The worldwide knowledge of the ecology and natural history of the masarid wasps, those wasps which bee-like provision their nest cells with pollen and nectar, is synthesized and discussed putting into context the investigations concerning nesting and flower visiting by southern African masarids conducted by the present author. Masarids are found mostly to favour warm to hot areas with relatively low rainfall and open scrubby vegetation. At the generic level the masarids of the Nearctic, Neotropical and Australian regions are distinct from each other and from those of the Palaearctic and Afrotropical regions combined. No species are shared between regions. Southern Africa is apparently the area of greatest species diversity. In this region, at least, there is a high incidence of narrow endemism. Masarids are associated with a relatively small range of plant families. Where sufficient records are available distinct major preferences are shown between zoogeographical regions. Relatedness of plant preferences between zoogeographical regions is apparent when relatedness of plant taxa is considered. Within a region there is marked overlap in masarid generic preferences for flower families. At the specific level there is marked oligolecty and narrow polylecty. The majority of nesting studies indicate that nest construction, egg laying and provisioning are performed by a single female per nest, however, nest sharing has been alledged for two species. No parasitic masarids have been recorded. Egg laying precedes provisioning. Mass provisioning is the rule. According to species, nests are sited in the ground, in non-friable soil or friable soil, in earthen vertical banks, on stones or on plants. Seven nest types are defined. Three bonding agents, water, nectar and self-generated silk are used. Masarids are evaluated as potential pollinators of their forage plants in southern Africa. The "masarid pollination syndrome", though less broad is shown to fall within that designated melittophily. The case studies considered make it clear that, whereas the masarids visiting some flower groups are members of a guild of potential pollinators, the masarids visiting others are probably their most important pollinators. Increasing land utilization is shown to threaten the existence of narrowly endmic masarid species.

"April 2004" / Bibliography: leaves 152-171. / xv, 171 leaves : ill., maps ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, School of Earth and Environmental Sciences, Discipline of Environmental Biology, 2004

A striking feature of hymenopterans societies is the absence of male workers. Foraging, nest building, brood care and all other activities required for the functioning of the colony are carried out by the females. These behaviours of the females and the implied cooperation and altruism have led social insect researchers to focus almost exclusively on the female members of hymenopterans societies. As a consequence, males have remained relatively neglected. The imbalance in the attention paid by researchers to females and males has been even more striking in the case of the extensively studied primitively eusocial wasp Rosalinda marginata. I have therefore focussed most of my attention to male R. marginata but of course, wherever possible and appropriate, I have compared the males with the females. Since almost nothing was known about R. marginata males, I began by obtaining basic information on the natural history and behaviour of the males. Male R. marginata are smaller in size and are different in the shape of the head as compared to the females. By conducting a three year survey, I found that just as nests of this species are found throughout the year, so are the males. The presence of males throughout the year has implications for the evolution of sociality by making available the opportunity to mate and found new nests, for females eclosing at any time of the year. Unlike the females who spend all their life on their nests, males stay on their natal nests only for one to twelve days (mean ± sd: 6.0 ± 2.6, N = 55) and leave their natal nest once and for all, to lead a nomadic life. It is difficult to determine how long males live after leaving their natal nests. However, I maintained males in the laboratory with ad libitum food and found that under these conditions they can live up to 140 days (mean physiological life span ± SD: 61.3 ± 28.0, N = 106). Like all eusocial hymenopterans males, R. marginata males also do not take part in any colony maintenance activities. They however, occasionally perform the following behaviours: solicit food, antennae nest, antennae another wasp, feed self, snatch food (from females), fan wings, body jerk and wing jerk, dominance and subordinate behaviours. Females of course perform all of these behaviours and many more. But there are no behaviours which are restricted to the males. Borrowing methods used by ecologists to measure species richness and diversity, I have computed the behavioral richness and diversity of male and female R. marginata. As expected, female behaviour is richer and more diverse compared to males. Comparing what the males did during their short stay on the nest and what females did during their long stay on the nest, I found that males did not forage or feed larvae (although I recorded one male feeding larvae thrice on one occasion when