Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / There have been many cases reported of amphibian populations declining. These are often due to anthropogenic factors such as habitat destruction and pollution. However, some declines have not had an obvious cause and many of these have been investigated and found to be due to pathogenic disease. Batrachochytrium dendrobatidis is a recently described pathogen of frogs. The population declines that have been associated with chytridiomycosis have occurred in relatively undisturbed areas such as national parks. The declines tend to occur at higher altitudes or in colder climates. This is thought to be because of the frog immune system being slower at lower temperatures. Chyt1id fungus has been found in frog populations throughout the world. Little research has been carried out in Africa although, chytridiomycosis has already been seen in Kenya and South Africa. In this project frogs were sampled from selected transects in the Western Cape and three sites in the Northern Cape. The effect of altitude on the occurrence of infection was tested in the Western Cape. It was found that 18 frogs were infected in the Western Cape and the effect of altitude was not significant. Large numbers of dead and dying frogs were found in two of the Northern Cape sites and the incidence of chytridiomycosis was high in these populations. Chytrid was found in two Bufogariepensis from the Eastern Cape and in Xenopus petersii from Kasanka National Park, Zambia. More research is needed on chytridiomycosis in these populations. The frogs in the Western Cape seem to survive with chytrid fungus infection whereas, the frogs in the Northern Cape are dying. This suggests another factor acting on the Northern Cape frog populations.

abstract: Riparian systems in the arid southwest are heavily altered and, based on relative land-area, provision a disproportionately high number of native wildlife. Amphibians and reptiles are collectively the most threatened vertebrate taxa and, in the Sonoran Desert, are often reliant on riparian habitat. The link between amphibians and environmental water characteristics, as well as the association between lizards and habitat structure, make herpetofauna good organisms for which to examine the effects of environmental change. My objective was to relate capture rates of a fossorial anuran and lizard abundance to aspects of native, invaded, and shrub-encroached riparian habitats in order to forecast the potential winners and losers of riparian habitat xerification and invasion. I measured habitat and monitored herpetofauna at 18 sites near the confluence of the San Pedro River and Gila River in Pinal County, Arizona in 2016 and 2017. Sites were divided into three categories based on dominant tree genus; Populus-Salix, Prosopis, and Tamarix, which represented native riparia, xeric riparia, and invaded riparia, respectively. Habitat measurements indicated that sites varied significantly in structure, and that dominant tree species was a useful descriptor of habitat physiognomy. Results from herpetofauna trapping demonstrated that Scaphiopus couchii, a fossorial anuran, occupy Prosopis sites at a much higher rate than at Tamarix sites, which were almost completely avoided. S. couchii was also found to be closely tied to xero-riparian habitat components present at Prosopis sites and soil analyses indicate that aspects of soil moisture and texture play an important role in the partitioning of this species across altered riparian habitats. Lizard abundance was found to be significantly lower in Tamarix habitat, with the majority of captures attributed to the generalist whiptail Aspidoscelis tigris. Additionally, more than half of lizard species that were analyzed displayed a negative association to Tamarix habitat. Of the three habitat types considered, Populus-Salix supported the greatest abundance of lizards. Based on this study, the deleterious effects of xerfication on a riparian herpetofauna community may be lesser than those of Tamarix invasion. These two forms of riparian habitat shift often co-occur, with the ultimate cause being changes in hydrologic regime. This may imply that a bottom-up approach, wherein historic hydrology is restored to restore or maintain native habitats, to riverine management is appropriate for riparian herpetofauna conservation. / Dissertation/Thesis / Masters Thesis Applied Biological Sciences 2018

