Aspectos heterocrônicos encontrados na evolução do crânio dos Rhynchosauria foram estudados como uma contribuição ao melhor entendimento da evolução deste grupo, bem como dos vertebrados em geral; para o que partiu-se do material Triássico do estado Rio Grande do Sul. A Análise dos Componentes Principais incluiu 22 crânios e foi utilizada para estabelecer classes de idades; a Equação de Alometria para estabelecer as modificações alométricas durante a ontogenia. Uma análise incluiu espécimes de Hyperodapedon sp., Scaphonyx sulcognathus e o "Rincossauro de Mariante"; e outra apenas espécimes de Hyperodapedon sp. A Análise Estatística Multivariada indicou que a alteração da forma craniana depende fortemente do tamanho. As alterações nas diferentes regiões do crânio, refletidas nos coeficientes alométricos, são uma consequência da alteração do tamanho dos indivíduos. Na primeira análise, 73% das medidas apresentaram crescimento alométrico negativo em relação à medida comparativa padrão, enquanto que apenas 27% das medidas apresentaram crescimento alométrico positivo (em nenhuma foi isométrico). Na segunda análise, 91% das medidas apresentaram crescimento alométrico negativo, enquanto que apenas 9% do crescimento alométrico foi positivo (em nenhuma foi isométrico). Encontrou-se um mosaico heterocrônico nos caracteres cranianos, com processos pedomórficos e peramórficos. A análise que inclui o "Rincossauro de Mariante" e Scaphonyx sulcognathus permite concluir que durante a ontogenia dos espécimes, o crânio apresentava um crescimento alométrico positivo do rostro e do teto da região craniana propriamente dita, quando comparados com o comprimento total do crânio; indicando uma peramorfoclinal. Exceto as do rostro e do teto da região craniana, todas as demais medidas apresentaram alometria negativa quando comparadas ao comprimento total do crânio, refletindo uma pedomorfoclinal. A análise que exclui o "Rincossauro de Mariante" e Scaphonyx sulcognathus indica que, durante a ontogenia, o crânio apresentava um crescimento alométrico positivo apenas no rostro, evidenciado pelo aumento na pré-maxila. Duas das heterocronoclinais encontradas na evolução dos rincossauros são as mais usuais nos vertebrados como um todo, e devidas à progênese por um lado e à hipermorfose por outro. A neotenia no rostro, associada a uma provável progênese na mandíbula levou à formação do aspecto mais característico dos rincossauros, a pré-maxila curvada ventralmente e verticalizada. A peramorfoclinal por hipermorfose pode ter contribuído para a extinção do grupo. Nos vertebrados provavelmente estão sempre associados dois processos (progênese e hipermorfose), ou mais, como neotenia e aceleração no caso dos rincossauros. Os processos não são fenômenos isolados, mas sempre parte de heterocronoclinais; as quais podem resultar de respostas a diferentes solicitações ambientais, possibilitadas pela flexibilidade genotípica. As respostas podem ser aprofundadas, canalizadas e fixadas, levando ou ao surgimento de grupos novos (pedomorfoclinais por progênese) ou à extinção de outros (peramorfoclinais por hipermorfose). Processos heterocrônicos podem originar mudanças graduais, mas não necessariamente; o que permite concluir que nem sempre o registro fóssil é realmente "incompleto", mas sim por vezes reflexo de algum tipo de evolução não gradual. Heterocronia não é uma teoria científica, mas uma metodologia que permite reconhecer padrões fenotípicos complexos resultantes da ontogenia. / Heterochronic aspects in the evolution of the Rhynchosauria skull were studied as a contribution to a better understanding of the evolution of this group, as well as of vertebrates as a whole; for what Triassic material from Rio Grande do Sul State was used. The Principal Components Analysis on 22 skulls sample was accomplished to establish age classes; and Allometry Equation to establish alometric changes during ontogeny. One analysis included Hyperodapedon sp., Scaphonyx sulcognathus and the "Rincossauro de Mariante" specimens, and the other only Hyperodapedon sp. Specimens. Multivariate Statistic Analysis showed that the skull shape changes was in accordance with size. The morphologic changes in the different skull regions reflected in the alometric coefficients are a consequence of individual size changes. In the first analysis, 73% of measurements showed a negative alometric growth when compared to the total skull length, whereas only 27% showed positive alometric growth (no one was isometric). In the second analysis, 91% of the measurements showed negative alometric growth when compared to the total skull length, whereas only 9% showed positive alometric growth (no one was isometric). The study showed a heterochronic mosaic of skull characters due to the association of paedomorphic and peramorphic heterochronic processes. The data taken from the analysis including the "Rincossauro de Mariante" and Scaphonyx sulcognathus, led to conclude that during ontogeny the skull presented a positive alometric growth of the rostrum and superior skull region compared to total skull length. This morphologic pattern, when analyzed in phylogenetic terms, reflects a peramorphocline. Excepting from