Recent work has shown that microorganisms are surprisingly like animals in having sophisticated behaviours such as cooperation, communication, and recognition, as well as many kinds of symbioses. Here we show first that the social amoeba Dictyostelium discoideum has a primitive farming symbiosis that includes dispersal and prudent harvesting of the crop. About one-third of wild-collected clones engage in husbandry of bacteria. Instead of consuming all bacteria in their patch, they stop feeding early and incorporate bacteria into their fruiting bodies. They then carry bacteria during spore dispersal and can seed a new food crop, which is a major advantage if edible bacteria are lacking at the new site. However, if they arrive at sites already containing appropriate bacteria, the costs of early feeding cessation are not compensated, which may account for the dichotomous nature of this farming symbiosis. We also observed farmer Dictyostelium discoideum clones carry bacteria that they do not use as food. We hypothesized that these bacteria may play a defensive role against other D. discoideum clones. In our second study, we investigated the impact of these bacteria-carrying farmers on non-farming D. discoideum clones. We found that the presence of farming clones reduces spore production in non-farmers. Furthermore, this effect increases with frequency of farming clones, demonstrating the vulnerability of non-farming clones to farmers though in this experiment we had not separated the effects of the farmer clone and the bacteria they carry. In our third study we exposed non-farmers to a filtered supernatant from the most common non-food carried bacterium, Burkholderia xenovorans . This supernatant is likely to carry whatever the bacteria are producing. We treated Dictyostelium clones at the beginning of the social stage and found that the supernatant enhanced spore production of farming clones and hurt spore production of non-farming clones. This study shows that the effects of the bacteria can be restricted to a filtered supernatant alone. This discovery of symbiosis of D. discoideum with bacteria, and its impact on social interactions among D. discoideum clones will provide a fertile ground for further experiments on the evolution of sociality.

Dictyostelids (Amoebozoa) are a group of social amoebae consisting of approximately 150 species, which are found in terrestrial habitats worldwide. They are divided into eight major clades based on molecular phylogeny, and within these clades are many species complexes. Some species are seemingly cosmopolitan in distribution, while others are geographically restricted. In this thesis dictyostelids were recovered from high latitude habitats (soils in Sweden and Iceland) as well as from the soles of shoes. Morphological characters and DNA sequence analyses were used to identify isolates that were recovered and delimit new species, as well as to investigate the monophyly of Dictyostelium aureostipes. Nine species were reported from Northern Sweden and four from Iceland. Among the isolates recorded in Sweden were two new species, described as D. barbibulus and Polysphondylium fuscans. P. fuscans was among the four species recovered from footwear, contributing evidence for anthropogenic transport of dictyostelids. Ecological patterns were assessed using linear regression and generalized linear models. The ecological analyses of dictyostelids recovered from Iceland indicate that these organisms are most frequently found in soils of near-neutral pH, but also exhibit a species richness peak in moderately acidic soils. These analyses indicate that in Iceland dictyostelid species richness decreases with altitude, and in the northern hemisphere the species richness increases with decreasing latitude. A three-region analysis of the D. aureostipes species complex indicated that this species is in fact made up of at least five phylogenetically distinct clades, and in light of this the group is in need of taxonomic revision. These results indicate that the dictyostelid species richness is higher than previously known, especially in high-latitude regions, and that even seemingly well-defined species may harbour cryptic diversity. Presently, species ranges may be expanding via anthropogenic dispersal but despite this, the dictyostelids are found to exhibit biogeographic trends well known from macroorganisms, such as a latitudinal gradient of species richness.

