Köln, University, Diss., 2003.

As abelhas da tribo Meliponini (abelhas sem ferrão) estão amplamente distribuídas pelas regiões tropicais do planeta, tendo um importante papel na polinização, sendo o gênero Melipona o que contém o maior número de espécies. A espécie Melipona marginata é uma das menores e mais ancestrais do grupo, e a exemplo de outras espécies nidifica em ocos de árvore. M. marginata, assim como outras espécies do gênero Melipona, vêm sofrendo com a destruição do seu ambiente natural, pelo desmatamento, sendo, aparentemente mais exigente que outras espécies quanto ao tamanho do fragmento florestal para se manter, devido a isso é encontrada apenas em fragmentos maiores, mais antigos e menos perturbados. Tendo em vista a perda de hábitat e o pouco conhecimento da biologia dessa espécie, este trabalho pretende analisar populações de M. marginata, através da técnica PCR+RFLP do DNA mitocondrial e marcadores microssatélites, visando contribuir para entendimento da estrutura populacional de M. marginata. Foram analisados 54 ninhos, provenientes dos estados de MG, SP, PR, SC e RS. Oito regiões do DNA mitocondrial foram amplificadas, e posteriormente digeridas com 12 enzimas de restrição. Foram detectados 14 haplótipos, sendo apenas um compartilhado. A população de SP apresentou o maior número de haplótipos. Os testes estatísticos demonstraram que as populações estão estruturadas e isoladas, não havendo fluxo gênico entre as populações. Já nas análises dos microssatélites foram analisados 4 locos, apresentando alta variabilidade genética, onde também foi verificado que as populações se encontram estruturadas. Os resultados obtidos podem ser explicados principalmente pela redução da área de floresta, mas, podem se dever a eventos antigos em conseqüência de mudanças climáticas ocorridas durante as últimas glaciações. / The tribe Meliponini (stingless bees) is present in all tropical regions of the world and has an important role in pollination. The genus Melipona has the highest number of species in the tribe. The specie Melipona marginata is considered the most ancestral within the genus, and like other species builds the nests in hollow of trees. Unfortunately several bee species have suffered with the devastation of their natural environment. M. marginata seems to be very dependent on the size of the forest fragment, being found only in the biggest, oldest and consequently more preserved ones. In view of the habitat loss and few biological knowledge about this specie, this research intended to analyse M. marginata populations molecularly, through mitochondrial DNA PCR-RFLP and microsatellite data., Fifty four colonies were analyzed, from MG, SP, PR, SC and RS states. Eight mitochondrial DNA regions were amplified and screened with 12 restriction enzymes. Fourteen haplotypes were verified and among them just one was shared. SP population showed the highest number of haplotypes. Statistic tests showed that the 5 populations were genetic structured and isolated, therefore not presenting gene flow. The 4 microsatellites loci analyzed showed high genetic variability. The statistics analysis indicated that the 5 populations are structure and isolated. These results can be explained mainly because the decrease of forest leading to population isolation, however we can not discard the hypothesis that this current scenario is a consequence of climatic changes occurred during the last glaciations.