rostrum and the superior skull region, every measurement showed negative alometric growth when compared to total length skull. This pattern reflects a paedomorphocline. The data taken on the analysis excluding "Rincossauro de Mariante" and Scaphonyx sulcognathus, allowed to conclude that during ontogeny the skull has had a positive alometric growth only in the rostrum, as showed mostly by the increasing of the premaxilla. Two of the heterochronoclines recognized in the evolution of rhynchosaurs are as well the most frequent among vertebrates as a whole; and due to progenesis for one hand, and hypermorphosis for the other. Neoteny in the rostrum, associated to a probable progenesis in mandible has led to the formation of the most characteristic aspect of upper Triassic rhynchosaurs, a premaxilla verticalized and ventrally bent. The peramorphocline due to hypermorphosis may have contributed to the extinction of the group. In vertebrates probably there are always associated at least two processes (progenesis and hypermorphosis), or even more; such as neoteny and acceleration in the case of rhynchosaurs. Processes are not isolated phenomena but always part of heterochronoclines, which may result of environmental solicitations, made possible thanks to the genotypic flexibility. Responses may be deepened, canalized and fixed, leading sometimes to the origin of new groups (paedomorphoclines due to progenesis); whereas in others may lead to extinction (peramorphoclines due to hypermorphosis). Heterochronic processes may give rise to gradual changes but not necessarily; so that the fossil record not always is "incomplete", but actually may reflect some kind of not gradual evolution. Heterochrony should be understood not as a scientific theory, but as a methodology permitting to recognize complex phenotypic patterns resulting from ontogeny.

Orientador: Célio Fernando Baptista Haddad / Banca: Rafael O. de Sá / Banca: Antonio Sebben / Resumo: O estudo comparativo da ontogenia fornece dados que podem servir tanto para elucidar alguns dos mecanismos de evolução, como para realizar reconstruções filogenética, principalmente porque permite a observação da diferenciação morfológica e temporal durante o desenvolvimento. O estudo das modificações do processo de ontogenia também fornece dados relativos aos processos de especiação. Neste sentido, os anfíbios podem ser considerados os melhores modelos para o estudo de ontogenia. Como eles têm embriões geralmente de vida livre, a aquisição da série de desenvolvimento necessário é relativamente fácil, quando comparado com qualquer outro animal vertebrado. Entre os anuros, por causa da diversidade de modos reprodutivos (que inclui desenvolvimento direto), os pipídeos oferecem uma rara oportunidade de investigar como o cuidado parental pode ter influenciado o desenvolvimento geral e, em particular, as rotas de desenvolvimento do esqueleto. Além disso, a família Pipidae também é um ramo que se diferenciou relativamente cedo na árvore de vida dos anfíbios anuros. Esta família apresenta uma combinação única de características plesiomórficas e exclusivamente derivadas, algumas das quais parecem representar adaptações a um estilo de vida aquático. O presente estudo descreve o desenvolvimento do crânio de Pipa arrabali e compara os dados disponíveis para outras espécies do grupo, incluindo outros Pipidae e Xenoanura. Para as análises morfológicas, foram utilizados 51 embriões em diferentes estágios de desenvolvimento. Para observar as estruturas esqueléticas os espécimes foram diafanizados e corados com uma técnica de dupla coloração para diferenciação de cartilagem e osso. O condrocrânio desta espécie é caracterizado pela redução ou ausência de várias estruturas, com uma concentração destas modificações nas regiões etmoidal e anterior. Quando comparado com outras espécies, parece óbvio / Abstract: The study of comparative ontogeny provides data that can serve both, to elucidate some of the mechanisms of evolution and phylogenetic reconstruction, mainly because it allows for the observation of morphological and temporal differentiation during development. The study of modifications of ontogenic process also permits that data relating to the process of speciation be observed. In this regard, frogs may be considered the best models for the study of ontogeny. As they generally have free living embryos, the acquisition of the necessary developmental series is relatively easy, when compared with many other vertebrates. Amongst frogs, because of the diversity of reproductive modes (which includes direct developers), pipids offer a rare opportunity to investigate how parental care may have influenced development in general and skeletal developmental pathways in particular. Besides, the family Pipidae is also a relatively early offshoot of the anuran tree of life, presenting a unique combination of plesiomorfic characteristics and exclusive derived characteristics, some of which seems to represent adaptations to an aquatic lifestyle. Here in, I describe the development of the cranium of Pipa arrabali and compare the data available to other species in the group, including other Pipidae and Xenoanura. For the morphological analyses, I used 51 embryos in different developmental stages. To observe the skeletal structures the specimens were cleared and double stained for cartilage and bone differentiation. The chondrocranium of this species is characterized by several reductions or absence of structures, with a concentration of these modifications in the ethmoidal and anterior regions. When compared to other species, it seems obvious that heterocrony is a pervasive mechanism for the ontogenetic modifications observed / Mestre