there seemed to be excess food on the nest). Males showed, dominance and subordinate behaviours and being solicited behaviour, significantly less often than females. On the other hand, males showed higher frequencies of feeding self and soliciting behaviour. However, these comparisons may not be fair because what males do in the first few days of their life is being compared with what females do throughout their life. Hence I truncated the female data at six days to make it comparable to the average age of the males on the nest. Even after doing so I found similar differences except that the males show similar rates of feed self and higher rates of subordinate behaviour compared to the young females. As mentioned in the beginning, absence of male workers is a striking feature of social Hymenoptera. I therefore naturally turned my attention to the possible reasons for this. As there has been much speculation on the ultimate, evolutionary explanations for why males do not work, I decided to investigate possible proximate explanations. To make my goal experimentally tractable, I decided to focus on the behaviour of feeding larvae as an example of work. In spite of the fact that R. marginata has been studied for over two decades, male R. marginata had never been seen to feed larvae, before my study. I advanced three hypotheses for why males do not feed larvae. (1) Males are incapable of feeding larvae. (2) Males do not feed larvae because they have insufficient access to food to satisfy themselves and to feed the larvae. (3) Males do not feed larvae because females perform the same task very efficiently. In a series of experiments designed to test these hypotheses, I showed that males are indeed capable of feeding larvae; they do so at small frequencies when given access to additional food and do so at impressive frequencies when deprived of females, provided with excess food and confronted with hungry larvae. Nevertheless, their ‘feed larva’ behaviour is less sophisticated and relatively inefficient compared to females. Thus I have shown male wasps can work, given an opportunity. In addition to being very satisfying, this result negates the preadaptation hypothesis which argues that male social Hymenoptera do not work because their solitary ancestors did not. In spite of what I have shown above, mating remains the main roleof the males. Therefore, I next turned my attention to a study of mating behaviour. It is well known that mating never takes place on the nest of R. marginata. Although some sporadic attempts had been made before, mating behaviour had never been observed in laboratory conditions before my study. In a series of trial-and-error pilot experiments, varying the age, cage size, number of wasps per cage, period of isolation from other wasps, lighting conditions etc., and with considerable help from enthusiastic volunteers among my lab-mates, I succeeded in observing mating behaviour in the laboratory. In the final, standardized experimental set up, we introduced a single male and a single female wasp, both isolated from their nests and other wasps for at least five days, into an aerated transparent plastic box and made observations for one hour. Using such an experimental set up, we first made a detailed qualitative description of mating behaviour. All behavioral interactions were initiated by the males. Males often attempted to mount the females but sometimes the females flew away, making the attempt to mount unsuccessful. On other occasions males successfully mounted the females, which involved climbing on the female and drumming and rubbing his antennae and abdomen on the corresponding parts of the female body. On some occasions mounting led to interlocking of the abdominal tips, a process we refer to as ‘conjugation’. Sometimes the conjugation lasted less than five seconds and during which the male remained on the back of the female; this was referred to as short conjugation (SC). But at other times the conjugation lasted for more than 20 seconds, and the male flipped on its back; this was Referred to as long conjugation (LC). I dissected all females, involved ineither LC or SC or both. Of the 47 pairs, 21 pairs mated successfully as judged by the transfer of sperm into the spermatheca. With the goal of developing a strategy to obtain live mated females for any future experiments, we attempted to determine the behavioral correlate/s of successful sperm transfer. It turns out that SC is inadequate for sperm transfer and LC is a good predictor of sperm transfer. Five to twenty days of age is optimal for mating for both males and females. There was a significant reduction in the probability of mating when one or both partners were younger or older than 5-20 days. We did not find any evidence for nestmate discrimination in the context of mating. This is not surprising because mating takes place away from the nest and active nestmate discrimination may therefore be unnecessary to avoid inbreeding. Under these experimental conditions neither body size of males and females nor ovarian conditions of the females appeared to influence mating success. I hope that my studies will enhance attention of future researchers to the males and will also facilitate experiments requiring mated wasps and permit the study of mate choice and other reproductive behaviours in this otherwise well- studied species.