As abelhas da tribo Meliponini (abelhas sem ferrão) estão amplamente distribuídas pelas regiões tropicais do planeta, tendo um importante papel na polinização, sendo o gênero Melipona o que contém o maior número de espécies. A espécie Melipona marginata é uma das menores e mais ancestrais do grupo, e a exemplo de outras espécies nidifica em ocos de árvore. M. marginata, assim como outras espécies do gênero Melipona, vêm sofrendo com a destruição do seu ambiente natural, pelo desmatamento, sendo, aparentemente mais exigente que outras espécies quanto ao tamanho do fragmento florestal para se manter, devido a isso é encontrada apenas em fragmentos maiores, mais antigos e menos perturbados. Tendo em vista a perda de hábitat e o pouco conhecimento da biologia dessa espécie, este trabalho pretende analisar populações de M. marginata, através da técnica PCR+RFLP do DNA mitocondrial e marcadores microssatélites, visando contribuir para entendimento da estrutura populacional de M. marginata. Foram analisados 54 ninhos, provenientes dos estados de MG, SP, PR, SC e RS. Oito regiões do DNA mitocondrial foram amplificadas, e posteriormente digeridas com 12 enzimas de restrição. Foram detectados 14 haplótipos, sendo apenas um compartilhado. A população de SP apresentou o maior número de haplótipos. Os testes estatísticos demonstraram que as populações estão estruturadas e isoladas, não havendo fluxo gênico entre as populações. Já nas análises dos microssatélites foram analisados 4 locos, apresentando alta variabilidade genética, onde também foi verificado que as populações se encontram estruturadas. Os resultados obtidos podem ser explicados principalmente pela redução da área de floresta, mas, podem se dever a eventos antigos em conseqüência de mudanças climáticas ocorridas durante as últimas glaciações. / The tribe Meliponini (stingless bees) is present in all tropical regions of the world and has an important role in pollination. The genus Melipona has the highest number of species in the tribe. The specie Melipona marginata is considered the most ancestral within the genus, and like other species builds the nests in hollow of trees. Unfortunately several bee species have suffered with the devastation of their natural environment. M. marginata seems to be very dependent on the size of the forest fragment, being found only in the biggest, oldest and consequently more preserved ones. In view of the habitat loss and few biological knowledge about this specie, this research intended to analyse M. marginata populations molecularly, through mitochondrial DNA PCR-RFLP and microsatellite data., Fifty four colonies were analyzed, from MG, SP, PR, SC and RS states. Eight mitochondrial DNA regions were amplified and screened with 12 restriction enzymes. Fourteen haplotypes were verified and among them just one was shared. SP population showed the highest number of haplotypes. Statistic tests showed that the 5 populations were genetic structured and isolated, therefore not presenting gene flow. The 4 microsatellites loci analyzed showed high genetic variability. The statistics analysis indicated that the 5 populations are structure and isolated. These results can be explained mainly because the decrease of forest leading to population isolation, however we can not discard the hypothesis that this current scenario is a consequence of climatic changes occurred during the last glaciations.

The south-west of Western Australia has a Mediterranean climate and flora endemic to this area, including the keystone species, jarrah (Eucalyptus marginata), have adapted to the droughted summer conditions. The introduction of an exotic soil borne pathogen, Phytophthora cinnamomi, has challenged the survival of this and many other species. The expectation might be that plants stressed by drought are more susceptible to disease and this study examined the development of disease caused by P. cinnamomi in E. marginata and the significance of water status to that development. Seedlings of E. marginata, clonal plants resistant to P. cinnamomi and clonal plants susceptible to P. cinnamomi, were subjected to different watering regimes in a number of field and glasshouse experiments. To determine the level of drought stress that could be imposed on container-grown E. marginata seedlings without killing them, a preliminary experiment progressively lowered the moisture levels of the substrate in their containers, until the plants reached wilting point, at which time moisture was restored to a predetermined droughted level and the process repeated. With each subsequent droughting the wilting point was lower until it was found that the seedlings could survive when only 5% of the moisture lost from container capacity to wilting point was restored. No deaths had occurred after seedlings had been maintained at this low level for 14 days (Chapter 2). Based on these findings, the level of droughting maintained in all experiments conducted under controlled glasshouse conditions was 10% restoration. After testing the appropriateness of underbark inoculation, and a zoospore inoculation method for which no wounding was necessary, a new, non-invasive stem inoculation technique was developed. Stems were moistened in a pre-treatment, then agar plugs colonized with P. cinnamomi mycelium were held against the stem with wads of wet cotton wool and bound in place with tape. This technique resulted in a high proportion of infection in E. marginata (Chapter 4) without the need for underbark inoculation or the use of zoospores (Chapter 3). It was successfully used in a large field trial in a rehabilitated bauxite mine site with 2-year-old E. marginata clonal plants, resistant to P. cinnamomi (Chapter 5). Inoculation