Division of labour is a hallmark of eusocial insects and is believed to be a major factor in their evolutionary success and ecological dominance. Division of labour can be of two kinds – reproductive division of labour where a minority of individuals are egg-layers or reproductives (kings and queens) and the majority are workers or non-reproductives involved mostly in non-reproductive tasks of the colony (workers). Kings/queens and workers are often referred to as separate castes within a social insect colony. There may be further non-reproductive division of labour within the worker caste, based on their morphology or age. In primitively eusocial organisms there is no morphological caste differentiation between the egg-layers and non-egg-layers resulting in greater flexibility in the social roles of individuals within a colony. This creates a very interesting scenario to study the mechanism of division of labour. Moreover our knowledge regarding division of labour especially non-reproductive division of labour is very limited for primitively eusocial organisms. In this thesis I have studied division of labour in a primitively eusocial wasp species, Ropalidia cyathiformis. R. cyathiformis is a tropical primitively eusocial wasp with a perennial nesting cycle. This species usually has a single dominant queen and several workers. I studied reproductive and non-reproductive division of labour, as well as the role of dominance behaviour in the regulation of both reproductive and non-reproductive activities. In addition to this I have also compared my findings with what is already known in the well-studied congeneric species, Ropalidia marginata. Reproductive division of labour To understand reproductive division of labour in R. cyathiformis, I studied queen succession, by experimentally removing the queen. When the queen was removed, one and only one individual increased her aggression and became the new queen of the colony, unchallenged by any other worker. Such a successor was referred to as a potential queen (PQ) until she lays her first egg. By removing the queen and successive PQs, I showed that there is not just one successor but a strict reproductive hierarchy of up to 3 PQs, who succeed the queen one after the other. Of many variables tested, I found that only the frequency of dominance behaviour was a significant predictor of whether or not an individual is part of the reproductive hierarchy and also of her position in the hierarchy. Dominance behaviour however does not perfectly predict the position of an individual in the reproductive hierarchy because I showed that an average of three more dominant individuals, are bypassed when an individual becomes the next queen or PQ. This was in contrast to the reproductive hierarchy in the congeneric Ropalidia marginata, where age rather than dominance behaviour was a predictor (though imperfect once again) of an individual’s position in the queue. Taken together, my results suggest that (a) these two sister species have evolved two rather different mechanisms of reproductive caste differentiation, (b) that neither of them strictly conform either to the so called “temperate” or “tropical” patterns of queen succession seen in most other species studied so far. Non-reproductive division of labour As mentioned above, non-reproductive division of labour in eusocial insects is based on either the morphology or the age of the individuals within the colony. Since there is no morphological castes present in primitively eusocial species, I focussed on the effect of age on division of labour in R. cyathiformis. I analysed the frequency as well as the probability of performance of four functionally significant tasks namely, two intranidal tasks – feed larva and build as well as two extranidal tasks – bring food and bring building material. I measured absolute as well as relative ages of the wasps. I found that there is an effect of age on division of labour. Age of first performance of the tasks indicated a clear sequence for the initiation of the tasks with intranidal tasks initiated before extranidal tasks. The frequency of task performance (FTP) and absolute age better explained the variation in the data as compared to probability of task performance (PTP) and relative age. This was in contrast to the pattern of age polyethism found in the congeneric species, Ropalidia marginata, where PTP and relative age better explains the variation in the data. This suggests a more flexible age-dependant division of labour in R. marginata and a rigid age polyethism in R cyathiformis. In addition I found that there was no clear-cut partitioning of the intranidal and extranidal tasks in R. cyathiformis, whereas in R. marginata, it has been shown that the frequency of the intranidal tasks decline with age while that of extranidal tasks increase with age. When taken together, I could say that R. marginata has