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A fundamental question in biology is: How is variation generated? At a basic level, the vast amount of variation and biodiversity is generated through a combination of genetic and environmental processes. Traditionally these processes were treated independently, but recently fields such as evolutionary development have worked to unify our understanding of these mechanisms and to investigate how these processes interact with each other to generate variation. Developmental plasticity provides a fantastic framework for studying how genetic and environmental (GxE) interactions shape and maintain natural variation. Butterflies and their wing color patterns have long been model systems for plasticity. This dissertation seeks to address the gxe mechanisms responsible for generating color variation in the Dogface butterfly, Zerene. Zerene is comprised of only two species Z. cesonia, the Southern Dogface, and Z. eurydice, the California Dogface, that differ in their color patterns. Z. cesonia also exhibits a seasonal plastic color pattern, where Z. eurydice does not. These features make the Zerene system an excellent model for disentangling the gxe processes contributing to variation both within and between species. Using an integrative approach these studies address the role of 1.) larval host plant divergence 2.) seasonal fluctuations and 3.) hybridization on the development of wing coloration variation. The findings of these studies contribute not only to our understanding of how butterflies generate their colors, but also to the wider knowledge base on how genetics and the environment influence the generation and maintenance of biological variation.

abstract: At the heart of every eusocial insect colony is a reproductive division of labor. This division can emerge through dominance interactions at the adult stage or through the production of distinct queen and worker castes at the larval stage. In both cases, this division depends on plasticity within an individual to develop reproductive characteristics or serve as a worker. In order to gain insight into the evolution of reproductive plasticity in the social insects, I investigated caste determination and dominance in the ant Harpegnathos saltator, a species that retains a number of ancestral characteristics. Treatment of worker larvae with a juvenile hormone (JH) analog induced late-instar larvae to develop as queens. At the colony level, workers must have a mechanism to regulate larval development to prevent queens from developing out of season. I identified a new behavior in H. saltator where workers bite larvae to inhibit queen determination. Workers could identify larval caste based on a chemical signal specific to queen-destined larvae, and the production of this signal was directly linked to increased JH levels. This association provides a connection between the physiological factors that induce queen development and the production of a caste-specific larval signal. In addition to caste determination at the larval stage, adult workers of H. saltator compete to establish a reproductive hierarchy. Unlike other social insects, dominance in H. saltator was not related to differences in JH or ecdysteroid levels. Instead, changes in brain levels of biogenic amines, particularly dopamine, were correlated with dominance and reproductive status. Receptor genes for dopamine were expressed in both the brain and ovaries of H. saltator, and this suggests that dopamine may coordinate changes in behavior at the neurological level with ovarian status. Together, these studies build on our understanding of reproductive plasticity in social insects and provide insight into the evolution of a reproductive division of labor. / Dissertation/Thesis / Ph.D. Biology 2012

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22281号 / 理博第4595号 / 新制||理||1659(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 沼田 英治, 准教授 森 哲, 教授 曽田 貞滋 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

