two species to sort out thereafter. The extent of segregation differed between strains, a characteristic that inter-species mixes share with intra-species mixes. In conclusion, the ability of cellular slime moulds to form multiclonal social groups in the wild suggests that one should look to factors in addition to close relatedness to understand the evolution of CSM social behaviour. The existence of fitness-related trade-offs between different traits indicates that individual-level selection can also contribute to the maintenance of chimaeric social groups.

Les amibes sociales sont des organismes eucaryotes présents dans le sol de presque toutes les zones climatiques. Ils sont remarquables pour leur passage d'un état unicellulaire à un état multicellulaire en réponse à la carence en nutriments. En période de carence, des millions de cellules forment des agrégats qui constituent chacun un nouvel organisme multicellulaire, contenant des spores, cellules reproductives, et des cellules de tige, cellules mortes qui favorisent la dispersion des spores. Ce comportement, de par le coût payé par les cellules de tige, a permis d'utiliser les amibes sociales en tant que système-modèle pour aborder des questions majeures de l'évolution de la coopération et de la multicellularité. Dans cette étude, nous examinons trois aspects différents du comportement des amibes sociales; agrégation, non-agrégation et compétition, et nous analysons comment ces aspects contribuent à notre compréhension de la coopération chez les amibes et systèmes microbiens en général.Nous avons exploré le fait bien connu mais négligé qu'en phase de carence nutritive, une fraction des cellules ne participent pas à la formation des agrégats pas et ne sont pas engagées dans le développement multicellulaire. Nous décrivons les facteurs phénotypiques et génétiques qui déterminent la fraction de cellules hors-agrégats chez D. discoideum. Les deux stratégies, d'agrégation et de non-agrégation, sont coûteuses ou bénéfiques d'un point de vue évolutif selon la durée de la phase de carence. Nous avons développé un modèle pour simuler ce processus. Nous proposons que le partitionnement de la population dans des états unicellulaire et multicellulaire est adaptative dans des environnements fluctuants avec une durée imprévisible des périodes de carence nutritive. Les amibes sociales sont donc situées à l'intersection de deux thèmes émergents en évolution microbienne, la coopération et le "placement des paris".Dans la deuxième partie, nous proposons un nouveau cadre pour aborder les observations a priori contradictoires de la diversité génétique dans les populations naturelles d'amibes sociales et une faible diversité nécessaire pour la coopération. Nous proposons que le cycle de vie complexe des amibes sociales fournit plusieurs points de compétition qui peut servir à la fois comme stabilisateur de la diversité et de la coopération. Nous explorons cette hypothèse expérimentalement avec un modèle en analysant la compétition entre 6 isolats naturels de D. discoideum. Notre simulation-modèle indique que la compétition à différents stades du cycle de vie peut conduire à l'exclusion des "gagnants sociaux". Toutefois nous n'avons pas réussi à expliquer la coexistence à long terme de souches génétiquement distinctes. Bien que préliminaires, nos résultats soulignent l'importance d'intégrer l'écologie des espèces dans les études de coopération microbienne.Enfin, nous nous concentrons sur une nouvelle dynamique d'agrégation chez P. pallidum observée dans notre laboratoire. L'agrégation est un processus au niveau de la population au cours duquel la population se divise en nombreuses sous-populations (agrégats) qui font face à la sélection de manière indépendante. Un tel fractionnement de la population peut avoir de fortes conséquences évolutives du point de vue de la coopération qui n'ont pas encore été explorées expérimentalement. Nous décrivons la dynamique des populations qualitativement et proposons plusieurs mesures quantitatives de partitionnement de la population en agrégats. Nos résultats préliminaires suggèrent qu'il existe une préférence pour les agrégats d'une certaine taille, mais qu'il n'existe aucune organisation spatiale des agrégats. / Social amoebae are eukaryotic organisms that inhabit soil of almost every climate zone. They are remarkable for their switch from unicellularity to multicellularity as an adaptation to starvation. When starved, millions of single cells aggregate and form a multicellular fruiting body, which contains reproductive spore cells and dead stalk cells, which help in spore dispersion. This costly behavior made social amoebae a model system for addressing major questions of the evolution of cooperation and multicellularity. In this study we look at three different aspects of social amoebae behavior; aggregation, non-aggregation and competition, and ask how they contribute to our understanding of cooperation in social amoebae and microbial systems in general.We explored the known but neglected observation that, upon starvation, not all cells aggregate and engage in multicellular development. We describe phenotypically and genetically non-aggregating cell proportion in D. discoideum species. Both aggregating and non-aggregating strategy are costly or beneficial depending on duration of starvation. With our computational model we propose that partitioning the population into unicellular and multicellular states is adaptive in fluctuating environments with unpredicted duration of starvation periods. Social amoebae may therefore lie at the intersection of cooperation and bet-hedging. In the second part, we provide a new framework for addressing the contrasting observations of high genetic diversity in natural populations of social amoebae and experimentally suggested low diversity-high relatedness required for cooperation. We propose that complex life cycle of social amoebae provides multiple competition points that can possibly play an important role in maintaining diversity and cooperation. We explore this experimentally and computationally by looking at competition over the whole life cycle between 6 natural isolates of D. discoideum. Our simulation model indicates that competition at different stages of the life cycle can lead to exclusion of “social winners”. Though we failed to explain strain coexistence. Although preliminary, our results emphasize the importance of integrating species ecology in cooperative studies.Finally, we focus on a new aggregation dynamics in P. pallidum species observed in our lab. Aggregation is a population level process during which population gets divided into numerous subpopulations/aggregates that face selection independently. Such population partitioning can have strong evolutionary consequences on cooperation that have not yet been explored experimentally. We describe the population dynamics qualitatively and propose several quantitative measurements of population partitioning into aggregates. Our preliminary results suggest that there is a preference for aggregates of certain size, but there is no spatial organization of aggregates.