Orientador: João Martins Pizauro Junior / Resumo: O desenvolvimento dos anfíbios é caracterizado por uma série de eventos que possibilitam a esses animais a ocupação dos ambientes aquático e terrestre. Nesse sentido, a supressão do sistema imunológico é necessária para que esses eventos ocorram de forma ordenada, evitando então, respostas autoimunes. Por outro lado essa imunossupressão durante a metamorfose pode tornar os animas mais susceptíveis a algumas doenças, dentre elas, a causada pelo fungo Batrachochytrium dendrobatidis (Bd), que é considerado o principal patógeno de anfíbios da atualidade. Nesse contexto, a produção de anticorpos da classe IgY anti-Bd, extraídos de gemas de ovos de galinhas poedeiras infectadas com o referido fungo, pode agregar valor ao ovo, auxiliar no combate a quitridiomicose, diminuição da perda da produção e dispersão do patógeno por criadouros, diminuindo também o uso de antibióticos no tratamento e profilaxia da quitridiomicose, aumentando o nível de segurança da carne e valorizando o produto final. No presente estudo a imunização das aves com o antígeno desativado fez com que o sistema humoral das mesmas respondesse a indução contra Bd viabilizando a produção e purificação de imunoglobulinas da classe IgY específicas e de alta avidez contra o fungo. A transferência passiva das imunoglobulinas purificadas das gemas apresentou resultados promissores, onde os girinos de rã-touro (Lithobates catesbeianus) tratados não apresentaram danos deletérios quanto à sanidade animal dos mesmos, mantendo ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The development of amphibians is characterized by a series of events that enable these animals to occupy aquatic and terrestrial environments. In this sense, suppression of the immune system is necessary for these events to occur in an orderly manner, avoiding autoimmune responses. On the other hand, this immunosuppression during metamorphosis may make the animals more susceptible to some diseases, among them, the one caused by the fungus Batrachochytrium dendrobatidis (Bd), which is considered the main amphibian pathogen today. In this context, the production of anti-Bd IgY class antibodies, extracted from egg yolks of laying hens infected with this fungus, may add value to the egg, assist in the fight against chytridiomycosis, decrease production loss and pathogen dispersion by breeding facilities, reduce the use of antibiotics in the treatment and prophylaxis of chytridiomycosis, increasing the safety level of the meat and valuing the final product. In the present study, the immunization of hens with the deactivated antigen caused their humoral system to respond to induction against Bd, enabling the production and purification of specific and highly avid fungal IgY class immunoglobulins. Passive transfer of purified immunoglobulins from yolk showed promising results, where treated bullfrog tadpoles (Lithobates catesbeianus) showed no deleterious damage to animal health, keeping the morphology and counting of blood cells within normal range. These results enable the develop... (Complete abstract click electronic access below) / Doutor

Wildlife in captivity has a long history of benefiting global conservation goals. Captive animals can raise awareness and appreciation for the conservation of endangered species. Additionally, captive animals can be used as source populations to reintroduce animals back to the wild or to supplement existing wild populations. The rapid increase in amphibian species threatened with imminent extinction has necessitated the creation of dozens of captive-breeding programs. The focus of this dissertation has integrated topics across the spectrum of animals in captivity and the wild, and the results provide useful recommendations for conservation action. First, I describe how market pressures over a 28-year period are causing meteoric increases in the prices of amphibians sold in the pet trade, indicating a high risk of overexploitation. Pet amphibians may facilitate greater understanding and appreciation of amphibians, but the pet trade must be sustainable. Improving amphibian husbandry will increase the number of captive-bred animals available in the pet trade, and it will allow greater production of threatened species for reintroductions. Secondly, by performing a systematic review of husbandry for 289 amphibian species native to the US, I identified a critical lack in taxon-specific husbandry and developed husbandry research prioritizations. Next, I used a combination of laboratory and field studies to examine domestication processes in amphibians by comparing defensive behaviors in two species of captive-bred and wild poison frog. Captive-bred amphibians had significantly reduced defensive behaviors compared to wild conspecifics, likely resulting from habitation processes related to their husbandry. Finally, I performed three reintroductions of the critically endangered Wyoming Toad (Anaxyrus baxteri) in Wyoming, US. I demonstrated how providing a transitionary period, called a soft-release, to captive-bred toads moving to a novel, wild environment can improve reintroduction success. My work illustrates how improving our understanding of the nexus between captivity and the wild can improve conservation action for endangered species.

