Globally, amphibian species have been in decline and a wide range of factors have been purported to be driving the decline. The Gulf Coastal Plain of Florida has a high degree of endemism and rarity and the biodiversity in the region includes a diverse suite of amphibian species. Degradation of habitat has been considered by many to be a major part of amphibian declines, however amphibian declines are complex and in many cases multiple factors are occurring in concert. My dissertation research examined aspects of habitat ecology and occupancy for two rare amphibians, Florida Bog Frog (Rana okaloosae) (Chapter 1, 2, and 3) and Reticulated Flatwoods Salamander (Ambystoma bishopi) (Chapter 5), that are both restricted to the Northern Gulf Coastal Plain. Further, for R. okaloosae I examined the influence of a sympatric congener, Bronze Frog (R. clamitans clamitans), on microhabitat selection (Chapter 1) and growth of tadpoles (Chapter 4). My overall goal was to be able to elucidate factors that limit the geographic range of R. okaloosae and A. bishopi and to identify habitat characteristics that managers could maintain or create to conserve or increase populations of these species. My first chapter examined the microhabitat relationships between R. okaloosae and R. c. clamitans. Rana okaloosae is endemic to northwestern Florida and is sympatric with R. c. clamitans, a more common and widely distributed congener. Further, the two species appeared to be syntopic, have overlapping breeding seasons, and are known to hybridize. The objectives of this chapter were to assess the microhabitat selection of both species and to assess differences in microhabitat use of males of both species during the breeding season. My modeling of habitat selection and comparison of variables used by each species suggests that males of these species select different resources when calling. Therefore, these sympatric ranids select for different resources at a fine scale, however there does appear to be some overlap among some selected habitat characteristics. In Chapter 2, I assessed the habitat use of R. okaloosae at multiple spatial scales. I surveyed for R. okaloosae and evaluated habitat characteristics at used sites and sites where I had no detections to develop among- and within-stream habitat models for R. okaloosae. Rana okaloosae used habitats with high amounts of emergent vegetation at both the among-stream scale and the within-stream scale. Emergent vegetation appears frequently in models of anuran habitat selection, particularly those that occur in fire-dominated landscapes. Further understanding the habitat requirements of R. okaloosae will allow land managers to use appropriate management activities (e.g., prescribed fire) that will increase emergent vegetation and potentially restore habitat that may help increase populations of R. okaloosae. In Chapter 3, I conducted aural surveys for R. okaloosae at two different spatial scales: range-wide and stream-level scales to understand how occupancy and colonization of R. okaloosae may be influenced by scale. My results suggest that at both spatial scales occupancy of R. okaloosae was best described by the presence of mixed forest wetlands at survey sites. At the range-wide scale, colonization and detection were constant across years, however, at the stream-level scale, colonization was predicted by the number of years since last fire and detection was best predicted by the additive combination of relative humidity and temperature. Occupancy of R. okaloosae was patchy at the range-wide and at the stream-level scales and colonization was low at both scales, while derived estimates of local extinction were moderately high. While R. okaloosae still occur in 3 watersheds where they were initially observed in the 1980's, one of the three watersheds appears to be very isolated and detections there are becoming very infrequent. In Chapter 4, I experimentally evaluated the effects of R. c. clamitans tadpoles on R. okaloosae tadpoles. My results suggest that there was limited influence of R. c. clamitans on R. okaloosae. Conversely, it appeared that Rana c. clamitans was more susceptible to intraspecific competition than interspecific competition. The lack of a strong competitive effect of Rana c. clamitans on Rana okaloosae suggests that competitive interactions among tadpoles may have a limited effect at the densities I examined. In Chapter 5, our objectives were to evaluate a suite of within-pool factors (i.e., vegetation structure, water