a more strongly developed age polyethism as compared to R. cyathiformis. This study also shows an evolution of age polyethism with R. cyathiformis behaving more like a typical primitively eusocial species while R. marginata more like a highly eusocial species. Role of dominance behaviour in reproductive and non-reproductive division of labour When reproductive regulation in R. cyathiformis was studied, I found that queens of this species target the potential queen (PQ) by showing the maximum frequency per hour of dominance behaviour to the PQ. The PQs on the other hand seem to show the maximum amount of dominance behaviour towards newborns (wasps of age class 0-5 days). Queens seem to regulate only reproductive activities and not the non-reproductive activities as there was no difference in the frequency of both feed larva and bring food behaviour in the colony even after removing the queen. It also appears that dominance behaviour is not used to signal hunger or regulate foraging as there was no significant correlation between the frequency per hour of bring food behaviour and dominance behaviour received. Moreover the foragers do not receive more aggression than other wasps in the colony from either the queen, PQ or intranidal workers. I also found a significant positive correlation between the frequency per hour of bring food behaviour and feed larva behaviour implying that foraging might be a self-regulated process in this species. Hence in R. cyathiformis it appears that dominance behaviour is used only for regulation of reproductive division of labour and not for regulation of non-reproductive division of labour. This was in contrast to the congeneric species, R. marginata where the opposite has been shown to be true; the reproductive regulation is achieved by means of pheromones produced by the queen and work organisation follows a decentralised self-organised manner with intranidal workers signalling or regulating foragers using dominance behaviour. Comparison with Ropalidia marginata Ropalidia cyathiformis and Ropalidia marginata, although congeneric species co-existing in the same habitat, have evolved very different mechanisms for division of labour. R. marginata exhibiting features such as 1) presence of a docile queen 2) reproductive regulation by means of pheromones 3) strongly developed and flexible age polyethism 4) decentralised work organisation seem to be more similar to a highly eusocial organism than to a primitively eusocial species. R. cyathiformis on the other hand seems to exhibit several features typical to a primitively eusocial species, such as 1) presence of a dominant queen 2) reproductive regulation by physical means 3) relatively weak and rigid age polyethism 4) self-regulatory method of work organisation. Hence the two species seem to be at two different stages of evolution with R. marginata appearing to be intermediate between primitively and highly eusocial species.

Reproduction is the avenue for gaining direct fitness. But in certain species some individuals do not reproduce, instead gain indirect fitness by helping relatives to reproduce; the prime examples for this come from the worker caste of social insects like ants, bees and wasps. For explaining such a perplexing paradox, also known as altruism, W.D. Hamilton proposed that individuals can gain fitness in two ways: directly, by reproducing (direct fitness), and indirectly, by helping relatives to reproduce (indirect fitness). Indirect fitness has since been the main focus for explaining the evolution of workers while usually overlooking the fact that workers can also gain direct fitness. One of the avenues for gaining direct fitness by workers is nest foundation and we have studied this phenomenon in a primitively eusocial wasp Ropalidia marginata. We found that workers routinely leave their natal nests to initiate new nests either alone or with a few other wasps. Before leaving their natal nests, such workers prepare in several ways for nest foundation, like enhancing their nutrient reserves and forming outside nest aggregations to engage in dominance interactions. Next, we investigated the emergence of cooperation and division of labour in newly founded nests and how these affect the productivities of the new nests. We found that while two wasps are sufficient for the emergence of cooperation and reproductive division of labour (DOL), it takes three wasps for non-reproductive DOL to emerge; cooperation and reproductive DOL are not sufficient for increasing colony productivity which comes about only with the addition of non-reproductive DOL. Finally, we found that it is ageing and nutrition, and not work done towards gaining indirect fitness that affect workers’ potential of gaining future direct fitness by independent reproduction via nest foundation, in other words, current indirect fitness is not incompatible with future direct fitness.