Aspectos heterocrônicos encontrados na evolução do crânio dos Rhynchosauria foram estudados como uma contribuição ao melhor entendimento da evolução deste grupo, bem como dos vertebrados em geral; para o que partiu-se do material Triássico do estado Rio Grande do Sul. A Análise dos Componentes Principais incluiu 22 crânios e foi utilizada para estabelecer classes de idades; a Equação de Alometria para estabelecer as modificações alométricas durante a ontogenia. Uma análise incluiu espécimes de Hyperodapedon sp., Scaphonyx sulcognathus e o "Rincossauro de Mariante"; e outra apenas espécimes de Hyperodapedon sp. A Análise Estatística Multivariada indicou que a alteração da forma craniana depende fortemente do tamanho. As alterações nas diferentes regiões do crânio, refletidas nos coeficientes alométricos, são uma consequência da alteração do tamanho dos indivíduos. Na primeira análise, 73% das medidas apresentaram crescimento alométrico negativo em relação à medida comparativa padrão, enquanto que apenas 27% das medidas apresentaram crescimento alométrico positivo (em nenhuma foi isométrico). Na segunda análise, 91% das medidas apresentaram crescimento alométrico negativo, enquanto que apenas 9% do crescimento alométrico foi positivo (em nenhuma foi isométrico). Encontrou-se um mosaico heterocrônico nos caracteres cranianos, com processos pedomórficos e peramórficos. A análise que inclui o "Rincossauro de Mariante" e Scaphonyx sulcognathus permite concluir que durante a ontogenia dos espécimes, o crânio apresentava um crescimento alométrico positivo do rostro e do teto da região craniana propriamente dita, quando comparados com o comprimento total do crânio; indicando uma peramorfoclinal. Exceto as do rostro e do teto da região craniana, todas as demais medidas apresentaram alometria negativa quando comparadas ao comprimento total do crânio, refletindo uma pedomorfoclinal. A análise que exclui o "Rincossauro de Mariante" e Scaphonyx sulcognathus indica que, durante a ontogenia, o crânio apresentava um crescimento alométrico positivo apenas no rostro, evidenciado pelo aumento na pré-maxila. Duas das heterocronoclinais encontradas na evolução dos rincossauros são as mais usuais nos vertebrados como um todo, e devidas à progênese por um lado e à hipermorfose por outro. A neotenia no rostro, associada a uma provável progênese na mandíbula levou à formação do aspecto mais característico dos rincossauros, a pré-maxila curvada ventralmente e verticalizada. A peramorfoclinal por hipermorfose pode ter contribuído para a extinção do grupo. Nos vertebrados provavelmente estão sempre associados dois processos (progênese e hipermorfose), ou mais, como neotenia e aceleração no caso dos rincossauros. Os processos não são fenômenos isolados, mas sempre parte de heterocronoclinais; as quais podem resultar de respostas a diferentes solicitações ambientais, possibilitadas pela flexibilidade genotípica. As respostas podem ser aprofundadas, canalizadas e fixadas, levando ou ao surgimento de grupos novos (pedomorfoclinais por progênese) ou à extinção de outros (peramorfoclinais por hipermorfose). Processos heterocrônicos podem originar mudanças graduais, mas não necessariamente; o que permite concluir que nem sempre o registro fóssil é realmente "incompleto", mas sim por vezes reflexo de algum tipo de evolução não gradual. Heterocronia não é uma teoria científica, mas uma metodologia que permite reconhecer padrões fenotípicos complexos resultantes da ontogenia. / Heterochronic aspects in the evolution of the Rhynchosauria skull were studied as a contribution to a better understanding of the evolution of this group, as well as of vertebrates as a whole; for what Triassic material from Rio Grande do Sul State was used. The Principal Components Analysis on 22 skulls sample was accomplished to establish age classes; and Allometry Equation to establish alometric changes during ontogeny. One analysis included Hyperodapedon sp., Scaphonyx sulcognathus and the "Rincossauro de Mariante" specimens, and the other only Hyperodapedon sp. Specimens. Multivariate Statistic Analysis showed that the skull shape changes was in accordance with size. The morphologic changes in the different skull regions reflected in the alometric coefficients are a consequence of individual size changes. In the first analysis, 73% of measurements showed a negative alometric growth when compared to the total skull length, whereas only 27% showed positive alometric growth (no one was isometric). In the second analysis, 91% of the measurements showed negative alometric growth when compared to the total skull length, whereas only 9% showed positive alometric growth (no