Orientador: Célio Fernando Baptista Haddad / Resumo: As forças de seleção natural e sexual atuam sobre os indivíduos e podem os levar ao isolamento reprodutivo. O acúmulo de modificações evolutivas pode levar ao surgimento de novas entidades. Espécie é unidade basal em biologia; assim, com sua exuberante biodiversidade, a Mata Atlântica é uma floresta neotropical fundamental para estudos de processos evolutivos em anfíbios anuros. Reunindo os gêneros Cycloramphus, Thoropa e Zachaenus, a família Cycloramphidae agrupa espécies de anuros saxícolas e terrestres, todas endêmicas da Mata Atlântica brasileira. Pelos seus hábitos especializados de vida e reprodução, os cicloramfídeos formam um grupo atrativo para estudos evolutivos. Na presente tese são apresentados quatro capítulos que abordam três escalas distintas da evolução: a diversificação entre espécies, populações e indivíduos. São abordados: (1) a filogenia molecular multilocus de Cycloramphus-Zachaenus, demonstrando que a evolução da terrestrialidade no clado se correlaciona com o dimorfismo sexual em tamanho; (2) a diversificação fenotípica associada a diversificações genéticas em populações de C. boraceiensis e C. dubius, demonstrando que estas duas espécies-irmãs saxícolas apresentam zona de contato e hibridação; (3) o sistema de acasalamento promíscuo e as relações sociais em C. boraceiensis, demonstrando que tamanho das fêmeas e dos machos e parentesco genético influenciam a formação dos casais; e (4) o sistema de acasalamento poligínico com fidelidade na espécie saxíco... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Natural and sexual selection forces act on individuals and may lead to reproductive isolation. The increase in evolutionary modifications may lead to the emergence of new entities. Species is the basal unity in biology; thus, with its exuberant biodiversity, the Atlantic rainforest is a fundamental Neotropical forest for studies of evolutionary processes in anuran amphibians. Comprising the genera Cycloramphus, Thoropa, and Zachaenus, the family Cycloramphidae gathers saxicolous and terrestrial anuran species, all endemic to the Brazilian Atlantic rainforest. Because of their specialized life and reproductive habits, cycloramphids are an attractive group for evolutionary studies. In the present thesis we show four chapters that address three distinct scales of evolution: the diversification among species, populations, and individuals. We goaled: (1) the multilocus molecular phylogeny of Cycloramphus-Zachaenus, demonstrating that the evolution of terrestriality in the clade is correlated with the sexual size dimorphism; (2) the phenotypic diversification associated with the genetic diversifications in populations of C. boraceiensis and C. dubius, demonstrating that these two saxicolous sister species show a contact zone and hybridization; (3) the promiscuous mating system and the social relations in C. boraceiensis, demonstrating that female and male sizes, and mate relatedness influence pair formation; and (4) the polygynic mating system with fidelity in the saxicolous specie... (Complete abstract click electronic access below) / Doutor

Triclosan (TCS) and triclocarban (TCC) are antimicrobials found in U.S. surface waters. This dissertation assessed the effects of TCS and TCC on early development and investigated their potential to bioaccumulate using Xenopus laevis as a model. The effects of TCS on metamorphosis were also investigated. For 0-week tadpoles, LC50 values for TCS and TCC were 0.87 mg/L and 4.22 mg/L, respectively, and both compounds caused a significant stunting of growth. For 4-week tadpoles, the LC50 values for TCS and TCC were 0.22 mg/L and 0.066 mg/L; and for 8-week tadpoles, the LC50 values were 0.46 mg/L and 0.13 mg/L. Both compounds accumulated in Xenopus. For TCS, wet weight bioaccumulation factors (BAFs) for 0-, 4- and 8-week old tadpoles were 23.6x, 1350x and 143x, respectively. Lipid weight BAFs were 83.5x, 19792x and 8548x. For TCC, wet weight BAFs for 0-, 4- and 8-week old tadpoles were 23.4x, 1156x and 1310x. Lipid weight BAFs were 101x, 8639x and 20942x. For the time-to-metamorphosis study, TCS showed an increase in weight and snout-vent length in all treatments. Exposed tadpoles metamorphosed approximately 10 days sooner than control tadpoles. For the hind limb study, although there was no difference in weight, snout-vent length, or hind limb length, the highest treatment was more developed compared to the control. There were no differences in tail resorption rates between the treatments and controls. At relevant concentrations, neither TCS nor TCC were lethal to Xenopus prior to metamorphosis. Exposure to relatively high doses of both compounds resulted in stunted growth, which would most likely not be evident at lower concentrations. TCS and TCC accumulated in Xenopus, indicating that the compound has the potential to bioaccumulate through trophic levels. Although TCS may increase the rate of metamorphosis in terms of developmental stage, it did not disrupt thyroid function and metamorphosis in regards to limb development and tail resorption.