Reproductive division of labor is the most distinctive characteristic of the social Hymenoptera; some individuals reproduce and others forego their own reproduction to raise non-descendant offspring. In species where females are reproductively totipotent and lack morphologically distinct castes, there is potential for reproductive conflict because more than one female in a colony may attempt direct reproduction. I focused my dissertation research on a subtropical population of the primitively eusocial paper wasp, Mischocyttarus mexicanus, to investigate the initiation, establishment, and development of the colony before the emergence of adult offspring. Female M. mexicanus exhibit variation in behavior and task performance, and switch between reproductive and non-reproductive roles. These changes in behavior and reproduction may be influenced by social context. In three studies, I investigated the role of social context on reproduction, behavior, and brain structure. In the first study, I tested the role of body size, reproductive potential, and immediate egg-laying potential on the reproductive tactic employed by females. I found that large females either became solitary foundresses or became part of a group-initiated colony. In contrast, small females left their natal colony and pursued joining other colonies. This joiner tactic is unique to this population and has not been observed in temperate zone populations. I also found that subordinate females had the potential to lay eggs if given the opportunity. This suggests an incentive to remain in a colony for future opportunities of direct reproduction. In the second study, I investigated the effect of three variables on non-nestmate acceptance: non-nestmate age, stage of colony development, and non-nestmate aggressive behavior. I demonstrated that non-nestmate acceptance was context-dependent. Both non-nestmate age and stage of colony development had an effect on the proportion of accepted non-nestmates. Although, non-nestmate aggressive behavior did not affect non-nestmate acceptance, it did trigger an aggressive response from colony nestmates. In the third study, I assessed the relationship of Mushroom Bodies (MB) volume, the brain neuropils associated with learning and memory, to environmental conditions and social interactions. I compared MB volume of newly-established colonies initiated by solitary foundresses to groups of foundresses. In addition, I performed laboratory experiments to differentiate between the effect of environmental conditions and social interactions. I found a positive relationship between MB volume and environmental conditions including light intensity and foraging experience. In contrast to previous studies, I found no association between MB volume and social interactions. Ovary development was positively correlated with MB development. This result suggests that although reproductive dominance is established in newly-initiated colonies, social dominance may not yet be established. In summary, my studies found an effect of social context on behavior, adoption of reproductive tactics and brain structure in colonies of M. mexicanus during the offspring pre-emergence phase.

Altruism in its extreme form is seen in social insects where most individuals give up their own reproduction and work to rear the offspring of their queen. The origin and evolution of such sterile worker castes remains a major unsolved problem in evolutionary biology. Primitively eusocial polistine wasps are an attractive model system for investigating this phenomenon. Ropalidia marginata (Lep.) (Hymenoptera: Vespidae) is one such tropical primitively eusocial wasp, in which new nests are initiated either by a single foundress or by a group of female wasps. Worker behaviour in Ropalidia marginata cannot be satisfactorily explained by the haplodiploidy hypothesis due to the existence of polyandry and serial polygyny which reduce intra-colony genetic relatedness to levels lower than the value expected between a solitary foundress and her offspring. Besides, wasps appear to move frequently between newly initiated nests, perhaps further reducing intracolony genetic relatedness. To study social organization and examine the possibility of kin recognition and task specialization under conditions of low intra-colony relatedness, genetically mixed colonies were created by introducing alien one-day old wasps onto recipient nests. As a first step I have tried to determine the factors that influence the acceptance of foreign wasps onto established colonies. I have introduced wasps between 1 to 20 days of age from donor colonies located at least 10 km away onto 12 different recipient colonies, observed these wasps for a period of 10 hours and later dissected them to examine their ovarian condition. Observations were carried out in the blind i.e. the observer was unaware of the identity of the wasps. Wasps upto 6 days of age were accepted by the alien nests. Older wasps may have been rejected because their relatively better ovarian condition may have been perceived as a reproductive threat to the recipient nest. Alternatively, younger wasps may have been accepted because they may be more easily moulded to the desired roles or due to some other correlate of age per se independent of ovarian condition. Although ovarian condition appeared to influence the probability of acceptance, it was not statistically significant in the presence of age in multiple regression models, making a favourable case for the 'ease of moulding hypothesis' or 'age per se hypothesis' over the 'reproductive threat hypothesis'. In any case these findings gave me a method to create genetically mixed colonies. On 12 different nests Ropalidia marginata, I similarly introduced one-day old wasps and thus created genetically mixed colonies. Such an introduction simulates the eclosion of distantly related individuals which is quite common on nests of R. marginata due to the presence of serial polygyny. About 7 such wasps were introduced per colony and the introductions were so arranged as to matched with natural eclosions on the recipient nest. After 7 days following the last introduction, colonies were observed for 20 hours each. Alien wasps became well integrated and performed most of the behaviours and tasks shown by the natal wasps. There was no evidence of kin recognition or task specialization between natal and introduced wasps. The introduced wasps also sometimes became replacement queens. In an attempt to test the costs in terms of brood rearing efficiency, of living in such genetically variable groups, I created kin and non-kin pairs of wasps in plastic containers. They were provided with ad libitum food, water and building material. The nests initiated were monitored till an adult offspring eclosed. There were no detectable differences in either the productivities or the developmental periods of immature stages in the kin and nonkin pairs suggesting that there is no apparent cost of living with unrelated or distantly related individuals. To compare the extent of cooperation between the two wasps in kin and non-kin pairs, I conducted behavioural observations on 12 pairs each of kin and nonkin wasps. I found no difference in the rates at which the non-egg layers brought food and pulp, fed larvae and built the nest in the kin and nonkin pairs suggesting that cooperative nest building and brood rearing was common to the kin as well as non-kin pairs. The results reported here strengthen the idea that factors other than genetic relatedness must play a prominent role in the maintenance of worker behaviour in Ropalidia marginata.