A proteína fosfatase do tipo-1 (PP1) é uma das principais proteínas serina/treonina fosfatases (PSTPs) e desempenha papeis fisiológicos tão diversos quanto importantes, tais como a regulação do metabolismo de carboidratos e do ciclo celular. A holoenzima PP1 é constituída por uma subunidade catalítica conservada (PP1c) que está associada a subunidades não-catalíticas que modulam sua localização subcelular, especificidade de substrato e atividade enzimática. Mais de 100 proteínas que interagem com a PP1c já foram identificadas em distintos organismos eucarióticos. Proteínas que interagem com a PP1c são, portanto, a chave para compreender os diferentes papéis biológicos da PP1. A subunidade catalítica da PP1 da ameba social Dictyostelium discoideum (DdPP1c) é codificada por um gene em cópia única o qual é expresso ao longo de todo o ciclo de vida desse organismo. Algumas proteínas que interagem e possivelmente modulam a atividade da PP1 de D. discoideum já foram identificadas, utilizando-se tanto buscas por similaridades na sequência genômica deste microorganismo como ensaios utilizando o sistema de duplo-híbrido em leveduras, utilizando-se a PP1c como isca. Com esta última abordagem, foram selecionados mais de 25 clones distintos de cDNA que codificam proteínas que potencialmente interagem com a DdPP1c, após varreduras de bibliotecas de cDNA de diferentes estágios de desenvolvimento de D. discoideum. Neste trabalho, nós confirmamos que o produto protéico de 11 destes clones interagem com a isca DdPP1c com base em novos ensaios de duplo-híbrido. Os demais clones codificam proteínas que não interagem com DdPP1c ou promovem auto-ativação do gene repórter. Selecionamos para estudos adicionais um clone do gene DDB_G0269300 cujo produto protéico predito de 423 de aminoácidos não tem função ainda conhecida. A sequência codificadora completa de DDB_G0269300 foi clonada para realização de novos ensaios de duplo-híbrido em leveduras, os quais confirmaram a especificidade de sua interação com DdPP1c. A proteína recombinante rDDB_G0269300 foi obtida com sucesso em bactérias, possibilitando a obtenção de anticorpos policlonais em camundongos. O anti-soro anti-rDDB_G0269300 é aparentemente específico no reconhecimento da proteína correspondente em extratos celulares de D. discoideum coletados em 12h e 16 da fase de desenvolvimento. Estes resultados coincidem com dados obtidos através de RT-qPCR que mostram aumento nos níveis dos transcritos de DDB_G0269300 entre 8h e 12h da fase de desenvolvimento, o que é indicativo da sua importância desta proteína durante esta fase do ciclo de vida de Dictyostelium como uma potencial parceira molecular da DdPP1c / Protein phosphatase type-1 (PP1) is a major protein serine/threonine phosphatase (PSTP) which plays as diverse as important physiological roles, such as regulation of carbohydrate metabolism and of cell cycle. The PP1 holoenzyme comprises a conserved catalytic subunit (PP1c) associated with non-catalytic subunits that modulate its subcellular localization, substrate specificity and enzymatic activity. More than 100 proteins that interact with PP1c have been identified in different eukaryotic organisms. Therefore proteins that interact with PP1c are key to the understanding of PP1 different biological roles. The catalytic subunit of PP1 the social amoeba Dictyostelium discoideum (DdPP1c) is encoded by a single copy gene which is expressed throughout the life cycle of this organism. Some proteins that interact with and possibly modulate the activity of D. discoideum PP1 have been identified, using both similarity searches in the genome sequence of this microorganism as yeast two-hybrid screenings using PP1c as bait. With the latter approach, we have selected more than 25 distinct cDNA clones encoding proteins that potentially interact with DdPP1c after screening D. discoideum cDNA libraries from different developmental stages. In this study, we confirmed that the protein product from 11 of these clones interact with the bait DdPP1c based on two-hybrid assays. The other clones encode proteins that either does not interact or promote self-activation of the reporter gene. The clone related to DDB_G0269300 gene that encodes a predicted protein of 423 amino acids with unknown function was selected for further studies. DDB_G0269300 full-length coding sequence was cloned and new yeast two-hybrid assays were performed confirming the specificity of the interaction with DdPP1c. The recombinant protein rDDB_G0269300 was successfully obtained in bacteria and further used for polyclonal antibodies production in mice. The antiserum anti-rDDB_G0269300 is apparently specific for recognition of the corresponding protein in D. discoideum cell extracts collected after 12h and 16h of development. These results agree with RT-qPCR data showing that the levels of DDB_G0269300 transcripts are increased between 8 h and 12 h during the development, which is indicative of its importance during this phase in Dictyostelium life cycle as a DdPP1c potential molecular partner.