A diferenciação morfofisiológica entre rainhas e operárias de Apis mellifera decorre da alimentação recebida durante o desenvolvimento larval, que estimula o aumento da produção de Hormônio Juvenil naslarvas que originarão rainhas. Dentre as diversas diferenças morfológicas entre operárias e rainhas encontramse estruturas especializadas para a coleta de pólen e própolis, localizadas na região da tíbia e do basitarso das pernas posteriores de operárias. A diferenciação das pernas tem início entre o quarto e o quinto estágio do desenvolvimento larval. Utilizandose Microscopia Eletrônica de Varredura o presente trabalho relata a presença das cerdas formando as estruturas castaespecíficas na fase de pupa de olho marrom. A partir de estudos de hibridação de microarrays de cDNA com amostras de RNA de A. mellifera de diversas fases do desenvolvimento larval, foram encontrados 91 genes com ortólogos conhecidos em Drosophila, diferencialmente expressos entre rainhas e operárias no período crítico da diferenciação de castas. Destes, cinco estão relacionados com o desenvolvimento de apêndices: ataxin2 (atx2), cryptocephal (crc), dachshund (dac), grunge (gug) e Retinoic and fat acid Binding Protein (RfaBP). O perfil destes genes, e ainda, ultrabithorax (ubx), distalless(dll) e abdominalA (abdA) (estes porsuassuasfunções durante a diferenciação das pernas de insetos) foram analisados por RTPCR em Tempo Real em pernas posteriores de operárias e rainhas desde o quarto estágio larval até o estágio de pupa de olho branco. Apenas ubx e abdA foram encontrados mais expressos em operárias ao final do desenvolvimento larval e início do desenvolvimento pupal. Estudossimilares dos genes abdA, dac, dll e ubx nossegmentos das pernas de pupas de olho branco indicam a tíbia como domínio de expressão de dac. Imunolocalizações utilizando um anticorpo contra um epitopo conservado entre Ubx e AbdA, FP6.87, em pernas posteriores de prépupas de operárias e rainhasrevelam a presença destas proteínas na tíbia apenas de operárias e diferencialmente localizadas no basitarso de operárias e rainhas. Os dados acima apresentados apontam Ubx, um gene Hox, como pontochave na regulação da formação das estruturas castaespecíficas. / Diphenism in the honey bee, Apis mellifera,resultsfromdifferential feeding of female larvae. Among the morphological differences, the hind legs of workers have structures that is used for carrying pollen and propolis, e.g. the corbicula, while the queens hind legslack thisstructures. The corbicula is an expanded region of the tibia deprived of bristles, which has a single bristle in the middle that seems to have a sensorial function. Using scanning electronic microscopy, we found that the leg structures and bristles of the corbicula are already formed in browneyed pupa. Microarray analysis has demonstrated that five of 240 differentiallyexpressed genesin developing castes are potentially related to the caste differences in leg development (ataxin2, cryptocephal, dachshund, grunge and Retinoic and fat acid Binding Protein). Using qPCR, we analyzed the expression of abdominalA, ataxin2, cryptocephal, grunge, Retinoic and fat acid Binding Protein and ultrabithorax genes during hind leg development. cryptocephal, ataxin2, grunge and Retinoic and fat acid Binding Protein genes, which are involved in imaginal disc elongation and bristle formation and are inhibited by juvenile hormone, were not found to be differentially expressed. However, ultrabithorax and abdominalA are over expressed in workersin the early pupalstage. By using immunohistochemistry, Ubx was localized in the tibia and basitarsus of prepupae of workers and in the basitarsus of pre pupae of queens. The pattern of Ubx expression suggests that this Hox gene is a key player in leg structuresformation and caste differentiation in A.mellifera.