Understanding wetland responses to human perturbations is essential to the effective management of Florida's surface and ground water resources. Southwest Florida Water Management District (SWFWMD) Rules (Chapter 40D-2.301(c) FAC) prohibit adverse environmental effects to wetlands, fish and wildlife caused by groundwater withdrawal. Numerous studies have documented the responses of biological attributes across taxa and regions to human disturbance. Biological assessment can provide information about ecological condition. Based on long-term monitoring conducted by the SWFWMD, the anthropogenic changes observed on the Starkey Wellfield are attributed to groundwater withdrawal. Biological indicators are species, species assemblages, or communities whose presence, abundance, and condition are indicative of a particular set of environmental conditions. Monitoring early indicators of ecosystem stress may shorten response time by shifting attention to the relatively quick response of sensitive species. Species used to assess biological condition should be abundant and tractable elements of the system that provide an early, diagnosis. Regulatory requirements within 40D-2 F.A.C. dictate an extensive analysis be conducted twice yearly on wetlands within all wellfields. This quantitative analysis provides information on the wetland plant community through the collection of eighteen categorized vegetative and physical variables. Because of the size of the area in which monitoring is required and the large number of wetlands, a rapid qualitative monitoring method was developed using vegetation and physical variables to classify wetlands into one of three categories based on their perceived health. Wetland plants have many characteristics suited to assessments of biological condition including their diversity, taxonomy, distribution, relative immobility, well developed sampling protocols, and, for herbaceous species, their moderate sensitivity to disturbance (U.S. EPA 2002, Doherty et al. 2000). Because amphibians occupy both aquatic and terrestrial habitats in their life history, have physiological adaptations and specific microhabitat requirements, they are considered to be extremely sensitive to environmental perturbations and excellent barometers of the health of the aquatic and terrestrial habitats in which they reside (Vitt et al. 1990, Wake 1998, Blaustein 1994, Blaustein et al. 1994). The purpose of my study was to 1) compare a qualitative method of wetland vegetation monitoring to a quantitative method, 2) document the reproductive success of anurans, and 3) compare anuran reproductive success to the vegetation monitoring results on the J. B. Starkey Wellfield (SWF). The results are published in chapters, with each chapter addressing one of the topics stated above. The results show a rapid, qualitative measure of wetland health is useful for the determination of severely affected wetlands. The anuran reproductive success reflected similar results. The results show that wetlands can be categorized based solely on amphibian reproductive success variables. The anuran categorization, qualitative vegetative categorization, and quantitative vegetative categorization overlap on the high and low success wetlands. The low degree of overlap observed in the intermediate category could be attributed to fish predation in a wetland otherwise suited for amphibian reproduction, natural variability in the two years of anuran data collected or lag time inherent in vegetative monitoring. Strong correlative evidence suggests hydroperiod regulates anuran reproductive success on the J. B. Starkey Wellfield. The average length of inundation was correlated with the number of tadpoles captured per unit effort and the number of tadpole species captured per year (R=0.73, p<.01; R=0.70, p<.05). The average Julian date of inundation at which breeding attempts stopped and no tadpoles were observed was weeks within the published breeding season for many species. I detected a correlation between the number of species calling in each wetland and the number of tadpole species captured per year (R=0.87, p<.001) suggesting call censuses may be used at this site to estimate anuran reproductive success if enough well-timed observations are made. These findings will allow resource managers and regulators to evaluate and possibly refine land management practices, including existing monitoring methods, and water policy to meet the needs of resident amphibians at the J.B. Starkey Wellfield.