level, and an index to presence of fish) that could influence occupancy of breeding wetlands by larval flatwoods salamanders on Eglin Air Force Base in Florida, USA. Site occupancy over a 4 year period was best described by a model that incorporated high herbaceous vegetation cover and open canopy cover. Detection probability was assessed, but it varied among years and was not included in the model. Our study suggests that managing the breeding habitat of flatwoods salamander for open canopies and dense herbaceous vegetation may contribute to this species' recovery. In conclusion, Chapters 1-3 of my dissertation contribute to a growing understanding about the habitat ecology of R. okaloosae. I have evaluated habitat use of R. okaloosae at multiple spatial scales. At the finest spatial scale R. okaloosae selected for sites that had an abundance of cover probably decreasing their risk of predation (Chapter 1). Similarly, in Chapter 2 at two spatial scales, among and within-streams, R. okaloosae selected for emergent vegetation. Finally, at the broadest spatial scale, range-wide, R. okaloosae were found to be associated with mixed forest wetlands (Chapter 3). I did not find strong support for competition between R. okaloosae and R. c. clamitans tadpoles, although there was some evidence of asymmetric competition (Chapter 4). Further, adult males of each species did not select the same habitat characteristics for calling sites, so there appeared to be some resource partitioning (Chapter 1). Finally, the presence of A. bishopi larvae was found to be associated with herbaceous vegetation and moderate amounts of canopy cover (Chapter 5). Results from Chapter 2 and 5 suggest that both R. okaloosae and A. bishopi are associated with habitat conditions that are likely a result of fire penetrating wetland areas. / Ph. D.

In contrast to mammals, some fish and amphibians have retained the ability to regenerate complex body structures or organs, such as the limb, the tail, the eye lens or even parts of the heart. One major difference in the response to injury is the appearance of a mesenchymal growth zone or blastema in these regenerative species instead of the scarring seen in mammals. This blastema is thought to largely derive from the dedifferentiation of various functional cell types, such as skeletal muscle, skin and cartilage. In the case of multinucleated skeletal muscle fibres, cell cycle re-entry into S-phase as well as fragmentation into mononucleated progenitors is observed both in vitro and in vivo. In order to identify molecules that initiate dedifferentiation of cells at the wound site in amphibians we have established a cellular assay with a cultured newt myogenic cell line. Using this assay we have found a serum activity that stimulates cell cycle re-entry in differentiated multinucleated newt myotubes. The activity is present in serum of all mammalian species tested so far and, interestingly, thrombin proteolysis amplifies the activity from both serum and plasma. We think this serum factor provides a link between wounding and regeneration and its identification will be a key step in understanding the remarkable differences in wound healing between mammals and amphibians. In the course of this PhD thesis we have characterized the serum factor as a thermo-labile, pH- and proteinase K-sensitive, high molecular weight protein that is resistant to denaturing conditions such as SDS, urea or organic solvents. Surprisingly, under denaturing conditions the activity behaves as a low molecular weight protein that displays charge heterogeneity on isoelectric focusing. Using these characteristics of the serum factor we have performed a systematic investigation of commonly used protein chromatography modes and separation techniques to develop a successful purification procedure. After four column chromatography steps -- cation exchange, hydrophobic interaction, heparin affinity and size exclusion chromatography under denaturing conditions -- we have achieved a 2,000-fold purification starting from a commercially available Crude Bovine Thrombin preparation. This represents about 40,000-fold purification over bovine serum. Silver stained gels of the most purified fractions revealed ten major protein bands. In order to finally identify the cell cycle re-entry factor, we are currently analyzing the purification by quantitative mass spectrometry by correlating the abundance of tryptic peptides with activity in sequential fractions across a chromatography run.