Amphibian populations are declining globally due to a litany of factors including pollution, disease, climate change, and most importantly, habitat destruction. As most amphibian life histories involve their populations being recruitment limited, focusing on the mechanism behind breeding habitat selection will reveal useful cues that managers may use to increase abundance and breeding success. Though there are many theoretical models that describe the distribution of animals in response to a resource, the ideal free distribution (IFD) theory has not yet been applied to amphibian settling decisions. Through this application of the IFD, I have found that a population of wood frogs (Lithobates sylvaticus) in Patuxent National Wildlife Refuge select vernal pools that are large, deep, and hold water into the summer months to breed from 2010-2015. This information will provide managers with the ability to predict sites where wood frogs will breed in the future, as well as describe the cues that wood frogs are cueing in on so we can protect, alter, or create ideal breeding habitat.

The present thesis is a contribution to unravel the molecular mechanisms that underlie urodele regeneration. Urodele amphibians (newts and salamanders) are among the few vertebrates with the remarkable ability to regenerate lost body appendages, like the limbs and the tail. Urodele tail and limb regeneration occurs via blastemal epimorphic regeneration. A blastema is a mound of progenitor cells that accumulates at the amputation plane and eventually gives rise to the missing structures. It is known today that dedifferentiating muscle fibers at the amputation plane contribute to the blastema cell pool, but how this process occurs on the cellular and molecular level is hardly understood, which is in part due to the lack of molecular methods to test gene function in urodeles. Furthermore, little is known about how coordinated growth and patterning occurs during urodele regeneration, and if the patterning mechanisms in regeneration are related to the ones in development. The goal of this study was to better understand these processes on the molecular level. To address these questions, I first established several methods in our model systems, which are the mexican salamander Ambystoma mexicanum (axolotl) and a cell line derived from the newt Notophthalmus viridescens. In order to monitor gene expression on a cellular level during regeneration, I worked out a good in situ hybridization protocol on axolotl tissue cryosections. To be able to test gene function, I established electroporation conditions to both overexpress genes in the cultured newt cells and to deliver morpholinos into axolotl cells in vivo and newt cells in culture. I demonstrate here that morpholinos are an effective tool to downregulate protein expression in urodele cells in vivo and in culture. Testing the role of two candidate genes in muscle fiber dedifferentiation, the homeobox containing transcription factor Msx1 and Rad, a GTP-binding protein of a new Ras-related protein family, revealed that neither seems to play a major role in muscle dedifferentiation, both in culture and in vivo. In addition to testing gene function I have examined the muscle dedifferentiation process in more detail. I show here that dedifferentiating muscle fiber nuclei undergo morphological changes that are likely due to chromatin remodeling events. I also demonstrate that the axolotl spinal cord expresses embryonic dorsoventral (d/v) patterning markers of the neural tube. The transcription factors Msx1, Pax7 and Pax6 are expressed in their respective d/v domains in both the differentiated and the regenerating axolotl spinal cord. Furthermore, the secreted signaling molecule sonic hedgehog (Shh) is expressed in the floor plate in both the differentiated and the regenerating cord. Using a chemical inhibitor (cyclopamine) and an activator of the hedgehog pathway, I discovered that hedgehog signaling is required for overall tail regeneration. Blocking hedgehog signaling does not only result in d/v patterning defects of the regenerating spinal cord, but it also strongly reduces blastema cell proliferation. In addition, I identified cartilage and putative muscle progenitor cells in the blastema, marked by the expression of the transcription factors Sox9 and Pax7, respectively. Both progenitor populations are reduced in the blastema in the absence of hedgehog signaling. The continuous expression of marker genes for embryonic progenitor cell domains in the mature axolotl may be related to their ability to regenerate.