one was isometric). The study showed a heterochronic mosaic of skull characters due to the association of paedomorphic and peramorphic heterochronic processes. The data taken from the analysis including the "Rincossauro de Mariante" and Scaphonyx sulcognathus, led to conclude that during ontogeny the skull presented a positive alometric growth of the rostrum and superior skull region compared to total skull length. This morphologic pattern, when analyzed in phylogenetic terms, reflects a peramorphocline. Excepting from rostrum and the superior skull region, every measurement showed negative alometric growth when compared to total length skull. This pattern reflects a paedomorphocline. The data taken on the analysis excluding "Rincossauro de Mariante" and Scaphonyx sulcognathus, allowed to conclude that during ontogeny the skull has had a positive alometric growth only in the rostrum, as showed mostly by the increasing of the premaxilla. Two of the heterochronoclines recognized in the evolution of rhynchosaurs are as well the most frequent among vertebrates as a whole; and due to progenesis for one hand, and hypermorphosis for the other. Neoteny in the rostrum, associated to a probable progenesis in mandible has led to the formation of the most characteristic aspect of upper Triassic rhynchosaurs, a premaxilla verticalized and ventrally bent. The peramorphocline due to hypermorphosis may have contributed to the extinction of the group. In vertebrates probably there are always associated at least two processes (progenesis and hypermorphosis), or even more; such as neoteny and acceleration in the case of rhynchosaurs. Processes are not isolated phenomena but always part of heterochronoclines, which may result of environmental solicitations, made possible thanks to the genotypic flexibility. Responses may be deepened, canalized and fixed, leading sometimes to the origin of new groups (paedomorphoclines due to progenesis); whereas in others may lead to extinction (peramorphoclines due to hypermorphosis). Heterochronic processes may give rise to gradual changes but not necessarily; so that the fossil record not always is "incomplete", but actually may reflect some kind of not gradual evolution. Heterochrony should be understood not as a scientific theory, but as a methodology permitting to recognize complex phenotypic patterns resulting from ontogeny.

A key question in biology is how changes in gene function or regulation produce new morphologies during evolution. The nectar spur is an evolutionarily labile structure known to influence speciation in a broad range of angiosperm taxa. Here, the genetic basis of nectar spur development, and the evolution of differences in nectar spur morphology, is investigated in Linaria vulgaris and two closely related species of orchid, the primitively longer-spurred Dactylorhiza fuchsii, and more derived short-spurred D. viridis (Orchidinae, Orchidaceae). Despite considerable morphological and phylogenetic differences, nectar spur ontogeny is fundamentally similar in each of the study species, proceeding from an abaxial bulge formed on the ventral petal relatively late in petal morphogenesis. However, spur development is progenetically curtailed in the short-spurred orchid D. viridis. In each case spur development involves class 1 KNOTTED1-like homeobox (KNOX) proteins. KNOX gene expression is not restricted to the spur-bearing petal, indicating that additional components are required to define nectar spur position, e.g. canonical ABC genes, determinants of floral zygomorphy, and additional (currently unknown) factors. However, constitutive expression of class 1 KNOX proteins in transgenic tobacco produces flowers with ectopic outgrowths on the petals, indicating that KNOX proteins alone are, to some degree, capable of inducing structures similar to nectar spurs in a heterologous host. Interestingly, KNOX gene expression is high in the ovary of all study taxa, suggesting that KNOX proteins may also have been involved in the evolution of this key angiosperm feature. Although principally involved in maintaining indeterminacy in the shoot apical meristem (SAM), members of the KNOX gene family have been co-opted in the evolution and development of compound leaves where they suppress differentiation and extend the morphogenetic potential of the leaf. A similar model is presented here to explain the role of KNOX proteins in nectar spur development. Co-option of KNOX gene expression to the maturing perianth delays cellular differentiation, facilitating the development of the nectar spur but requiring additional, unknown factors, to determine nectar spur fate. As facilitators of nectar spur development, changes in the spatio-temporal patterns of KNOX gene expression may alter the potential for nectar spur development and explain the critical length differences observed between the orchids D. fuchsii and D. viridis (and among other angiosperm taxa). Taken together, the available data indicate that KNOX genes confer a meristematic state upon plant tissues in a variety of morphogenetic contexts, making the gene family a potentially versatile tool to mediate a wide variety of evolutionary transformations.