Les urodèles amphibiens, dont fait partie l’axolotl (Ambystoma mexicanum), ont la capacité de régénérer leurs organes et membres suite à une amputation, tout au long de leur vie. La patte est l’organe dont le processus de régénération est le mieux caractérisé et ce dernier est divisé en deux phases principales. La première est la phase de préparation et commence immédiatement suite à l’amputation. Elle renferme des étapes essentielles au processus de régénération comme la guérison de la plaie et la formation d’une coiffe apicale ectodermique. Par la suite, les fibroblastes du derme et certaines cellules musculaires vont revenir à un état pluripotent via un processus appelé dédifférenciation cellulaire. Une fois dédifférenciées, ces cellules migrent et s’accumulent sous la coiffe apicale pour former le blastème. Lors de la phase de redéveloppement, les cellules du blastème se divisent puis se redifférencient pour régénérer la partie amputée. Fait intéressant, la régénération d’un membre ou la guérison d’une plaie chez l’axolotl ne mène jamais à la formation d’une cicatrice. Afin d’en apprendre plus sur le contrôle moléculaire de la régénération, les gènes Heat-shock protein-70 (Hsp-70) et Transforming growth factor-β1 (Tgf-β1) ont été sélectionnés. Ces gènes jouent un rôle important dans la réponse au stress et lors de la guérison des plaies chez les mammifères. HSP-70 est une chaperonne moléculaire qui est produite pour maintenir l’intégrité des protéines cellulaires lorsqu’un stress se présente. TGF-β1 est une cytokine produite suite à une blessure qui active la réponse inflammatoire et qui stimule la fermeture de la plaie chez les amniotes. Les résultats présentés dans cette thèse démontrent que Hsp-70 est exprimé et régulé lors du développement et de la régénération du membre chez l’axolotl. D’autre part, nos expériences ont mené à l’isolation de la séquence codante pour Tgf-β1 chez l’axolotl. Nos résultats montrent que Tgf-β1 est exprimé spécifiquement lors de la phase de préparation dans le membre en régénération. De plus, le blocage de la voie des Tgf-β avec l’inhibiteur pharmacologique SB-431542, lors de la régénération, mène à l’inhibition du processus. Ceci démontre que la signalisation via la voie des Tgf-β est essentielle à la régénération du membre chez l’axolotl. / Urodele amphibians, such as the axolotl (Ambystoma mexicanum), have the unique ability, among vertebrates, to perfectly regenerate many parts of their body throughout their life. Among the complex structures that can be regenerated, the limb is the most widely studied. Limb regeneration is divided in two main phases. The preparation phase, which begins right after amputation, includes wound healing and the formation of an apical ectodermal cap. During this phase, dermal fibroblasts and muscle cells will lose their characteristics and become pluripotent through a process called cellular dedifferentiation. The dedifferentiated cells migrate and accumulate under the apical ectodermal cap to form the blastema. During the redevelopment phase, the cells in the blastema proliferate and redifferentiate to regenerate the lost structures. It is interesting to highlight the fact that regeneration never leads to scar formation in the axolotl. In order to learn more about the molecular control of limb regeneration, the genes Heat-shock protein-70 (Hsp-70) and Transforming growth factor-β1 (Tgf- β1) were selected for their important roles in stress response and wound healing in mammals. HSP-70 is a molecular chaperone which is produced to protect cellular proteins when the cell faces a stress. TGF-β1 is a cytokine produced after wounding that activates the inflammatory response and stimulates wound closure in amniotes. Results presented in this thesis show that Hsp-70 is expressed and regulated during limb development and regeneration in the axolotl. We were also able to isolate the cDNA coding for axolotl Tgf-β1 and our results show that this gene is expressed specifically during the preparation phase of limb regeneration. Treatment of regenerating axolotls with a specific inhibitor of Tgf-β signalling, SB-431542, led to complete inhibition of regeneration. This directly implies that Tgf-β signalling is essential for limb regeneration in axolotl.