The documented decline of amphibian populations over the past two decades has increased attention towards amphibian conservation. Much of this attention has been focused on testing hypotheses as to the causal factors of these declines, however providing convincing data to support any of these hypotheses has proved difficult. The testing of these hypotheses and the implementation of endangered species recovery programs has been restricted by a lack of knowledge of the ecology and population demography of amphibian species that have suffered dramatic declines. This thesis presents aspects of the research phase of the recovery program for the Southern Corroboree Frog, Pseudophryne corroboree, a species that declined to very low numbers during the early 1980's. In particular, this research aimed to determine the distribution, abundance, population dynamics and demography of this rare species. A complete reassessment of the conservation status of P. corroboree was undertaken and the nature of the persistence of this species across the landscape was analysed. Temporal trends in abundance and its relationship with population size were also investigated. Early life-history survivorship and recruitment to metamorphosis were studied at the scale of individual nest sites and populations, and the adult male population age structure and annual mortality were investigated using skeletochronology. The shout/response survey technique was used to survey and monitor the number of breeding male P. corroboree during this study. This method was found to provide consistent results when the surveys were conducted over a short (two week) period during the peak breeding season in January. Neither time of day, nor the number of males present at a pool, was found to influence the level of responsiveness of male P. corroboree to the shout/response technique. Variation in the number of responding males to the shout/response technique through the breeding season, assessed at a single site over two seasons, was unimodal with the peak responding period occurring during the last two weeks of January during both the 1998 and the 1999 breeding seasons. A systematic survey covering 213 sites across the entire historic distribution of P. corroboree found this species to be persisting at 79 sites. The majority of these sites were in the northwestern portion of the species former range, around the Jagungal Wilderness area, while no extant sites were found in the south-eastern portion of the species former range in the Smiggin Holes and Perisher Blue ski resorts area. The overall abundance of males at persistent sites was extremely low, with 92 percent of sites having fewer than ten responding males. Only one site was found to support greater than fifty responding males. A logistic regression analysis found the persistence of P. corroboree to be associated with increased number of pools within a site, decreased distance to nearest extant population and geographic position (latitude and longitude) in the landscape. While annual variation was observed in the number of breeding males for individual sites, there was no overall trend for an increase or decrease in the number of males, regardless of population size. The average annual extinction rate for local populations was five percent during this study, with those populations becoming extinct having very few breeding males (between one and three) during the previous season. Embryonic and tadpole survivorship was monitored for individual nests at three sites across three years. Recruitment to metamorphosis for P. corroboree was characterised by high variation in survivorship between nest sites, populations and years, while overall recruitment for nest sites was skewed towards lower survivorship. Average nest survivorship to metamorphosis across all sites and years was ten percent but the skewed nature of this survivorship meant that the majority of nest sites attained very low or no survivorship. The low proportion of nest sites that did attain high survivorship provided the greatest contribution to overall recruitment. The levels of embryonic and tadpole mortality observed in this study would be providing a considerable contribution to the regulation of current population sizes. The greatest level of early life-history mortality was observed during the late autumn/winter egg and tadpole stage, with high survivorship during the summer and early autumn egg stage and the post-winter tadpole stage. The estimated sex ratio for seven populations, based on the number of eggs within male nest sites, indicated that for most populations, regardless of population size, there was a greater proportion of females to males. In general, the estimated sex ratio of smaller populations showed greater annual variation and had a lower average number of females to males than the single large population. Tadpole surveys conducted across remnant populations during both 1998 and 1999 found recruitment to metamorphosis to be very low for the majority of populations. A third of all populations during both years attained no recruitment to metamorphosis, with those populations that did attain recruitment typically having fewer than 20 tadpoles. While sites with more frogs generally recruited more tadpoles, there was no strong relationship between population size and the number of tadpoles recruited per male at the scale of either pool or site. There was also no significant difference in recruitment levels between the two years. Tadpole surveys across breeding pools within the single large population also found very low tadpole abundance. There was no strong relationship between the number of male frogs at a pool and the number of tadpoles per male and there was no significant difference in tadpole abundance between the two years. Based on the low density of males at pools and sites (typically less than five), and the skewed nature of nest survivorship identified from monitoring individual nest sites, it seems likely that both deterministic and stochastic factors are influencing recruitment levels in remnant populations of P. corroboree. This study determined that adult male P. corroboree could be accurately aged using the technique of skeletochronology, and this technique was used to determine the adult male population age structure for three populations. The results indicated that adult male P. corroboree can reach sexual maturity from metamorphosis in three years, but the majority of individuals take four years. The oldest individual identified in this study was nine years old from metamorphosis. The adult male age structure at the single large site showed very little annual variation, whereas the two smaller populations showed highly pulsed age structures from one year to the next. The size of adult males was found to be a poor predictor of age. Annual adult male survivorship, calculated by following cohorts from one year to the next, was 55 percent. Based on this calculation of annual adult male survivorship, it seems likely that the initial decline in P. corroboree involved increased levels of adult mortality. The results of this study indicate that the persistence of. corroboree in the wild is precarious in the short-term. For this reason, it is recommended that efforts be undertaken to secure this species ex situ. Attempts to increase population numbers in the wild would greatly benefit from determining the factor(s) that have caused the decline in this species, however, failure to do so should not preclude field experimental management aimed at developing technique to increase the size of remnant populations. This is because it is likely that small population stochasticity is contributing to the current regulation of population size and it is possible that the factors that caused the decline in this species cannot be removed from the environment.