was in late spring after the winter and spring rainfall. This timing was to allow comparison of disease development in stressed plants under normal droughted summer conditions compared with itsdevelopment in non-stressed, irrigated plants. However, two months after inoculation, the area was deluged with unseasonal and abnormally heavy summer rainfall, negating any difference in the treatments and causing an outbreak of P. cinnamomi in the soil from an adjacent infested site. This resulted in the infection and death of some noninoculated control clones. Monitoring of the site continued for twelve months and the advance of P. cinnamomi at the site was mapped. To test the effect of drought on the expression of P. cinnamomi under more controlled conditions, a series of glasshouse experiments was set up that simulated two possible summer conditions; drought or drought followed by abnormally high summer rainfall. These experiments utilised E. marginata seedlings and clonal plants, some resistant and some susceptible to P. cinnamomi. Plants were inoculated with P. cinnamomi prior to or after droughting. Results were compared to those of control plants that had not experienced water deficit. In both seedlings and clonal plants, the greatest extent of colonization was found in plants which had experienced no water deficit. These results indicated that drought stress played a role in inhibiting the in planta development of P. cinnamomi in all genotypes (Chapter 8). This finding was consistent for both clones, susceptible and resistant to P. cinnamomi. Most recoveries were made from non-stressed clonal plants, resistant to P. cinnamomi (Chapter 6) and more colonization was found in non-stressed clonal plants, susceptible to P. cinnamomi (Chapter 7), than was recorded for droughted plants. The results of the field trial showed that P. cinnamomi was not recovered from some inoculated stems, which had obvious lesions, when segments were plated onto selective agar. This led to an intensive in vitro investigation into improved methods of recovery. Dark brown exudates from some segments of inoculated stems stained the surrounding agar onto which they were plated, suggesting the presence of phenolic compounds. Recovery of the pathogen from stems increased by about 10% when segments were first soaked in distilled water to leach out the phenolic compounds, then replated onto agar. Other recovery methods were also tested, including (1) baiting with Pimelea ferruginea leaves floated on the surface of water or soil filtrate, in which the infected stem segments were immersed and (2) the application of different light and temperature regimes. It was clearly shown that exudates from infected stems of field grown E. marginata inhibited the outgrowth of P. cinnamomi onto the agar. To counter the possible toxic effect that oxidized phenolics had on the growth of the P. cinnamomi, an antioxidant was added to the agar. P. cinnamomi was grown on media whichincorporated exudates from infected stems and different concentrations of ascorbic acid, with and without adjusted pH levels. There was a pronounced pH effect, with less growth on media with lower pH and no significant increase in growth of the mycelium with increased ascorbic acid concentration on pH adjusted agar (Chapter 9). The inhibitory effect of the exudates from the stem segments led to an investigation of the possibility that, if seedlings to be planted in the rehabilitation process could be pre-treated with phenolic compounds to render them more resistant, they may have an advantage when establishing in areas where there was a potential threat of P. cinnamomi. E. marginata seeds were germinated and the seedlings grown hydroponically in a constant temperature growth room. Different concentrations of synthetic catechol, a phenolic compound naturally occurring in E. marginata, were added to the nutrient solution. Roots remained immersed in the catechol solutions for three days, before being inoculated at the root tip with zoospores of P. cinnamomi. Roots in higher concentrations of catechol were less colonized than those in lower concentrations, indicating an increased resistance to the pathogen (Chapter 10). Further work is required to determine if seedlings treated before being planted in areas threatened by an outbreak of P. cinnamomi have a greater capacity for survival, and for how long the protection persists. The improved recovery of P. cinnamomi from infected plants is important for accurate assessment of the spread of the disease in an area and for the subsequent implementation of management strategies of containment and control. An outbreak of P. cinnamomi can impact on the revegetation of rehabilitated mine sites and the aetiology of the pathogen in mine sites needs to be more fully understood. The interaction of plant defences with the invasive pathogen has been examined in a range of environments in the field, the glasshouse, in a hydroponics system and in vitro. The results indicate that summer droughting increases the resistance of E. marginata to P. cinnamomi. However, more work is required to understand the mechanisms involved. The study also indicates that clones of E. marginata, selected as resistant to P. cinnamomi, are not resistant under all conditions and that environmental interactions should be further investigated. Lastly, for effective management strategies to be implemented it is critical that the pathogen can be confidently isolated from plants. It was shown that exudates from infected hosts inhibit the recovery of P. cinnamomi. Recovery methods that can overcome these inhibitory compounds are required. The findings invite further research into the complexity of host-pathogen relationships.