Les poissons-perroquets, séparés en deux tribus les Scarini et les Sparisomatini, se sont diversifiés au sein des Labridae à partir d’ancêtres carnivores. L’un des facteurs essentiels pour expliquer cette diversification est leur exploitation d’une nouvelle niche trophique : les microorganismes. La plupart des Sparisomatini paissent des macroalgues pour y extraire les micro-organismes épiphytiques tandis que les poissons-perroquets brouteurs (tous les Scarini, certains Sparisoma) raclent ou creusent le substrat rocheux pour y récolter les microorganismes épilithiques et endolithiques. Ces changements de comportement alimentaire se sont accompagnés de nombreuses évolutions dentaires avec notamment la présence de plaques dentaires chez les brouteurs mais aussi certains paisseurs. Les phylogénies moléculaires ont contredit le précédent scénario évolutif sur la denture des poissons-perroquets qui stipulait l’apparition progressive de plaques dentaires à partir de dentures à dents non recouvertes d’os (dents libre s). Cette thèse se propose de réexaminer l’évolution de la denture chez les poissons -perroquets en se basant sur les progrès phylogénétiques mais aussi techniques (microtomographie 3D), tout en faisant le lien avec leur régime et leur fonction écologique. / Parrotfishes, divided into the tribes Scarini and Sparisomatini , are labrid fishes that arouse from carnivorous ancestors. One of the main factors that explain this radiation is the exploitation of microorganisms as a food source. While most Sparisomatini browse macroalgae to get epiphytic microorganisms, grazing parrotfishes (all Scarini and some Sparisoma) scrap or excavate hard substrate to obtain epilithic and endolithic microorganisms. Changes in feeding behaviour are associated with dentition specialization with notably dental plates in grazing but also some browsing parrotfishes. Gene-based phylogenies contradicted the previous evolutionary scenario about parrotfish dentition evolution, which states the progressive emergence of dental plates from non-coalesced dentitions. This thesis manuscript aims to re-examine the evolution of parrotfish dentitions in the light of the new phylogenies by using X-ray 3D microtomography, and to link this evolution with diet and ecology.

Diferenças no timing da melanização e esclerotização do exoesqueleto são evidentes quando se compara a morfologia externa de abelhas de hábitos sociais e as solitárias. A esta diferença convencionamos chamar de heterocronia da maturação cuticular, o termo heterocronia significando variações no tempo relativo, ou ritmo, de um evento ontogenético em relação ao ancestral ou entre taxons. Propusemos que as abelhas sociais, que após a ecdise permanecem na colônia por vários dias, alcançariam a maturidade de alguns sistemas orgânicos, entre eles o tegumento, muito mais tarde que as espécies de abelhas solitárias que ao emergir partem imediatamente para atividades extra-nidais. Neste contexto, o objetivo deste trabalho consistiu em testar esta hipótese utilizando o tegumento em maturação das espécies de abelhas sociais, Apis mellifera e Frieseomelitta varia, e da espécie solitária Centris analis, em estudos comparativos de expressão gênica, ultraestrutura e quantificação de hidrocarbonetos cuticulares (CHCs). Para isto utilizamos sequenciamento de mRNA (RNA-seq), microscopia eletrônica de transmissão (MET) e cromatografia de gás e espectrometria de massas (CG/MS). Os perfis de expressão de genes da via de melanização/esclerotização cuticular (ebony e tan) diferenciaram as espécies sociais da solitária, assim como a expressão de genes com função na via de metabolismo de quitina (Cda5, Idgf4 e chitooligosacchariodolytic-domain-like) e de genes codificadores de proteínas estruturais da cutícula (CPR14, CPR17, CPR18, CPR25, CPR23, CPR26, Apd-3 e Apd-like). Genes com função na regulação da maturação cuticular (FTZ-F1, E74, Hr46 e Hr4) se mostraram co-expressos nas espécies sociais e os perfis de expressão destes genes, exceto Hr46, e de outros reguladores (Ethr, Hr38, Rickets e Ptx-1) também diferenciaram as espécies sociais da solitária. Ressaltamos em nossas análises os genes do ciclo circadiano, cuja expressão tem relação com a deposição de quitina cuticular, além de genes de vias de pigmentação não melanínicas. As análises de MET, abrangendo outras três espécies de abelhas (Bombus brasilienses: primitivamente eussocial; Euglossa cordata: facultativamente social; Tetrapedia diversipes: solitária), mostraram diferenças consistentes entre a ultraestrutura e espessura das cutículas das espécies sociais e solitárias, o que reforçou nossos resultados de RNA-seq. A quantificação absoluta dos CHCs diferenciou as abelhas sociais da solitária, consistente com a hipótese de heterocronia da maturação cuticular e com os perfis de expressão de genes envolvidos na biossíntese de CHCs. Assim, além de desvendar transcriptomas de tegumento de três espécies de abelhas, a comparação da expressão gênica aliada à análise de ultraestrutura da cutícula e quantificação de CHCs levaram à caracterização de diferenças no processo de maturação cuticular entre as espécies sociais e solitárias / Differences in the timing of exoskeleton melanization and sclerotization processes are evident when comparing the external morphology of social and solitary bee species. Such differences may constitute a relevant example of cuticular maturation heterochrony, this term referring to a genetic change in timing of an ontogenetic process relative to an ancestor or between taxons. We proposed that social bees, which remain protected inside