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.

Ropalidia marginata is characterized as a primitively eusocial wasp due to the absence of morphological differentiation between the queen and worker castes. Unlike other primitively eusocial wasps, however, the queen in this species is a docile individual, who does not use aggression to regulate worker reproduction, and does not act as the central pacemaker of her colony. However, if the queen dies or is experimentally removed, one of the workers steps up her aggression immensely within minutes, and if the queen is not replaced, she develops her ovaries, reduces aggression and takes over as the new queen of the colony. We call her the potential queen (PQ). When I started my work on R. marginata, two very intriguing questions were demanding to be answered, which had developed from work done by my immediate seniors in the lab. I decided to pursue both of these for my thesis. My work has been enriched by inputs from several collaborators and colleagues - I couldn’t have done all of it by myself. So, henceforth, I will be using the word “we”, instead of the first person singular to describe the work that has gone into this thesis. Question 1: Is there a designated successor to the queen in R. marginata? My senior Sujata P. Kardile has shown in her thesis, that in R. cyathiformis, a primitively eusocial wasp very closely related to R. marginata, the queen is always succeeded by the next most aggressive individual in the colony, and so the PQ is easily predictable in the presence of the queen. However, in R. marginata, the PQ appears to be an unspecialized individual, who cannot be predicted in the presence of the queen by using age, ovarian profile or behaviour as the yardsticks. However, the PQ becomes evident within minutes after queen removal. The swiftness with which the PQ is established led us to believe that perhaps the successor to the queen in R. marginata is known to the wasps, though we cannot identify her in the presence of the queen. We designed an experiment to check for the presence of such a ”cryptic successor” in R. marginata. Our experiments involved splitting a normal, queen-right nest into two halves separated by a wire mesh partition, so that the wasps could not move across the mesh. Earlier we had used this set-up to demonstrate that a PQ always establishes herself on the queen-less fragment of the nest. So, to test if there is a cryptic successor, we allowed a PQ to establish herself on the queen-less fragment, and then exchanged the queen and the PQ (designated as PQ1) between the two sides. There is a 50% probability that the cryptic successor, if present, would be on the queen-less side in the beginning. Then, upon exchange, she should be able to hold her position on the other side easily. On the other hand, if the cryptic successor is first on the queen-right side, then, upon exchange, she should take over as the PQ (PQ2), and PQ1 should not be able to hold her status. The cryptic successor hypothesis had two predictions: (i) the PQ1 would lose to a PQ2 in about half the cases, and (ii) there would never be a PQ3. We obtained a PQ2 in 5 out of 8 cases, and we never had a PQ3. So we could conclude that there is indeed one individual who is the designated successor to the queen in R. marginata. Since we could not identify her in the presence of the queen, we call her the cryptic successor. The cryptic successor did not receive even a single act of aggression from the PQ1, or from any other individual in the colony. Thus we conclude that she is acceptable to all the wasps in the colony. We next used the more sophisticated and rigorous method of network analysis to check if the PQ could be predicted due to some unique position she might be holding in the social network on her colony. Since this was a first study in a primitively eusocial insect using network tools, we began by characterizing the social networks of queen-right and queen-less colonies of R. marginata, and compared them with the R. cyathiformis networks to see how different the R. marginata society is from a typical primitively eusocial one. The R. marginata social networks based on dominant-subordinate interactions were low in their centrality measure as compared to the R. cyathiformis networks. However, in both the species, the queen-less networks were highly centralized, star-shaped networks with the PQs at the centre. Neither the queens, nor the PQs were key individuals in the queen-right colonies, but it is interesting to note that the removal of an insignificant node, the queen, resulted in a major change in the network architecture, converting the de-centralized queen-right network into a highly centralized one. Such centralized star-shaped networks are unique, and to our knowledge, the first ever described, in any social system. When we removed the queen from the data set (in silico removal), the resulting network was similar in centrality to the queen-right networks. We then did a comparative analysis of the positional importance of the PQs of the two species, and tried to see if we could use this as a tool