Phosphite is currently used for the management of Phytophthora cinnamomi in native plant communities. A greater understanding of how phosphite affects the host-pathogen interaction is required in order to determine the most effective treatment. This thesis aimed to investigate the effects of applied phosphite concentration on phytotoxicity, in planta concentration of phosphite, disease development and anatomical responses of Eucalyptus marginata. Spraying the foliage to run-off with 7.5 and 10 g phosphite/L led to the development of severe leaf necrosis within 7 days, with greater than 60% of the leaf area damaged. Moderate phytotoxicity was observed after treatment with 5 g phosphite/L. In planta concentration of phosphite in stems, lignotubers and roots did not differ significantly between applied concentrations of phosphite. Stem tissue contained the largest concentration of phosphite at one week after spraying, with approximately 210 and 420 µg phosphite/g dry weight detected after treatment with 5 and 10 g phosphite/L, respectively. In a subsequent field trial, the applied concentration of phosphite was found to affect the duration of effectiveness of phosphite in protecting E. marginata seedlings from stem colonisation by P. cinnamomi. Plants were wound-inoculated with P. cinnamomi at 6-monthly intervals after spraying with phosphite. The 2.5 and 5 g phosphite/L treatments were effective against colonisation by P. cinnamomi when inoculated 0 and 6 months after spraying, but only the 5 g phosphite/L treatment inhibited P. cinnamomi within 12 months of spraying. Phosphite had no effect on colonisation by P. cinnamomi when plants were inoculated at 17 months after spraying. The in planta concentration of phosphite detected in the leaves, stems and roots of plants treated with 5 g phosphite/L did not differ significantly between the time of harvest or tissue type at 0.2 and 6 months after spraying. P. cinnamomi remained viable in plants treated with phosphite.Treatment with 2.5 and 5 g phosphite/L when P. cinnamomi was well established in the stems was ineffective at preventing the death of E. marginata. Between 45 and 89% of plants were girdled on the day of spraying. Spraying plants with 2.5 and 5 g phosphite/L when conditions were less favourable for the pathogen reduced the mortality of E. marginata for up to 10 months. E. marginata seedlings responded to damage by P. cinnamomi with the production of kino veins and woundwood. Bark lesions were in the process of being sloughed off by 7 months after inoculation in plants that remained alive. In plants of a resistant (RR) clonal line and susceptible (SS) clonal line, phosphite treatment inhibited lesion extension in stems, but lesions did not indicate the amount of stem colonised by P. cinnamomi. The pathogen was isolated from up to 17 cm beyond the lesion front in the RR clonal line. Treatments that reduced the mortality of E. marginata were 5 g phosphite/L in the RR clonal line (RR/5) and 10 g phosphite/L in the SS clonal line (SS/10). Uninoculated plants were wounded with liquid nitrogen to determine the microscopic responses to injury in the absence of the pathogen. Wound closure was achieved within 21 days of wounding, with callus formation and vascular cambium regeneration. A wound periderm separated wounded tissue from healthy tissue, adjacent to a lignified boundary zone. Two types of phellem were observed – thin-walled phellem (TnP) and thick-walled phellem (TkP). The first-formed TnP layers contained variable-shaped cells, while subsequent layers were more cubical in shape. Multiple TnP layers developed up to 42 days after wounding, with TkP cells sandwiched between the TnP layers. Genotype and phosphite treatment did not affect the wound responses. Inoculated plants with a restricted lesion extension also formed a wound periderm to separate damaged tissue from healthy tissue. Phosphite treatment stimulated the responses to P. cinnamomi in both clonal lines. Early development of the wound periderm was visible by 6 days after phosphite treatment. It waspreceded by the formation of a ligno-suberised boundary zone in the cambial zone and in phloem parenchyma cells existing prior to injury. Suberin was not detected in the SS/0 treatment. TnP layers completely surrounded lesioned tissue in plants still alive by 24 days after phosphite treatment. Extensive callus production was evident in the SS/10, RR/5 and RR/10 treatments. Temperature affected the post-inoculation efficacy of phosphite and anatomical responses of E. marginata. At 20°C, lesion extension was restricted in both clonal lines of E. marginata, irrespective of phosphite treatment. Greater than 70% of inoculated plants in all treatments produced a ligno-suberised boundary zone at 20°C and between 30 and 70% formed a wound periderm. At 28°C, lesion extension was reduced in phosphite-treated plants at 7 days after treatment. However, lesions continued to extend up to 5 mm per day in the SS clonal line and very few SS plants formed a wound periderm at the lesion front. This contrasted with the strong responses to abiotic wounding observed in uninoculated SS plants at 28°C. The most extensive responses to P. cinnamomi were detected in the RR/5 treatment at 28°C, with a ligno-suberised boundary zone and differentiated TnP of a wound periderm observed in greater than 70% of plants. This treatment resulted in significantly less girdled plants than all other treatments at 28°C, including the RR/0 treatment. At 23 and 24°C, there was no significant difference in acropetal lesion extension or circumferential lesion spread between clonal lines. The inoculation technique and environmental conditions may have resulted in too high a disease pressure for a full expression of resistance in the RR clonal line. This thesis demonstrates that phosphite has the potential to enhance the resistance of young E. marginata and enable them to survive infection by P. cinnamomi. However, its effectiveness is dependent upon a number of factors, including host resistance, environmental conditions, the applied phosphite concentration and the timing of application.