The (Plethodon nettingi) Cheat Mountain Salamander is a rare and endangered species that relies heavily on (Picea rubens) Red Spruce for habitat. P. rubens communities on the Cheat Mountain range in West Virginia have been disturbed by fires and logging, and regeneration of P. rubens stands are central to the survival of the P. netting. A supervised and unsupervised landscape classification of three Landsat images over the past 26 years was conducted to analyze change in P. rubens communities on Cheat Mountain Range. Change detection results revealed that from 1986-2012 a 52% growth increase of P. rubens stands, 18% loss, and 29% stayed the same over the last 26 years. P. rubens stands are vital habitat to the rare and endangered P. netting and regrowth of P. rubens is vital in restoring the habitat of the salamander on the Cheat Mountain. The regrowth of P. rubens on the Cheat Mountain range is critical to the survival of the P. nettingi. Identifying critical forest as it relates to salamander habitat is essential for conservation efforts. Since not all P. rubens stands are of equal significance to the P. nettingi, it is important to identify and map those that adhere to their stringent habitat needs as defined by forest fragmentation, aspect, slope, and lithology. I used spatial analysis and remote sensing techniques to define critical forest characteristics by applying a forest fragmentation model utilizing morphological image analysis, northeast and southwest aspects, moderate slopes, and limestone lithology. Patches were ranked based on this quantitative model and key P. rubens stands identified using spatial statistics. The results could aid in prioritizing research areas as well as conservation planning in regards to P. rubens and the P. nettingi. In this study, the MaxEnt modeling framework was used to predict habitat suitability for P. rubens under current conditions and under two future climate change scenarios. P. rubens distribution data was acquired from the U.S Geological Survey. Both the IPCC A1B and A2 emission scenarios of the HadCM3 global circulation model were projected to years 2040-2069 and 2070-2099. Results showed that a substantial decline in the suitability of future P. rubens habitat on the Cheat Mountain is likely under both climate change scenarios, particularly at lower elevations. By the end of the century, P. rubens is likely to be extirpated from the Cheat Mountain Range. By the end of century, the A1B and A2 scenarios predict the average habitat suitability for P. rubens on Cheat Mountain will be 0.0002 and 0.00004 respectively. Conservation as well as species migration efforts for P. rubens should be focused on areas such as Cheat Mountain to preserve this vital habitat.

The use of Light Detection and Ranging (LiDAR) information is gaining popularity, however its use has been limited in deciduous forests. This thesis describes two studies using LiDAR data in an Eastern Kentucky deciduous forest. The first study quantifies vertical error of LiDAR derived digital elevation models (DEMs) which describe the forests terrain. The study uses a new method which eliminates Global Positioning System (GPS) error. The study found that slope and slope variability both significantly affect DEM error and should be taken in to account when using LiDAR derived DEMs. The second study uses LiDAR derived forest vegetation and terrain metrics to predict terrestrial Plethodontid salamander abundance across the forest. This study used night time visual encounter surveys coupled with zero-inflation modeling to predict salamander abundance based on environmental covariates. We focused on two salamander species, Plethodon glutinosus and Plethodon kentucki. Our methods produced two different best fit models for the two species. Plethodon glutinosus included vegetation height standard deviation and water flow accumulation covariates, while Plethodon kentucki included only canopy cover as a covariate. These methods are applicable to many different species and can be very useful for focusing management efforts and understanding species distributions across the landscape.