to predict the PQ in the queen-right network. In R. cyathiformis, the PQs had consistently high ranks (mostly rank 2) in the network based on the degree index, while the PQs in R. marginata had random ranks in the hierarchy. However, since the PQs are known not to have unique ranks in the dominance hierarchies, we repeated the analysis using data on all interactions from the Q-PQ exchange experiments described above. Neither the cryptic successors nor the losers occupied any unique ranks in the all interactions networks. Thus the successors in R. marginata are truly cryptic, even in their social networks. Since R. marginata is known to be more evolved than typical primitively eusocial species, it is likely that the queen’s successor is identified by the wasps through some subtle cue like smell, and so we cannot identify her using the methods that are adequate for the identification of the PQ in a typical primitively eusocial species like R. cyathiformis. Question 2: How does the queen signal her presence and reproductive status to her workers or, how do the workers perceive the presence of their queen? The fact that in spite of her docility, the queen in R. marginata manages to maintain complete reproductive monopoly in her colony, gives rise to the obvious question of how she suppresses worker reproduction. The most attractive hypothesis is that she uses a pheromone like queens of highly eusocial species. My senior A. Sumana had shown that the queen pheromone, if present, is not a volatile substance. She also showed that the queen interacts at a very low rate with her workers, and so they cannot possibly perceive her by means of direct interactions. Since the PQ steps up her aggression within minutes of queen removal, we used her as a proxy to know how soon the queen’s absence is felt in the colony. We built a model to delineate the relationship between the decay time of the pheromone (td), the average age of the queen’s signal present with the PQ (ta), and the average realization time (tr); where tr = td − ta. Using Dijkstra’s algorithm, we showed that the queen could interact faster with the PQ by using relay interactions. Then using experimental data from 50 colonies, we obtained a ta of 102.9 minutes. The td was 340 minutes, and so we obtained a tr of 237.1 minutes; which meant that the PQ should not perceive the queen’s absence within 237 minutes of queen removal, if the queen pheromone is transmitted by a relay mechanism. However, from our experimental data, we had obtained a tr of 30 minutes. So we concluded that physical interactions, both direct and indirect were inadequate for the workers to perceive their queen. As we had ruled out physical interactions, we then wanted to check if it is possible that the queen applies her pheromone to the nest material, from where it is perceived by the workers when they walk or sit on the nest, or antennate the nest surface. The “rub abdomen behaviour (RA)” has been observed to be quite typical of R. marginata queens, and is not very common in the workers of the species. RA involves rubbing the ventral side of the tip of the abdomen or dragging it on the nest surface while walking. We thought that the queen might be using this behaviour to apply her pheromone on the nest material. So we characterized this behaviour using focal behaviour sampling, and found that the queen rubs her abdomen on the nest once in every 23 minutes. Since the observed tr is 30 minutes, it is quite likely that the queen uses the rub abdomen behaviour to apply her pheromone on the nest. The next step was to check for the source of the queen pheromone. We looked for glands that open near the base of the sting, and the Dufour’s gland was a good choice, as it is known to be involved in the recognition of egg-laying workers in the honeybees. We performed a bioassay in the blind using the crude extract of the Dufour’s gland (prepared in Ringer’s solution) from the queen. The Dufour’s gland extract of a randomly chosen worker and the solvent were used as controls. We found that the PQ responds to the queen’s Dufour’s gland extract by lowering her aggression to 65% of what she was showing on queen removal and before the application of the extract. However, the PQ did not change her behaviour significantly when the worker’s extract or Ringer’s solution was applied. The PQ’s reduction of aggression on application of the queen’s extract mimicked the reaction of PQ’s when the queen is re-introduced on the nest some time after removal. So we hypothesize that the Dufour’s gland is the source of the queen pheromone (signal) in R. marginata. This thesis has opened up newer questions pertaining to the power of the queen and the intricacies of the succession to power in R. marginata. For example, we need to pursue chemical analyses of the Dufour’s gland extract of R. marginata to have conclusive proof of it’s being the source of the queen pheromone. But that is perhaps suitable topic for my juniors in the lab, who can continue the tradition of beginning with questions opened up by their seniors!

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.