This thesis has looked at the proximate mechanisms by which eusociality is maintained in colonies of the primitively eusocial wasp Ropalidia marginata. Unlike other typical primitively eusocial species, the R, marginata queen is remarkably docile and non-interactive and hence cannot possibly use aggression to maintain her status. Recent evidence hints at pheromonal queen signalling through the Dufour’s gland. Hence, queen-worker difference in Dufour’s gland composition has been studied in details. Queens and workers differ with respect to overall composition of Dufour’s glands, categories of compounds, and individual compounds as well. The Dufour’s gland compounds may be having a bouquet effect in queen signalling, with individual compounds being less important than the overall composition. The queen pheromone also appears to be an honest signal of fertility, as compounds that differ consistently between queens and workers are correlated with ovarian development of queens, and solitary foundresses and potential queens, who are intermediate between queens and workers in ovarian development, are intermediate in their Dufour’s gland profile as well. When the queen is removed from a colony, one of the workers (potential queen, PQ) shows high aggression, and if the queen is not returned, goes on to become the next queen of the colony. The aggression of PQ comes down as a function of time since queen removal, and correlated with this, the ovaries of PQ increase. Dufour’s gland profile of PQ is similar to workers immediately after queen removal, but comes closer to queens with passage of time. This hints at an interesting transition in maintenance of eusociality from “queen control” by aggression to “queen signal” by pheromone during the queen establishment phase. It has generally been assumed that one set of chemicals can carry multiple information, namely queen signal and colony signal. Initial statistical analysis of chemical composition data showed that perhaps both caste and colony signals can be conveyed by the Dufour’s gland compounds, but detailed analysis cast some doubt on this, as the Dufour’s gland compounds could not be separated into non-overlapping subsets with respect to importance in caste and colony discrimination. A bioassay showed that the wasps do not make colony discrimination from Dufour’s gland compounds. This suggests that the ability to statistically differentiate groups of organisms from their chemical profiles does not guarantee similar discrimination by the organisms themselves, emphasising the need for bioassays to resolve such issues.