the colony for many days before initiating outside nest activities, would reach the maturity of some organic systems, such as the integument (epidermis and cuticle), later than solitary bees, which start such activities immediately after ecdysis. We tested this hypothesis in a comparative study of the developing integument of eusocial bees, Apis mellifera and Frieseomelitta varia, and the solitary bee Centris analis. Using RNA-seq, we verified that the expression profiles of genes involved in cuticular melanization and sclerotization (ebony and tan), chitin deposition and organization (Cda5, Idgf4, chitooligosacchariodolytic-domain-like), and cuticle formation (CPR14, CPR17, CPR18, CPR25, CPR23, CPE26, Apd-3, Apd-like) were positively, correlated between the two eusocial species, but not between the eusocial and the solitary species. Some of the genes with roles in regulating exoskeleton maturation (FTZ-F1, E74, Hr46, Hr4) were co-expressed only in the eusocial species. The expression profiles of these genes (except Hr46) and other regulatory genes (Ethr, Hr38, Rickets, Ptx-1) were also positively correlated exclusively in the eusocial bees. We also highlighted the expression of genes involved in non-melanin pigment production and the expression of circadian rhythm genes that could be related to chitin layers deposition. Transmission electron microscopy analysis of the integument of the two eusocial and the solitary bee species, in addition to other three bee species (the primitively eusocial Bombus brasilienses; the facultatively social Euglossa cordata; the solitary bee Tetrapedia diversipes), showed differences in cuticle ultrastructure and thickness, thus supporting the RNA-seq data. In agreement with our hypothesis, CHC quantifications were consistent with the expression levels of genes involved in CHC biosynthesis, thus differentiating the superficial cuticle layer of the eusocial and solitary species. Together, the integument transcriptomes, ultrastructure, and CHC quantification allowed us to characterize differences in the timing of cuticle maturation in social and solitary bees

Diferenças no timing da melanização e esclerotização do exoesqueleto são evidentes quando se compara a morfologia externa de abelhas de hábitos sociais e as solitárias. A esta diferença convencionamos chamar de heterocronia da maturação cuticular, o termo heterocronia significando variações no tempo relativo, ou ritmo, de um evento ontogenético em relação ao ancestral ou entre taxons. Propusemos que as abelhas sociais, que após a ecdise permanecem na colônia por vários dias, alcançariam a maturidade de alguns sistemas orgânicos, entre eles o tegumento, muito mais tarde que as espécies de abelhas solitárias que ao emergir partem imediatamente para atividades extra-nidais. Neste contexto, o objetivo deste trabalho consistiu em testar esta hipótese utilizando o tegumento em maturação das espécies de abelhas sociais, Apis mellifera e Frieseomelitta varia, e da espécie solitária Centris analis, em estudos comparativos de expressão gênica, ultraestrutura e quantificação de hidrocarbonetos cuticulares (CHCs). Para isto utilizamos sequenciamento de mRNA (RNA-seq), microscopia eletrônica de transmissão (MET) e cromatografia de gás e espectrometria de massas (CG/MS). Os perfis de expressão de genes da via de melanização/esclerotização cuticular (ebony e tan) diferenciaram as espécies sociais da solitária, assim como a expressão de genes com função na via de metabolismo de quitina (Cda5, Idgf4 e chitooligosacchariodolytic-domain-like) e de genes codificadores de proteínas estruturais da cutícula (CPR14, CPR17, CPR18, CPR25, CPR23, CPR26, Apd-3 e Apd-like). Genes com função na regulação da maturação cuticular (FTZ-F1, E74, Hr46 e Hr4) se mostraram co-expressos nas espécies sociais e os perfis de expressão destes genes, exceto Hr46, e de outros reguladores (Ethr, Hr38, Rickets e Ptx-1) também diferenciaram as espécies sociais da solitária. Ressaltamos em nossas análises os genes do ciclo circadiano, cuja expressão tem relação com a deposição de quitina cuticular, além de genes de vias de pigmentação não melanínicas. As análises de MET, abrangendo outras três espécies de abelhas (Bombus brasilienses: primitivamente eussocial; Euglossa cordata: facultativamente social; Tetrapedia diversipes: solitária), mostraram diferenças consistentes entre a ultraestrutura e espessura das cutículas das espécies sociais e solitárias, o que reforçou nossos resultados de RNA-seq. A quantificação absoluta dos CHCs diferenciou as abelhas sociais da solitária, consistente com a hipótese de heterocronia da maturação cuticular e com os perfis de expressão de genes envolvidos na biossíntese de CHCs. Assim, além de desvendar transcriptomas de tegumento de três espécies de abelhas, a comparação da expressão gênica aliada à análise de ultraestrutura da cutícula e quantificação de CHCs levaram à caracterização de diferenças no processo de maturação cuticular entre as espécies sociais e solitárias / Differences in the timing of exoskeleton melanization and sclerotization processes are evident when comparing the external morphology of social and solitary bee species. Such differences may constitute a relevant example of cuticular maturation heterochrony, this term referring to a genetic change in timing of an ontogenetic process relative to an ancestor or between taxons. We proposed that social bees, which remain protected inside the colony for many days before initiating outside nest activities, would reach the maturity of some organic systems, such as the integument (epidermis and