A proteína fosfatase do tipo-1 (PP1) é uma das principais proteínas serina/treonina fosfatases (PSTPs) e desempenha papeis fisiológicos tão diversos quanto importantes, tais como a regulação do metabolismo de carboidratos e do ciclo celular. A holoenzima PP1 é constituída por uma subunidade catalítica conservada (PP1c) que está associada a subunidades não-catalíticas que modulam sua localização subcelular, especificidade de substrato e atividade enzimática. Mais de 100 proteínas que interagem com a PP1c já foram identificadas em distintos organismos eucarióticos. Proteínas que interagem com a PP1c são, portanto, a chave para compreender os diferentes papéis biológicos da PP1. A subunidade catalítica da PP1 da ameba social Dictyostelium discoideum (DdPP1c) é codificada por um gene em cópia única o qual é expresso ao longo de todo o ciclo de vida desse organismo. Algumas proteínas que interagem e possivelmente modulam a atividade da PP1 de D. discoideum já foram identificadas, utilizando-se tanto buscas por similaridades na sequência genômica deste microorganismo como ensaios utilizando o sistema de duplo-híbrido em leveduras, utilizando-se a PP1c como isca. Com esta última abordagem, foram selecionados mais de 25 clones distintos de cDNA que codificam proteínas que potencialmente interagem com a DdPP1c, após varreduras de bibliotecas de cDNA de diferentes estágios de desenvolvimento de D. discoideum. Neste trabalho, nós confirmamos que o produto protéico de 11 destes clones interagem com a isca DdPP1c com base em novos ensaios de duplo-híbrido. Os demais clones codificam proteínas que não interagem com DdPP1c ou promovem auto-ativação do gene repórter. Selecionamos para estudos adicionais um clone do gene DDB_G0269300 cujo produto protéico predito de 423 de aminoácidos não tem função ainda conhecida. A sequência codificadora completa de DDB_G0269300 foi clonada para realização de novos ensaios de duplo-híbrido em leveduras, os quais confirmaram a especificidade de sua interação com DdPP1c. A proteína recombinante rDDB_G0269300 foi obtida com sucesso em bactérias, possibilitando a obtenção de anticorpos policlonais em camundongos. O anti-soro anti-rDDB_G0269300 é aparentemente específico no reconhecimento da proteína correspondente em extratos celulares de D. discoideum coletados em 12h e 16 da fase de desenvolvimento. Estes resultados coincidem com dados obtidos através de RT-qPCR que mostram aumento nos níveis dos transcritos de DDB_G0269300 entre 8h e 12h da fase de desenvolvimento, o que é indicativo da sua importância desta proteína durante esta fase do ciclo de vida de Dictyostelium como uma potencial parceira molecular da DdPP1c / Protein phosphatase type-1 (PP1) is a major protein serine/threonine phosphatase (PSTP) which plays as diverse as important physiological roles, such as regulation of carbohydrate metabolism and of cell cycle. The PP1 holoenzyme comprises a conserved catalytic subunit (PP1c) associated with non-catalytic subunits that modulate its subcellular localization, substrate specificity and enzymatic activity. More than 100 proteins that interact with PP1c have been identified in different eukaryotic organisms. Therefore proteins that interact with PP1c are key to the understanding of PP1 different biological roles. The catalytic subunit of PP1 the social amoeba Dictyostelium discoideum (DdPP1c) is encoded by a single copy gene which is expressed throughout the life cycle of this organism. Some proteins that interact with and possibly modulate the activity of D. discoideum PP1 have been identified, using both similarity searches in the genome sequence of this microorganism as yeast two-hybrid screenings using PP1c as bait. With the latter approach, we have selected more than 25 distinct cDNA clones encoding proteins that potentially interact with DdPP1c after screening D. discoideum cDNA libraries from different developmental stages. In this study, we confirmed that the protein product from 11 of these clones interact with the bait DdPP1c based on two-hybrid assays. The other clones encode proteins that either does not interact or promote self-activation of the reporter gene. The clone related to DDB_G0269300 gene that encodes a predicted protein of 423 amino acids with unknown function was selected for further studies. DDB_G0269300 full-length coding sequence was cloned and new yeast two-hybrid assays were performed confirming the specificity of the interaction with DdPP1c. The recombinant protein rDDB_G0269300 was successfully obtained in bacteria and further used for polyclonal antibodies production in mice. The antiserum anti-rDDB_G0269300 is apparently specific for recognition of the corresponding protein in D. discoideum cell extracts collected after 12h and 16h of development. These results agree with RT-qPCR data showing that the levels of DDB_G0269300 transcripts are increased between 8 h and 12 h during the development, which is indicative of its importance during this phase in Dictyostelium life cycle as a DdPP1c potential molecular partner.

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.