In societies where all individuals are reproductively totipotent and yet, at a given time only one of them reproduces, it is interesting to examine the factor(s) that may influence and predict who will be the reproductive. I am investigating various behavioural, morphological and physiological parameters in the primitively eusocial wasp Ropalidia marginata, and their role in determining the current reproductive and her future successors. In several group-living species, especially in primitively eusocial ones, a strong link between behavioural dominance and reproductive dominance is observed. Hence, I am also investigating the possible determinants of behavioural dominance in R. marginata. I have carried out my study on artificially constituted pairs of wasps as well as in natural colonies in laboratory cages, which represent the founding phase and the established phase in the colony cycle, respectively. Chapter 1: Behavioural and Reproductive Dominance in Pairs of R. marginata Age and body size had no effect on behavioural dominance in pairs of R. marginata, whereas prior experience of behavioural dominance affected future dominance status, indicating presence of winner- and loser-effects. Dominance ranks are relatively stable. This is different from what has been found in colonies, where dominance ranks sometimes change on a daily basis. Body size had no effect, whereas age and behavioural dominance had a significant effect on reproductive dominance in pairs, with older individuals and more dominant individuals having a higher probability of becoming the reproductive. Since no relationship was found between age and behavioural dominance, we predict that the underlying mechanisms by which age and behavioural dominance affect reproductive dominance and independent of each other. This study gives a clear indication that age and behavioural dominance are important variables that determine the reproductive individual during the founding phase of the colony. Chapter 2: Comparison of Dominance Indices and Recommendations for their Use When several individuals interact with each other as in colonies, in a differential and sometimes in a preferential manner, it is difficult to attribute dominance ranks to individuals. Dominance indices are employed to simplify these interactions and rank individuals in dominance hierarchies. Since the rationale behind using a particular dominance index is seldom given in behavioural literature, a comparison of three dominance indices was carried out in second part of the thesis. Each index was gauged on how similar are its ranks as compared to other two indices. Indices were also compared based on the number of untied or unique ranks they attributed. The index that gave least number of ties in ranks was assumed to be better than others. In addition to data from R. marginata colonies, I used data from R. cyathiformis colonies (a congeneric species which behaves more like a typical primitively eusocial species), and artificial data sets, to increase variability in the interaction patterns. We found that each of the indices had their own advantages and disadvantages. In species like R. marginata and R. cyathiformis, where only a few pairs show interactions, and among those who do, very few show reversals, Frequency-based Dominance Index (FDI) is the recommended index of choice. Studies like these will help in understanding how dominance indices operate under certain situations before applying them to construct hierarchies. Chapter 3: Behavioural and Reproductive Dominance in Colonies of R. marginata Age does not affect behavioural dominance, whereas winner and loser effects exist in colonies of R. marginata, just as in pairs. When analysed in detail, I found that colonies of R. marginata showed fewer proportion of pairs interacting, and lower frequency/hour/pair of dominance-subordinate interactions as compared to experimentally paired individuals (from 1st chapter). However, the dominance displays and behaviours were much more intense and severe in colonies. After dominance hierarchies are already established in colonies, frequent need to show dominance behaviour may not arise, due to familiarity between interacting individuals. However, since individuals are possibly aware of each others’ strengths due to past interactions, dominance behaviours are much more severe when contests do happen. My results show that there might be some similarities in terms of determinants of behavioural dominance between pairs and colonies, but the expression of behavioural dominance is quite different. From earlier work it was already known that if the queen/reproductive of the colony disappears or is experimentally removed, one of the individuals shows extreme levels of aggression. This individual, referred to as the potential queen (PQ), will go on to become the next queen of the colony. Her behavioural profile, from the emergence till she establishes herself as the next queen have been well studied earlier. What was not known were the factor(s) that determine the identity of the PQ. It was also unclear what happens when the queen as well as the PQ are both removed, simultaneously or in quick succession. To test whether there is a longer reproductive hierarchy in R. marginata, the queen and the first potential queen of a nest were removed. I found that successive potential queens emerged as readily as the first potential queen, and with dominance profiles comparable to the first PQ, indicating that a reproductive hierarchy indeed exists, at least up to five PQ’s. It was also found that these potential queens were acceptable to all other individuals, as there was not a single act of behavioural dominance directed toward any potential queen. It was also observed that all PQs went on to become queens if the previous queen or PQ was not returned. When tested for various morphological, physiological, behavioural and life history traits (factors possibly influencing the position of an individual in the reproductive hierarchy), we found that age is the only variable that emerges as an important predictor of reproductive succession, with older animals having a higher chance to succeed as next queens of the colony, although even age is not an absolute predictor. Unlike in the pairs, in colonies of R. marginata behavioural dominance is not a good predictor of an individual’s ability to be the queen or the potential queens. The four most important findings of my study are: (i) the first demonstration of winner and loser effects in social insects; (ii) the demonstration that behavioural dominance influences reproductive dominance in pairs but not in colonies; (iii) demonstration of a long reproductive queue among individuals of a colony; and (iv) discovering that age is an important predictor of the identity of the queen and the future queens of the colony. I believe these findings will add significantly to our growing knowledge of the social biology of R. marginata. Finally, my work shows that pairs of R. marginata, representing the founding phase of the colony, behave more like a typical primitively eusocial species, whereas colonies which represent the established phase of the colony cycle behave more like highly eusocial species. Finding the characters of two different forms of sociality in the same species in different phases of the colony cycle makes R. marginata an excellent model system to study evolution of eusociality.