Hardwood bottomland ecosystems provide critical habitat for various wildlife among numerous ecosystem services. Since the 1800s, these forested wetlands have been logged and drained for agriculture. The federal government passed a series of legislative acts that protected wetlands and provided monetary support for restoration. The Wetlands Reserve Program (WRP) was established in 1990 with the goal of restoring ecological function in wetlands. Although several studies have measured plant and wildlife responses to WRP restorations, no standard protocol has been developed to monitor the state of ecological restoration at sites. Index of biotic integrity (IBI) models are commonly used to evaluate ecological function by assigning scores derived from biological characteristics measured at disturbed sites and comparing them with reference sites. Therefore, the objectives of my study were to: (1) characterize vegetation, amphibian and bird communities among 17 WRP restoration and 4 reference bottomland sites, and (2) develop IBI models for these communities to use in monitoring ecological restoration. My study was conducted across 10 counties in western Tennessee from March – August 2008, and communities were measured using standard sampling techniques. I detected 15 amphibian and 95 bird species at bottomland WRP sites, which ranged 2 – 21 years old. Anurans were common among sites, but salamanders were only detected at reference sites containing mature forests. The bird community changed predictably in response to succession, with grassland birds dominating young restoration sites and scrub-shrub and forest birds dominating older restoration and reference sites. Vegetation structure was related to site age, and a good predictor of bird community composition. Variables retained in the vegetation IBI model included density of snags, logs and overstory trees, basal area, and percent vertical cover measured using a profile board. The bird IBI model contained relative abundance of bark feeding, branch nesting, and twig nesting guilds. Presence of salamanders was the only variable in the amphibian IBI model. My results indicate that the WRP is contributing to the regional biodiversity of western Tennessee. The IBI models that I developed can be used for monitoring ecological restoration in Tennessee hardwood bottomlands; however, their applicability outside this region should be validated.

Threats from man-made radionuclides include waste issues, increasing number of power plants, underground bomb testing, nuclear weapons, and “dirty bombs”. Until recently the ionizing radiation protection system focused on protecting humans with an implied protection of biota. However, goals of sustainable development and precautionary principles for human activity are leading to an inclusion of plant and animal populations in the protection system. From this perspective, the present thesis examines wetlands that function as sinks for the radionuclide 137Cs, and describes calculated and measured radiation doses to residing biota. Also, multi-level effects from exposure to low-dose rate ionizing radiation were studied. Accumulation of 137Cs after the Chernobyl accident fallout was studied in a riparian wetland with a mean activity concentration of 1 200 kBq m-2 in Sweden (paper I). A mass balance budget of 137Cs showed that the sedimentation of new material was balanced by the decay process of 137Cs in parts of the wetland (paper I). Frogs were identified as organisms of concern in this wetland. Internal radiation doses, based on whole body measurements of frogs, were estimated to be lower than external doses based on soil samples (paper II). Current dose models for biota resulted in a wide range of doses depending on different levels of conservatism in the models. Therefore, in situ measurements with frog-phantoms were found to provide valuable dose information (paper III). Measured doses using frog-phantoms were lower than calculated doses using several dose models. Although a dose conversion factor by FASSET was found to be useful for comparison with measurements in the field. A higher dose was measured to the phantom surface in comparison to inner parts, i.e. the sensitive skin of frogs receives the highest dose. Estimated and measured radiation doses to frogs were below suggested dose rate limits. Low-dose rate 137Cs exposure of eggs and tadpoles from three amphibian species, Scaphiopus holbrookii, Bufo terrestris, and Rana catesbeiana, showed no increased levels of strand breaks in red blood cells, and no effects on development, survival or growth up to metamorphosis (paper IV). The ecological factor larval density had a stronger effect on metamorphic traits than low-dose rate radiation. Higher levels of strand breaks were detected after an acute dose in R. catesbeiana than after a chronic dose supporting a dose rate limit for protection of amphibians rather than a dose limit (paper IV). Based on current knowledge, frogs in the contaminated wetland are probably not exposed to radiation doses from 137Cs that are harmful for the population. However, variations in sensitivity between populations and species, and adaptive responses have been shown for amphibians exposed to other stressors. This supports further research on effects of chronic low-dose rates of ionizing radiation on amphibians.