Evolution is not always straight-forward, as selection pressures may differ between different generations of the same species. This thesis focuses on the evolution of life history of the model species, the Green-veined White butterfly Pieris napi. In central Sweden P. napi has two generations per year. The directly developing summer generation is short-lived and time stressed, compared to the diapausing generation. In paper I polyandry, defined as female mating rate, was shown to differ between generations but was unaffected by environmental factors. In paper II both males and females of the direct developing generation were shown to eclose more immature than the diapausing generation, indicating larval time constraints. Consistent with this, diapausing males mated sooner than direct developers. Directly developing females, however, mated sooner after eclosion than diapausing females, even though they are more immature. This was shown to negatively affect fecundity, but can pay off when the season is short. Paper III shows that directly developing males have less sex pheromones at eclosion than diapausers, and the differences in sex pheromone production is consistent with developmental time constraints and the differences in mating system. In P. napi and other polyandrous butterflies, males transfer a large, nutritious ejaculate at mating. Large ejaculates confer advantages under sperm competition, but as they are costly, males should adjust ejaculate size to the risk of sperm competition. In paper IV we found that males transfer on average 20% larger spermatophores under high male competition than at low competition. The same effect could be observed if we added male sex pheromone to the air in a mating cage without male-male competition. Paper V shows that males of the two generations respond differently to an increase in male-male competition, with diapausing males transferring larger spermatophores than direct developers at high male competition risk. / <p>At the time of the doctoral defence the following paper was unpublished and had a status as follows: Paper 5: Submitted manuscript.</p>

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O dimorfismo sexual exibido por machos polifênicos em algumas espécies do gênero Ptychoderes envolve variação no rostro, antena e ventritos. A existência de polifenismo pode ser um importante componente no processo evolutivo por meio de novidades morfológicas e comportamentais. O objetivo desse estudo foi determinar a variação em caracteres morfométricos, polifenismo em machos, variação de estruturas com conhecido dimorfismo sexual, possíveis padrões alométricos e testar estas inferências para Ptychoderes através do mapeamento do dimorfismo sexual e de machos em uma reconstrução filogenética de Ptychoderes usando Mesquite 2.04. Foram medidas 23 variáveis morfométricas em 510 espécimes com as seguintes análises realizadas: análises de cluster, analises de componentes principais (PCA), analise de variáveis canônicas (AVC), análise de regressão por eixo maior reduzido (RMA). Cada tipo de dimorfismo foi mapeado em uma filogenia prévia como dois estados separados usando parcimônia. Para todas as espécies o dimorfismo sexual apresentou diferenças significativas entre os sexos com relação aos segmentos antenais (II- X).O compriemtno do rosto e ventrito V foram confirmados como indicativos de dimorfismo sexual (exceto em P. jordani). A única espécie em que não ocorreu machos polifenicos foi P. depressus. Nas outras espécies machos grandes e pequenos diferem significantemente para muitas variáveis com similaridades e diferenças. Na ACP, o primeiro componente (PC1) apresentou alta porcentagem de variância nos dados de todas as espécies; apresentou loadings de mesmo sinal sugerindo diferenças relacionadas ao tamanho para as espécies P. jordani, P. depressus, P. virgatus, P. mixtus e P. callosus e para as espécies P. viridanus, P. antiquus, P. elongates e P. nebulosus apresentou loadings positivos e negativos sugerindo diferenças relacionadas a forma (alometria). O PC2 apresentou loadings positivos e negativos para todas as espécies, um provável componente alométricos. A AVC confirmou os grupos: machos grandes, machos pequenos e fêmeas quando estes ocorreram. Nós encontramos diferentes padrões alométricos para todas as espécies com diferenças e semelhanças entre as espécies. Todos esses resultados confirmam a hipótese de polifenismo em machos e dimorfismo sexual para Ptychhoderes. A análise dos padrões alométricos para o dimorfismo sexual revelou alometria positiva para o comprimento do rostro (CR1) em machos e fêmeas, com os ventritos apenas em machos. Padrões alométricos