cuticle), later than solitary bees, which start such activities immediately after ecdysis. We tested this hypothesis in a comparative study of the developing integument of eusocial bees, Apis mellifera and Frieseomelitta varia, and the solitary bee Centris analis. Using RNA-seq, we verified that the expression profiles of genes involved in cuticular melanization and sclerotization (ebony and tan), chitin deposition and organization (Cda5, Idgf4, chitooligosacchariodolytic-domain-like), and cuticle formation (CPR14, CPR17, CPR18, CPR25, CPR23, CPE26, Apd-3, Apd-like) were positively, correlated between the two eusocial species, but not between the eusocial and the solitary species. Some of the genes with roles in regulating exoskeleton maturation (FTZ-F1, E74, Hr46, Hr4) were co-expressed only in the eusocial species. The expression profiles of these genes (except Hr46) and other regulatory genes (Ethr, Hr38, Rickets, Ptx-1) were also positively correlated exclusively in the eusocial bees. We also highlighted the expression of genes involved in non-melanin pigment production and the expression of circadian rhythm genes that could be related to chitin layers deposition. Transmission electron microscopy analysis of the integument of the two eusocial and the solitary bee species, in addition to other three bee species (the primitively eusocial Bombus brasilienses; the facultatively social Euglossa cordata; the solitary bee Tetrapedia diversipes), showed differences in cuticle ultrastructure and thickness, thus supporting the RNA-seq data. In agreement with our hypothesis, CHC quantifications were consistent with the expression levels of genes involved in CHC biosynthesis, thus differentiating the superficial cuticle layer of the eusocial and solitary species. Together, the integument transcriptomes, ultrastructure, and CHC quantification allowed us to characterize differences in the timing of cuticle maturation in social and solitary bees

Skeletal (cartilaginous, bony, and dental) tissues undoubtedly exemplify the key innovation of vertebrates. Among all recent vertebrates, the most numerous and successful lineage is represented by the Ray-finned fishes that, accordingly, exhibit amazing variety of skeletal architectures and phenotypic adaptations. In order to depict fundamental principles of fish cranial skeletogenesis the developmental formation of skeletal architectures was described, compared and analyzed using members of early branching fish lineages, that exemplify very different strategies of skeletogenesis. While the Senegal bichirs and the Tropical gars are heavily armored forms with massive exoskeleton and hyperossified dental structures covering the whole oropharyngeal region, the European sterlets, on the contrary, possess mostly cartilaginous skeleton and reduce their dental structures during early development. Whole analysis is underpinned by the Northen pike, teleostean species with lightened skeletal architecture with comparable number of cranial elements. The present study represents the first complex comparative analysis of their skeletogenesis and odontogenesis. This allowed to define developmental strategies founding different lineage-specific skeletal architecture of vertebrates. Comparative description of...

Le développement dentaire est essentiellement étudié chez la souris, modèle mammifère le plus commun en biologie. Cependant, contrairement à la majorité des mammifères, les souris ne remplacent pas leurs dents. Ainsi, les mécanismes impliqués dans le remplacement dentaire mammalien sont encore inconnus. Au cours de cette thèse, nous nous sommes intéressés au développement et remplacement dentaire mammalien en utilisant le lapin Oryctolagus cuniculus comme modèle d’étude. Le lapin étant déjà séquencé, utilisé en recherche biomédicale avec une période de gestation courte et remplaçant ses dents, il semblait être un modèle pertinent en odontologie. Le lapin était un modèle méconnu du point de vue du développement dentaire, nous avons donc d’abord réalisé une étude histo-morphologique afin de caractériser la mise en place des dents déciduales et permanentes. Des reconstructions 3D des tissus mous ont été réalisés à différents stades embryonnaires afin d’obtenir une chronologie du développement et remplacement dentaire. Cette chronologie commence aux premières observations morphologiques de l’initiation du développement des premières dents jusqu’à la minéralisation des dernières dents à se développer.Puis, suite à l’identification dans la bibliographie de gènes candidats potentiellement impliqués dans le remplacement dentaire, nous avons étudiés les profils d’expressions de ces gènes afin de mieux comprendre la régulation spatio-temporelle du remplacement dentaire chez le lapin. Nous avons ensuite replacé nos résultats sur le développement chez le lapin dans un contexte évolutif. Ainsi, nous avons réalisé une étude d’anatomie comparée chez les lagomorphes actuels et quelques fossiles afin d’identifier des variations morphologiques dentaires au cours de leur histoire évolutive. Nous nous sommes particulièrement intéressé à la mise en place des cuspides au cours de l’odontogénèse ainsi qu’aux variations de la surface occlusale des dents supérieure tout au long de la vie des lapins et autres lagomorphes. En comparant les variations au cours de l’évolution avec celles observées lors de l’ontogénie dentaire chez le lapin nous avons identifié des processus d’hétérochronies du développement. Nous avons ainsi montré que la molaire actuelle du lapin suit un processus de péramorphose, donc de surdéveloppement, en