Les urodèles amphibiens, dont fait partie l’axolotl (Ambystoma mexicanum), ont la capacité de régénérer leurs organes et membres suite à une amputation, tout au long de leur vie. La patte est l’organe dont le processus de régénération est le mieux caractérisé et ce dernier est divisé en deux phases principales. La première est la phase de préparation et commence immédiatement suite à l’amputation. Elle renferme des étapes essentielles au processus de régénération comme la guérison de la plaie et la formation d’une coiffe apicale ectodermique. Par la suite, les fibroblastes du derme et certaines cellules musculaires vont revenir à un état pluripotent via un processus appelé dédifférenciation cellulaire. Une fois dédifférenciées, ces cellules migrent et s’accumulent sous la coiffe apicale pour former le blastème. Lors de la phase de redéveloppement, les cellules du blastème se divisent puis se redifférencient pour régénérer la partie amputée. Fait intéressant, la régénération d’un membre ou la guérison d’une plaie chez l’axolotl ne mène jamais à la formation d’une cicatrice. Afin d’en apprendre plus sur le contrôle moléculaire de la régénération, les gènes Heat-shock protein-70 (Hsp-70) et Transforming growth factor-β1 (Tgf-β1) ont été sélectionnés. Ces gènes jouent un rôle important dans la réponse au stress et lors de la guérison des plaies chez les mammifères. HSP-70 est une chaperonne moléculaire qui est produite pour maintenir l’intégrité des protéines cellulaires lorsqu’un stress se présente. TGF-β1 est une cytokine produite suite à une blessure qui active la réponse inflammatoire et qui stimule la fermeture de la plaie chez les amniotes. Les résultats présentés dans cette thèse démontrent que Hsp-70 est exprimé et régulé lors du développement et de la régénération du membre chez l’axolotl. D’autre part, nos expériences ont mené à l’isolation de la séquence codante pour Tgf-β1 chez l’axolotl. Nos résultats montrent que Tgf-β1 est exprimé spécifiquement lors de la phase de préparation dans le membre en régénération. De plus, le blocage de la voie des Tgf-β avec l’inhibiteur pharmacologique SB-431542, lors de la régénération, mène à l’inhibition du processus. Ceci démontre que la signalisation via la voie des Tgf-β est essentielle à la régénération du membre chez l’axolotl. / Urodele amphibians, such as the axolotl (Ambystoma mexicanum), have the unique ability, among vertebrates, to perfectly regenerate many parts of their body throughout their life. Among the complex structures that can be regenerated, the limb is the most widely studied. Limb regeneration is divided in two main phases. The preparation phase, which begins right after amputation, includes wound healing and the formation of an apical ectodermal cap. During this phase, dermal fibroblasts and muscle cells will lose their characteristics and become pluripotent through a process called cellular dedifferentiation. The dedifferentiated cells migrate and accumulate under the apical ectodermal cap to form the blastema. During the redevelopment phase, the cells in the blastema proliferate and redifferentiate to regenerate the lost structures. It is interesting to highlight the fact that regeneration never leads to scar formation in the axolotl. In order to learn more about the molecular control of limb regeneration, the genes Heat-shock protein-70 (Hsp-70) and Transforming growth factor-β1 (Tgf- β1) were selected for their important roles in stress response and wound healing in mammals. HSP-70 is a molecular chaperone which is produced to protect cellular proteins when the cell faces a stress. TGF-β1 is a cytokine produced after wounding that activates the inflammatory response and stimulates wound closure in amniotes. Results presented in this thesis show that Hsp-70 is expressed and regulated during limb development and regeneration in the axolotl. We were also able to isolate the cDNA coding for axolotl Tgf-β1 and our results show that this gene is expressed specifically during the preparation phase of limb regeneration. Treatment of regenerating axolotls with a specific inhibitor of Tgf-β signalling, SB-431542, led to complete inhibition of regeneration. This directly implies that Tgf-β signalling is essential for limb regeneration in axolotl.