positivos relacionados ao polifenismo nos antenômeros foram confirmados para os machos grandes e pequenos de quase todas as espécies exceto em P. nebulosus. O ancestral de clados na filogenia de Ptychoderes foi inferido para machos polifênicos (exceto P. depressus) com variáveis no rostro, antenas e ventritos indicativas de dimorfismo sexual com alometria positiva. Estes padrões poderiam estar ligados com o comportamento de proteção das fêemeas realizados por machos grandes durante a oviposição. / The sexual dimorphism exhibited by polyphenic males in some species of the Ptychoderes involves variation in rostrum, antennae and ventrites. The existence of polyphenism should be an important component in the evolutionary process via morphological and behavior novelties. The goal of this study was to determine the variation of monomorphic traits, polyphenism in males, variation of structures with known sexual dimorphism, possible allometric patterns and to test these predictions for Ptychoderes by mapping both male and sexual dimorphism in a phylogenetic reconstruction of the genus Ptychoderes using Mesquite 2.04. Twenty-three morphometric variables were measured in 510 specimens with the following analyses performed: Cluster analysis; Principal Component Analysis (PCA); Canonical Variance Analysis (CVA), analysis of regression of Reduced Major Axis (RMA). Each type of dimorphism was mapped on the previous phylogeny as a separate two-state using parsimony. For all species the sexual dimorphism provided significant differences between the sexes for antennal segments (II-X). The length of rostrum and ventrite V were confirmed as indication of sexual dimorphism (except P. jordani). The unique species without polyphenics males was P. depressus. The others species major and minor males differed significantly for many variables with similarities and differences. In the PCA, the first component (PC1) had a high percentage of the variance in the data for all species; present loadings of the same signal suggesting differences related to the size of the species P. jordani, P. depressus, P. virgatus, P. mixtus and P. callosus and for the species P. viridanus, P. antiquus, P. elongates and P. nebulosus the PC1 presented positive and negative loadings suggesting differences related to the shape (allometry). The PC2 showed both positive and negative loadings for all species, a probable allometric component. The CVA confirmed the groups: major males, minor males and female, when they occurred. We found different allometric patterns for all species, with similarities and differences between species. All these results confirm the hypothesis the polyphenism in males and sexual dimorphism for Ptychoderes. The analyses of allometric patterns for sexual dimorphism results positive allometry for rostral length (RL1) in males and females, with ventrites only for males. The positive allometric patterns related with polyphenism in the antennae were confirmed for larges and small males in almost all species, except in P. nebulosus. The ancestor of the clades in the Ptychoderes phylogeny was predicted to have polyphenic males (except P. depressus) with variables in the rostrum, antennae and ventrites indicative of the sexual dimorphism with positive allometry. These patterns should be linkage to the protection behavior of the female performed by large males during oviposition.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O dimorfismo sexual exibido por machos polifênicos em algumas espécies do gênero Ptychoderes envolve variação no rostro, antena e ventritos. A existência de polifenismo pode ser um importante componente no processo evolutivo por meio de novidades morfológicas e comportamentais. O objetivo desse estudo foi determinar a variação em caracteres morfométricos, polifenismo em machos, variação de estruturas com conhecido dimorfismo sexual, possíveis padrões alométricos e testar estas inferências para Ptychoderes através do mapeamento do dimorfismo sexual e de machos em uma reconstrução filogenética de Ptychoderes usando Mesquite 2.04. Foram medidas 23 variáveis morfométricas em 510 espécimes com as seguintes análises realizadas: análises de cluster, analises de componentes principais (PCA), analise de variáveis canônicas (AVC), análise de regressão por eixo maior reduzido (RMA). Cada tipo de dimorfismo foi mapeado em uma filogenia prévia como dois estados separados usando parcimônia. Para todas as espécies o dimorfismo sexual apresentou diferenças significativas entre os sexos com relação aos segmentos antenais (II- X).O compriemtno do rosto e ventrito V foram confirmados como indicativos de dimorfismo sexual (exceto em P. jordani). A única espécie em que não ocorreu machos polifenicos foi P. depressus. Nas outras espécies machos grandes e pequenos diferem significantemente para muitas variáveis com similaridades e diferenças. Na ACP, o primeiro componente (PC1) apresentou alta porcentagem de variância nos dados de todas as espécies; apresentou loadings de mesmo sinal sugerindo diferenças relacionadas ao tamanho para as espécies P. jordani, P. depressus, P. virgatus, P. mixtus e P. callosus e para as espécies P. viridanus, P. antiquus, P. elongates e P. nebulosus apresentou loadings positivos e negativos sugerindo diferenças relacionadas a forma (alometria). O PC2 apresentou loadings positivos e negativos para todas as espécies, um provável componente alométricos. A AVC confirmou os grupos: machos grandes, machos pequenos e fêmeas quando estes ocorreram. Nós encontramos diferentes padrões alométricos para todas as espécies com diferenças e semelhanças entre as espécies. Todos esses resultados confirmam a hipótese de polifenismo em machos e dimorfismo sexual para Ptychhoderes. A análise dos padrões alométricos para o dimorfismo sexual revelou alometria positiva para o comprimento do rostro (CR1) em machos e fêmeas, com os ventritos apenas em machos. Padrões alométricos positivos relacionados ao polifenismo nos antenômeros foram confirmados para os machos grandes e pequenos de quase todas as espécies exceto em P. nebulosus. O ancestral de clados na filogenia de Ptychoderes foi inferido para machos polifênicos (exceto P. depressus) com variáveis no rostro, antenas e ventritos indicativas de dimorfismo sexual com alometria positiva. Estes padrões poderiam estar ligados com o comportamento de proteção das fêemeas realizados por machos grandes durante a oviposição. / The sexual dimorphism exhibited by polyphenic males in some species of the Ptychoderes involves variation in rostrum, antennae and ventrites. The existence of polyphenism should be an important component in the evolutionary process via morphological and behavior novelties. The goal of this study was to determine the variation of monomorphic traits, polyphenism in males, variation of structures with known sexual dimorphism, possible allometric patterns and to test these predictions for Ptychoderes by mapping both male and sexual dimorphism in a phylogenetic reconstruction of the genus Ptychoderes using Mesquite 2.04. Twenty-three morphometric variables were measured in 510 specimens with the following analyses performed: Cluster analysis; Principal Component Analysis (PCA); Canonical Variance Analysis (CVA), analysis of regression of Reduced Major Axis (RMA). Each type of dimorphism was mapped on the previous phylogeny as a separate two-state using parsimony. For all species the sexual dimorphism provided significant differences between the sexes for antennal segments (II-X). The length of rostrum and ventrite V were confirmed as indication of sexual dimorphism (except P. jordani). The unique species without polyphenics males was P. depressus. The others species major and minor males differed significantly for many variables with similarities and differences. In the PCA, the first component (PC1) had a high percentage of the variance in the data for all species; present loadings of the same signal suggesting differences related to the size of the species P. jordani, P. depressus, P. virgatus, P. mixtus and P. callosus and for the species P. viridanus, P. antiquus, P. elongates and P. nebulosus the PC1 presented positive and negative loadings suggesting differences related to the shape (allometry). The PC2 showed both positive and negative loadings for all species, a probable allometric component. The CVA confirmed the groups: major males, minor males and female, when they occurred. We found different allometric patterns for all species, with similarities and differences between species. All these results confirm the hypothesis the polyphenism in males and sexual dimorphism for Ptychoderes. The analyses of allometric patterns for sexual dimorphism results positive allometry for rostral length (RL1) in males and females, with ventrites only for males. The positive allometric patterns related with polyphenism in the antennae were confirmed for larges and small males in almost all species, except in P. nebulosus. The ancestor of the clades in the Ptychoderes phylogeny was predicted to have polyphenic males (except P. depressus) with variables in the rostrum, antennae and ventrites indicative of the sexual dimorphism with positive allometry. These patterns should be linkage to the protection behavior of the female performed by large males during oviposition.