comparaison aux lagomorphes fossiles. Le lapin est ainsi un modèle animal prometteur en biologie du développement et en évolution afin de mieux comprendre la mise en place du remplacement dentaire mammalien ainsi que les variations de forme dentaire au cours de l’évolution des mammifères. / Tooth development is essentially studied in mice, the favorite mammalian model in biology. However, mice do not replace their teeth on contrary to numerous mammals. So, the mechanisms involved in mammalian tooth replacement are still unknown. In this thesis, we focused on mammalian dental development and replacement using the European rabbit Oryctolagus cuniculus as animal model. The European rabbit is already sequenced, used in biomedical field, has a short gestation time and replaced its teeth, so rabbit seemed to be a relevant model in dental research.Rabbit dental development was not defined, so we first performed a histo-morphological study to characterize the development of deciduous and permanent teeth. 3D soft tissue reconstructions were performed at different embryonic stages to obtain a chronology of tooth development and replacement. This chronology begins with the first morphological observations of the initiation of the development of the first tooth until the mineralization of the last tooth to develop.Then, we identified in the bibliography candidate genes potentially involved in dental replacement. We studied the expression profiles of these genes in order to better understand the spatio-temporal regulation of tooth replacement in rabbits.We then returned our results to rabbit development in an evolutionary context. Thus, we performed a comparative anatomy study in the current lagomorphs and some fossils in order to identify dental morphological variations during their evolutionary history. We were particularly interested in the setting of cusps during odontogenesis as well as in the variations of the occlusal surface of the upper cheek teeth throughout the life of rabbits and other lagomorphs. Comparing changes during evolution with those observed during dental ontogeny in rabbits allow us to identify heterochronous processes of development. We have shown that the current molar rabbit follows a process of peramorphosis, so an overdevelopment compared to fossil lagomorphs.The rabbit is thus a promising animal model in developmental and evolutionary biology to better understand the implementation of mammalian tooth replacement and tooth shape variations during mammalian evolution.

une délicate maison de bêtises est un recueil de poésie qui explore la tension entre rester et partir. La sujet lyrique évoquée dans les poèmes est tiraillée entre son envie de connexion, de relation, et son besoin de liberté. Écrit en vers libres, séparé en trois parties selon les affects de la sujet, le recueil explore les espaces, physiques et émotionnels, que l’on fuit puis réinvestit incessamment. L’essai s’intéresse au recueil Là où fuit le monde en lumière de Rose Eliceiry et analyse comment les lieux mis en place dans les poèmes interagissent avec la sujet lyrique. Il réfléchit à l’inscription de la douleur sur les corps, que ceux-ci soient humains ou urbains. S’appuyant notamment sur le concept de Thirdspace développé par le géographe Edward W. Soja, il invite à repenser les lieux au-delà de leur inscription physique et mentale, dans ce qu’ils peuvent produire comme espaces tiers. La compréhension de ces emplacements autres se construit également à partir des travaux de Michel Foucault, autour de ce qu’il nomme des hétérotopies et des hétérochronies. Entrant et sortant de ces espaces autres, la sujet lyrique d’Eliceiry est soumise aux délimitations des lieux qui l’accueillent ou la rejettent. Malgré l’importance de l’altérité au cœur de la poésie, cet essai étudie également la présence et la signification de multiples lieux communs insérés dans la parole poétique. Ces topoï littéraires ancrent l’œuvre et l’expérience de la sujet lyrique dans notre contemporanéité en évoquant à la fois certains enjeux de l’époque et différentes réalités sociales. / une délicate maison de bêtises is a book of poetry that explores the tension between staying and going. The lyrical subject evoked in the poems is torn between its desire for connection, for relationship, and its need for freedom. Written in free verses, separated into three parts according to the affects of the subject, the book explores places, physical and emotional, that we avoid and then constantly reinvest. This dissertation focuses on the book of poetry Là où fuit le monde en lumière of the Quebecoise poet Rose Eliceiry. It analyses how the places of the city interact with the lyrical subject. It explores the inscription of pain on bodies, whether they are human or urban. Based on the concept of Thirdspace developed by the geographer Edward W. Soja, it invites us to rethink places beyond their physical and mental inscription, in what they can produce as othering spaces. The understanding of these spaces is also built around the work of Michel Foucault, from what he calls heterotopias and heterochronies. Entering and leaving these other areas, the lyrical subject of Eliceiry is vulnerable to the boundaries of the places that welcome or reject her. Despite the importance of otherness at the heart of the poems, this essay also studies the presence and significance of multiple commonplaces inserted in the poetic speech. These literary topoi anchor the work and experience of the lyrical subject in our contemporaneity by evoking both certain issues of the time and different social realities.