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.

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.

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.

Vildbiet guldsandbi (Andrena marginata F.) har en central roll i jordbrukslandkapet som en viktig pollinatör av grödor och vilda växter. I Sverige hotas artens fortlevnad av biotopförlust, habitatfragmentering och födobrist. En av de viktigaste näringskällorna är växten ängsvädd (Succisa pratensis M.) som tidigare var vanligt förekommande men som nu minskar i sin utbredning. Syftet med denna studie var att utreda om populationsstorlekarna av guldsandbi och ängsvädd är relaterade. Guldsandbinas observationsbaserad populationsstorlek uppskattades samt att ängsväddens bestånd inventerades vid fyra lokaler i Övre Klarälvsdalen, i norra Värmland. Binas populationsstorlek uppskattades genom transektinventeringar mellan 30 juli och 11 augusti 2013. Under perioden observerades totalt 286 honor och per observationsdag noterades ett medelvärde på 40,86 (± 8,1 SE, 95% konfidensintervall; n=7) honor. Ängsväddpopulationen uppskattades till totalt 2328 växter från alla fyra områden. Det fanns inget statistiskt samband mellan populationsstorlekarna av ängsvädd och guldsandbin. En jämförelse mellan den för biet förväntade populationsstorleken, baserad på förekomst av näringsväxten, och den observerade populationsstorleken, korrigerad för inventeringsbias, visade bra överensstämmelse vid två av fyra områden (8,4% respektive 5,7% från det teoretiska värdet) och dålig överensstämmelse vid de andra två områdena. Det kan mycket väl vara så att det finns en relation mellan populationerna i Övre Klarälvsdalen, men man måste öka antalet lokaler och ta hänsyn till ytterligare miljöfaktorer som påverkar både bina och växterna för att kunna dra säkrare slutsatser om ett samband. / The wild-bee Andrena marginata has a key function as an important pollinator of crops and wild plants in agriculture landscapes. The species’ persistence is threatened in Sweden due to loss of habitat, fragmentation and food scarcity. One of the most important food sources is the plant Succisa pratensis, which previously was very common but now has declining populations. The purpose of this study was to analyse if there was a relation between the population sizes of A. marginata and S. pratensis. The population size of the bees was estimated and the plant population was surveyed at four sites in the upper valley of the River Klarälven, Värmland. Bee population size was estimated by visual transect surveys conducted between 30 July and 11 August 2013. In this period a total of 286 females was observed, with a mean of 40,86 (± 8,1 SE, 95% confidence interval; n=7) per day. The total population size of S. pratensis from the four sites was estimated to 2328 plants. There was no significant correlation between the population sizes of the bees and the host plant. A comparison between the bee’s predicted theoretical population size, based on the number of host plants, and the observed population size, corrected for observer bias, showed good agreement for two of the sites (within 8,4% and 5,7% of the theoretical value) and poor agreement at the other two sites. There may well be a relation between the bee and plant populations in the upper valley of Klarälven, but one needs to increase the number of sites and take into account additional environmental factors that affect both the bees and the plants to draw a firmer conclusion about such a relationship.