For most of the animals, if not all, finding their way to a particular place is crucial for survival. To address this challenge of way-finding, different animals have evolved with different homing strategies. Social hymenopterans like honey bees, ants and wasps are of special interest – foragers of these insects show excellent homing capabilities while having simple neural resources. In this study field, honey bees and ants (desert ants, in particular) are among the most studied animals. Compared to these insects, our understanding on the homing mechanisms of social wasp is rather poor. For my thesis, I have studied homing behaviour of the tropical social wasp Ropalidia marginata, a predator in their foraging habit. To begin with, first I had to know their typical foraging range, which I found to be within about 500 m from their nest. Forager wasps possess a surprisingly well-developed familiarity with their foraging landscape, apparently more intricate than honey bees and desert ants. They acquire this spatial familiarity through flying around the landscape before starting foraging for food. Compared to honey bees and desert ants, this learning period in wasps appears to be much longer – this can be attributed to the much higher density of the tropical landscape in which they have evolved. I have also found that, if needed, they can fly to a distance of about 1.5 km for foraging and can return to their nest even if passively displaced to familiar and unfamiliar places. To return from unfamiliar places, they probably use some sort of searching mechanisms – a skill that they improve with their age. Such searching behaviour is prevalent throughout other hymenopteran insects. I conclude that capability and mechanisms of spatial orientation, navigation and homing in animals are much influenced by their evolutionary origin and the environment in which they have evolved.

Altruism is defined as a trait in an individual that increases some other individual’s fitness at the expense of her own. Therefore, existence of such traits in a population is an evolutionary paradox, as natural selection should eliminate such a trait. Extreme altruism in the form of eusociality where individuals relinquish their own reproduction to help raise other’s offspring has been an enigma in evolutionary biology since Darwin. Primitively eusocial organisms provide one with a unique system to study the evolution and maintenance of altruism as in these kind of species most of the individuals are capable of developing their reproductive organs, although at a certain point in time, only one or a few individuals actually reproduce. Ropalidia marginata is a primitively eusocial wasp belonging to the insect order Hymenoptera, Family Vespidae. R. marginata colonies are monogynous, although serial polygyny is observed in a colony’s lifetime. Colony initiation happens either by single founding or multiple founding. Newly founded colonies may accept individuals from other colonies, but mature colonies seldom do. Production of males is irregular, and once eclosed, they generally leave their natal nest within a week. The haplodiploidy of Hymenopteran species, i.e. the males being haploid and the females diploid, make them uniquely genetically predisposed for eusociality to evolve as was shown by William Donald Hamilton in his kin selection theory. Primitvely eusocial Hymenopteran species, being susceptible to experimental manipulation, allows one to test the predictions of this theory. In this thesis I have addressed three aspects of the biology of R. marginata using microsatellite markers, which are the following: 1) Distribution of nestmate genetic relatedness in early founding (pre‐emergence) and mature ( post‐emergence colonies) and their comparison (Chapter 3) 2) Role of relatedness and fertility in predicting the queen’s successor (Chapter 4) 3) Genetic structure of populations (Chapter 5) CHAPTER 1. INTRODUCTION: This chapter gives a brief outline of the field of molecular ecology putting its techniques to the context of insect sociobiology. CHAPTER 2. METHODS: This chapter gives a general outline of the molecular genetic methods involved. In addition, the issue of the mutation process in R. marginata microsatellites has also been addressed. There are two main models of mutation for microsatellite evolution i.e. infinite alleles model (IAM) and the step‐wise mutation model (SMM). To understand the actual process of mutation in R. marginata, sets of alleles with continuous sizes were sequenced and aligned. This was repeated for several of the loci. Seven out of the nine loci genotyped revealed clear step‐like mutation pattern and was binned accordingly. Two loci were dropped as the actual nature of step‐sizes in these two loci was unclear. Therefore, the final dataset consisted of genotype for 7 loci. This