Newts have the incredible ability to regenerate many different organs and tissues as adults, including the limbs. Limb regeneration occurs via the dedifferentiation of stump tissue and the formation of a blastema, which provides the majority of cells for the regenerate. Despite all that we have learned about dedifferentiation and blastema formation, the cellular and molecular mechanisms underlying these processes are still poorly understood. We used representational difference analysis (RDA) to identify genes involved in the early dedifferentiation process in newt forelimb regeneration. Our analysis identified approximately 410 unique genes that were differentially regulated during this process. Microarray analysis was used to determine the expression profile of these genes throughout limb and tail regeneration. We used quantitative PCR (qPCR) to validate the expression of a subset of these genes [β-catenin, wntless, dapper, thymosin-β 4 (Tβ4), and thymosin-β 10/15 (Tβ10/15)] in regenerating limb and tail tissue, as well as in differentiating newt myoblasts. We also verified the expression of these genes in the regenerating newt limb using immunohistochemistry (IHC) and in situ hybridization (ISH). Finally, we performed a functional analysis on β-catenin, wntless, dapper, and Tβ4 by overexpressing these genes in mouse myoblasts to examine their effects on differentiation and potential roles in dedifferentiation. Quantitative PCR verified the expression of β-catenin, wntless, dapper, and Tβ4 during limb regeneration and IHC/ISH localized the β-catenin and Tβ4 proteins to the blastema during regeneration. Tβ10/15 was shown by qPCR to be expressed in the tail during regeneration. Overexpression of newt β-catenin, wntless, dapper, and Tβ4 in mouse myoblasts showed that each of these genes has an inhibitory effect on the differentiation of myoblasts into myotubes and, therefore, may play a role in promoting or maintaining the dedifferentiated state. Our work has identified a large number of genes with potential roles in regulating the dedifferentiation process during newt forelimb regeneration. We have also laid a framework from which much more work can be done by drawing on the genes we have identified and the microarray data, which indicate ideal follow-up candidates. Our analysis of specific genes has also increased our understanding of the molecular events occurring during the dedifferentiation process in the regenerating newt limb.

Background Molecular studies of appendage regeneration have been hindered by the lack of a stable and efficient means of transferring exogenous genes. We therefore sought an efficient integrating virus system that could be used to study limb and tail regeneration in salamanders. Results We show that replication-deficient foamy virus (FV) vectors efficiently transduce cells in two different regeneration models in cell culture and in vivo. Injection of EGFP-expressing FV but not lentivirus vector particles into regenerating limbs and tail resulted in widespread expression that persisted throughout regeneration and reamputation pointing to the utility of FV for analyzing adult phenotypes in non-mammalian models. Furthermore, tissue specific transgene expression is achieved using FV vectors during limb regeneration. Conclusions FV vectors are efficient mean of transferring genes into axolotl limb/tail and infection persists throughout regeneration and reamputation. This is a nontoxic method of delivering genes into axolotls in vivo/ in vitro and can potentially be applied to other salamander species.

Amphibians are in decline globally and a significantly greater percentage of ambystomatid salamander species are in decline relative to other species; habitat loss contributes significantly to this decline. The goals of this thesis is to better understand extinction risk in a marbled salamander (ambystoma opacum) population and how forestry effects extinction risk. To achieve this goal we first estimated an important life history parameter (Chapter 1) then used a metapopulation model to estimate population viability and determine what aspects of their life history put them most at risk (Chapter 2) and finally predicted extinction risk in response to hypothetical forestry scenarios (Chapter 3). In Chapter 1 we estimated one of the requisite parameters for the model, juvenile survival, based on 8 years of field data. We estimated juvenile survival probabilities (to first breeding) at 17% for males and 11% for females. To our knowledge, these are the first estimates for marbled salamanders that include both returning and dispersing individuals. In Chapter 2 we used a metapopulation model to estimate extinction risk and sensitivity of extinction risk to changes in vital rates and other model parameters. We found that although there is considerable uncertainty in our estimate it is likely that extinction risk is low at our study site. Sensitivity analysis revealed that small changes in adult survival lead to relatively large changes in persistence and the presence of an apparent threshold in reproductive failure probabilities beyond which extinction risk rapidly increased. In Chapter 3 we used the extinction risk and sensitivity estimates to model the effects of forestry on the metapopulation. We parameterized several different levels of impact of forestry on salamander survival; for each parameterization we calculated the extinction risk for 20 different forestry scenarios involving buffer size (30 to 300 meters) and complete or partial restrictions on cutting (5 different levels). We found for all but the most optimistic parameterizations large buffers (around 200 meters) with high restrictions on cutting within the buffer were necessary to maintain a low extinction risk. Overall we show that although the population at our intensively studied field site is unlikely to go extinct under present conditions small decreases in adult survival, small increases in catastrophe rate, and intensive forestry can all make extinction likely.

No description available.