Urodele amphibians have the remarkable ability to re-grow lost body parts. This regenerative response after injury in urodeles involves dedifferentiation of fully differentiated cells into proliferative cells. One well-studied example of this is the dedifferentiation of multinucleated muscle cells into mononucleate cells resembling their precursors, the myoblasts. To form these mononucleate cells the differentiated myotubes in vivo must re-enter and complete the cell cycle; they again proliferate and produce progeny. A key question is what factors induce the myotubes to re-enter the cell cycle and proliferate. Early events of cell cycle re-entry can be studied in the A1 cell line, a myogenic cell line isolated from the Notophthalmus viridescens hindlimb, which traverses cell cycle until G2 in response to serum. In particular, it was found that thrombin cleavage induces a factor in serum of all animals tested so far to promote S phase re-entry in A1 myotubes. We have used this S phase re-entry of the A1 cell line to purify the serum activity and developed a 5-step purification protocol that enriches the activity almost 2 000 fold over the starting material, or 40 000 fold over serum. To conveniently produce and test potential candidates for their ability to induce S phase re-entry in A1 myotubes, we also developed an overexpression- and purification system for emerging candidates. Candidates were then tested for this activity with or without prior incubation with thrombin. We identified Bmp proteins as the first pure molecules that were found in fractions across the purification of the activity and that could also induce cell cycle re-entry in a dose-dependent manner when recombinantly added to the A1 myotubes. Furthermore, this response could be blocked in a dose-dependent manner by the known bmp-inhibitor noggin. Finally, we showed that inhibition of Bmp signaling in vivo causes defects in axolotl tail regeneration.

Understanding how migration, genetic drift, and natural selection interact to maintain the genetic and phenotypic variation we observe in natural populations is a central goal of population genetics. Amphibians provide excellent model organisms for investigating the interplay between these evolutionary forces because amphibians are generally characterized by limited dispersal abilities, high philopatry, and are obligately associated with the areas around suitable habitats (e.g. breeding ponds). Thus, on relatively small geographic scales, the relative effects of all of these evolutionary forces can be studied together. Here, we study the interaction of migration, genetic drift, natural selection, and historical process in the ornate chorus frog (Pseudacris ornata). We report the development and characterization of 10 polymorphic microsatellite genetic markers. Number of alleles per locus ranged from 2 to 21 averaging 9.2 and expected heterozygosities ranged from 0.10 to 0.97 averaging 0.52. However, in an analysis of two populations, three locus-by-population comparisons exhibited significant heterozygote deficiencies and indicated that null alleles may be present some loci. Furthermore, we characterized genetic structure and historical biogeographic patterns in P. ornata using these microsatellite markers along with mitochondrial DNA sequence data. Our data indicate that in these frogs, migration may play a large role in determining population structure as pairwise estimates of FST were relatively small ranging from 0.04 to 0.12 (global FST = 0.083). Additionally, we observed an overall pattern of isolation-by-distance in neutral genetic markers across the species range. Moreover, our data suggest that the Apalachicola River basin does not impede gene flow in P. ornata as it does in many vertebrate taxa. Interestingly, we identified significant genetic structure between populations separated by only 6 km. However, this fine scale genetic structure was only present in the more urbanized of two widespread sampling localities. Finally, in this study, we demonstrated that there was a significant correlation between the frequency of green frogs and latitude. There was a higher frequency of green frogs in southern samples and a lower frequency of green frogs in northern samples. However, when we interpreted this phenotypic cline in light of the overall pattern of isolation-by-distance, it was apparent that the neutral evolutionary forces of genetic drift and migration could explain the cline, and the invocation of natural selection was not necessary.