A diferenciação morfofisiológica entre rainhas e operárias de Apis mellifera decorre da alimentação recebida durante o desenvolvimento larval, que estimula o aumento da produção de Hormônio Juvenil naslarvas que originarão rainhas. Dentre as diversas diferenças morfológicas entre operárias e rainhas encontramse estruturas especializadas para a coleta de pólen e própolis, localizadas na região da tíbia e do basitarso das pernas posteriores de operárias. A diferenciação das pernas tem início entre o quarto e o quinto estágio do desenvolvimento larval. Utilizandose Microscopia Eletrônica de Varredura o presente trabalho relata a presença das cerdas formando as estruturas castaespecíficas na fase de pupa de olho marrom. A partir de estudos de hibridação de microarrays de cDNA com amostras de RNA de A. mellifera de diversas fases do desenvolvimento larval, foram encontrados 91 genes com ortólogos conhecidos em Drosophila, diferencialmente expressos entre rainhas e operárias no período crítico da diferenciação de castas. Destes, cinco estão relacionados com o desenvolvimento de apêndices: ataxin2 (atx2), cryptocephal (crc), dachshund (dac), grunge (gug) e Retinoic and fat acid Binding Protein (RfaBP). O perfil destes genes, e ainda, ultrabithorax (ubx), distalless(dll) e abdominalA (abdA) (estes porsuassuasfunções durante a diferenciação das pernas de insetos) foram analisados por RTPCR em Tempo Real em pernas posteriores de operárias e rainhas desde o quarto estágio larval até o estágio de pupa de olho branco. Apenas ubx e abdA foram encontrados mais expressos em operárias ao final do desenvolvimento larval e início do desenvolvimento pupal. Estudossimilares dos genes abdA, dac, dll e ubx nossegmentos das pernas de pupas de olho branco indicam a tíbia como domínio de expressão de dac. Imunolocalizações utilizando um anticorpo contra um epitopo conservado entre Ubx e AbdA, FP6.87, em pernas posteriores de prépupas de operárias e rainhasrevelam a presença destas proteínas na tíbia apenas de operárias e diferencialmente localizadas no basitarso de operárias e rainhas. Os dados acima apresentados apontam Ubx, um gene Hox, como pontochave na regulação da formação das estruturas castaespecíficas. / Diphenism in the honey bee, Apis mellifera,resultsfromdifferential feeding of female larvae. Among the morphological differences, the hind legs of workers have structures that is used for carrying pollen and propolis, e.g. the corbicula, while the queens hind legslack thisstructures. The corbicula is an expanded region of the tibia deprived of bristles, which has a single bristle in the middle that seems to have a sensorial function. Using scanning electronic microscopy, we found that the leg structures and bristles of the corbicula are already formed in browneyed pupa. Microarray analysis has demonstrated that five of 240 differentiallyexpressed genesin developing castes are potentially related to the caste differences in leg development (ataxin2, cryptocephal, dachshund, grunge and Retinoic and fat acid Binding Protein). Using qPCR, we analyzed the expression of abdominalA, ataxin2, cryptocephal, grunge, Retinoic and fat acid Binding Protein and ultrabithorax genes during hind leg development. cryptocephal, ataxin2, grunge and Retinoic and fat acid Binding Protein genes, which are involved in imaginal disc elongation and bristle formation and are inhibited by juvenile hormone, were not found to be differentially expressed. However, ultrabithorax and abdominalA are over expressed in workersin the early pupalstage. By using immunohistochemistry, Ubx was localized in the tibia and basitarsus of prepupae of workers and in the basitarsus of pre pupae of queens. The pattern of Ubx expression suggests that this Hox gene is a key player in leg structuresformation and caste differentiation in A.mellifera.

Anelosimus studiosus exhibits two behavioral phenotypes: subsocial and social. This is the only documented spider inhabiting a temperate climate exhibiting social behavior. While the subsocial phenotype is most common throughout the range, the social behavior occurs in isolated pockets in northern latitudes. This study examines the origins of the social phenotype within a segment of the spider's range. Two hypotheses are tested: 1) pockets of social behavior represent a single origin or 2) pockets of social behavior represent local evolutions, thus leading to multiple origins of evolution. Microsatellite loci were used to determine genetic structure of the population and to estimate the origins of social behavior. All loci showed lower observed than expected heterozygosities and all populations show indications of high levels of inbreeding. A phylogeny indicates four of the six populations fall out by location, not phenotype. We propose these results reflect multiple local evolutions of the social strategy.