Humans are modifying the global landscape at an unprecedented scale and pace. As a result, species are declining and going extinct at an alarming rate. Here, I investigate two main aspects of species’ declines: what factors are contributing to their declines and how effective our conservation efforts have been. I assessed one of the main mechanisms for protecting species by looking at the Endangered Species Act (ESA) in the United States. I examined three separate indicators of species declines for different groups of species: range contractions in Canadian imperilled species, declines in abundance in global amphibian populations and increases in temporal variability in abundance in North American breeding birds. I found that change in recovery status of ESA listed species was only very weakly related to the number of years listed, number of years with a recovery plan, and funding. These tools combined explained very little of the variation in recovery status among species. Either these tools are not very effective in promoting species’ recovery, or species recovery data are so poor that it is impossible to tell whether the tools are effective or not. I examined patterns of species’ declines in three different groups in relation to a number of anthropogenic variables. I found high losses of Canadian imperiled bird, mammal, amphibian and reptile species in regions with high proportions of agricultural land cover. However, losses of imperiled species are significantly more strongly related to the proportion of the region treated with agricultural pesticides. This is consistent with the hypothesis that agricultural pesticide use, or something strongly collinear with it (perhaps intensive agriculture more generally), has contributed significantly to the decline of imperiled species in Canada. Global increases in UV radiation do not appear to be a major cause of amphibian population declines. At individual sites, temporal changes in amphibian abundance are not predictably related to changes in UV intensity. Variability in species’ abundance of North American breeding birds, after accounting for mean abundance, is not systematically higher in areas of high human-dominated land cover or climate change. Rather, it appears that areas with a high proportion of human-dominated cover come to have a higher proportion of highly abundant, and thus more variable, species.

Glyphosate-based herbicides are the dominant pesticide on the market and are utilized worldwide in both the agricultural and forestry industries. Their prevalence comes at a time when concern over the potential effects of pesticide application in amphibian spawning grounds is growing. The primary goal of this thesis was to determine if the glyphosate-based herbicide WeatherMax® has the potential to disrupt sexual differentiation and growth in the wood frog (Lithobates sylvaticus) in a pulse exposure at the predicted maximal environmental concentration (PMEC) of 2.88 mg acid equivalent per liter. This was carried out in laboratory, mesocosm and in-situ field exposures, in an attempt to determine how a potential disruption might vary between experimental environments. In this study, tadpoles from three split-wetlands targeted at the PMEC for WeatherMax were found to display no significant change in survival or growth, however gene expression of several genes involved in steroidogenesis during sexual differentiation (cyp19, cyp17, star, foxl2) were found to be affected. The effects on these genes appeared to be dependant on the exposure concentration of WeatherMax in each wetland, which varied even though all three wetlands were meant to target the PMEC. The wetland that was measured as having the highest herbicide concentration (PMEC 13) was found to have a female biased sex ratio. The results found in the field varied from those found in the more artificial exposures. In the laboratory the PMEC of WeatherMax experienced complete mortality, whereas in the mesocosms survival was not significantly affected. Sex ratios were unaffected in the laboratory, however at the PMEC there was a significant male bias in the mesocosms. The discrepancies in the results obtained from the different exposure types highlights the importance of real world exposures. That the same concentration that caused complete mortality in the laboratory caused sublethal effects in the field is of importance as it denotes that these endpoints may not be easily investigated in these synthetic exposures. This project is a part of the Long Term Experimental Wetlands Area (LEWA) and contributes to the body of information amassed therein on the impact of a glyphosate-based herbicide on amphibians in a wetland ecosystem.