Limited breeding success in captive breeding programs has necessitated the development of assisted reproductive technologies (ART) to preserve and increase genetic variation and population numbers of both captive and wild amphibian groups. ART has been shown to be successful in numerous anuran species, and current studies focus on the application of ART in ex-situ populations. The focus of this project is to show that linking in-situ and ex-situ amphibian populations through sperm cryopreservation, genome banking, and in-vitro fertilization is possible, with the goal of increasing gene diversity throughout groups in order to produce self-sustaining, wild populations in the future. Specific objectives include developing a sperm-cryopreservation methodology using sperm from the model species Anaxyrus fowleri, applying this protocol to the cryopreservation of spermatozoa from two other threatened anurans to determine protocol transmissibility, and linking in-situ and ex-situ populations of an endangered species using cryopreserved sperm form wild males to produce viable offspring.

Amidst the amphibian extinction crisis, in situ and ex situ amphibian species conservation initiatives utilize assisted reproductive technologies for optimal genetic management of captive and wild populations. Development of effective, simple, low-cost methods for obtaining gametes for artificial fertilization, sperm biobanking, or natural breeding alleviates obstacles for institutions or programs to apply reproductive technologies. Objectives herein include investigating the efficacy of alternative hormones for inducing spermiation, an alternative hormone administration route for inducing ovulation, exploring the physiological effects of hormone therapy, and utilizing sperm from deceased animals for biobanking.

<p> Per- and polyfluoroalkyl substances (PFAS) are a class of persistent environmental contaminants that have become ubiquitous, resulting in widespread exposure among humans and wildlife. Amphibians are regularly exposed in the field, making them susceptible to sublethal effects of PFAS exposure. In amphibians exposed to PFAS, deleterious effects have been observed, including reduction in body condition measured using the scaled mass index (SMI) and degraded hepatic condition, among others. PFAS may dysregulate lipid metabolism by altering signaling cascades regulated by peroxisome proliferator activated receptors (PPAR), but whether changes in energy stores can explain changes in amphibian SMI and/or hepatic condition remain underexplored. Since lipids are a critical energy reserve for anurans, understanding whether lipid metabolism is being perturbed is critical. The central objective of this thesis was to investigate the effect of PFAS on lipid homeostasis in <em>Xenopus laevis </em>tadpoles within the context of a PPAR mechanism of action (MOA), considering apical, molecular, and lipidomic endpoints. I conducted three studies: (a) a study to characterize SMI and the relative expression of the hepatic xPPARα/β/γ during metamorphosis, (b) a pharmaceutical exposure to assess the <em>in vivo</em> effects of xPPARα/β/γ agonism on hepatic gene expression for select downstream targets (<em>apoa5, fabp1, acox1,​ pck1</em>), and (c) a chronic PFAS exposure to investigate the effects of environmentally relevant concentrations (PFOS, PFHxS, PFOA, PFHxA at 0.5 ppb; binary mixture of PFOS:PFHxS at 1 ppb) on lipid homeostasis through apical endpoints (mass, snout vent length, SMI, hepatic condition), relative hepatic gene expression, and Multiple Reaction Monitoring (MRM) profiling of the hepatic lipidome for changes in relative class abundance. In study (a), I identified SMI and hepatic expression of <em>xPPARα/β/γ</em> is dynamic during late metamorphosis, indicating the potential for heightened susceptibility. However, in study (b), pharmaceutical agonists had no effect on <em>X. laevis</em> at high doses. For study (c), I did not observe effects on a majority of apical endpoints, including SMI, but detected a significant sex-specific reduction in hepatic condition for male<em> X. laevis</em> tadpoles exposed to single-chemical perfluorosulfonic acid (PFSA) treatments. For gene expression, I observed a transient downregulation for apolipoprotein-V (<em>apoa5</em>) at Nieuwkoop and Faber (NF) stage 62 for <em>X. laevis</em> tadpoles exposed to single-chemical perfluorocarboxylic acid (PFCA) treatments. Lipid profiling detected transient dysregulation of predominantly membrane lipids in-response to short-chain PFAS treatments at NF 58. Overall, our findings indicate PFAS may exert toxicity during anuran metamorphosis through multiple mechanisms of action (MOA) with sex-specific and developmental-stage specific outcomes.</p>

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