chapter also discusses the initial steps in data formatting and analysis. CHAPTER 3. GENETIC RELATEDNESS IN DIFFERENT STAGES OF COLONY DEVELOPMENT: In this chapter I have estimated nestmate genetic relatedness using seven polymorphic microsatellite loci in two different stages of colony development of the primitively eusocial wasp Ropalidiamarginata and compared them. In both kinds of nests the average colony relatedness was observed to be less than 0.75, i.e., what is expected for full sib females in Hymenoptera. Moreover, it was observed that the nestmates at the initial colony founding stage are on average less related to each other than in mature colonies. From this, one may postulate that the indirect component of inclusive fitness plays a relatively minor role than its direct component as individuals chose to leave a higher relatedness background in favour of a lower relatedness background. As newly founded colonies are relatively smaller in size than mature colonies, the probability of an individual wasp becoming the queen in this kind of colony is higher than in mature colonies. CHAPTER 4. TESTING THE ROLE OF RELATEDNESS AND FERTILITY IN PREDICTING THE QUEEN’S SUCCESSORS: R. marginata colonies are headed by docile queens. When this queen dies or is removed, one of the workers becomes extremely aggressive. She is known as the potential queen because within a few days she becomes the new queen of the colony and her aggression comes down. Predicting the successor in the presence of the queen has eluded most of the approaches attempted so far. The probability of an individual becoming the queen has been found to be uncorrelated with her body size, aggression, ovarian status or mating status. The only trend that has been observed till date, is a positive correlation with age, but the pattern is not perfect. However, the workers themselves seem to be perfectly aware of who their immediate successor going to be. In this chapter, I have tested several models of queen succession constructed in an inclusive fitness framework. These models have been tested both using relatedness alone as well as using fertility along with relatedness. Predictions of none of the models actually matched the observed sequence of successors. The wasps do not seem to be choosing their successor to maximize their inclusive fitness. CHAPTER 5. GENETIC STRUCTURE OF NATURAL POPULATIONS: I have also looked at the genetic structure of R. marginata populations in a large part of its natural distribution. I have used both F and R statistics to estimate the level of structuring and compared them. Both Fat as well Rst were found to be significantly larger than 0. Also Fis and Ris both were small and not significant suggesting lack of inbreeding. Rst was observed to be higher than Fst. Permutation test revealed a higher contribution of mutation in this structuring than migration, suggesting Rst to be a better measure of genetic structuring in this case. Similar pattern was observed with Anlysis of MOlecular VAriance. Pairwise Fst/(1‐Fst) values were found to be uncorrelated with distance, whereas barely significant trend was observed with Rst/(1‐Rst). The scatter across the trend line in both the cases suggested lack of migration drift equilibrium, with drift being more relative to migration. Higher level of structuring was observed at the level of the colony. However, colonies were rather outbred as was suggested by high and negative values of Fia and Ria values. This is not at all surprising as nestmates are related to each other. The pattern of isolation by distance at the colony level was similar to that observed in case of the populations. However, there was even higher degree of scattering of the individual points in this case. CHAPTER 6. CONCLUSIONS: Hamilton’s inclusive fitness theory has received a wide attention from and acceptance by sociobiologists, and relatedness have been measured in a wide variety of social insects. In this thesis relatedness in the context of colony founding was measured and compared with mature colonies. Also, several models constructed in an inclusive theory framework were experimentally tested. In both, support for indirect fitness was found wanting. The population genetic structure of R. marginata revealed that the sub populations are small in size and migration among them low. It also suggested significant contribution of colony level structuring on the population genetic structuring. Using more modern molecular genetic and statistical techniques, these and similar other questions can be addressed with higher precision and rigour, and such studies are expected to greatly advance our understanding of the basic premise of this thesis, i.e., how can eusociality evolve and be maintained? We hope that the current work will encourage others to ask such questions in other species.