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Programa de Doctorado en Estudios Medioambientales, Nature is complex and organisms commonly need to rapidly be able to detect and respond to environmental inputs in order to increase their survival odds. The ability of a given genotype to alter its morphology, behavior or development against changing environments is known as phenotypic plasticity, which is adaptive when the induced phenotypes confer increased fitness in the altered environment. Adaptive plasticity favors phenotypic diversity and increases population viability, as well as facilitates the maintenance of genetic variation reducing the severity of bottleneck events during rapid environmental change, and also by shielding genetic variants from selection. In particular, phenotypic plasticity is essential for species with low vagility and high philopatry, as is the case of most amphibian species. The life-cycle of amphibians is often a complex one that includes an aquatic larval stage that gives rise to a terrestrial stage through metamorphosis. Metamorphosis is thus a key ontogenetic switch point that entails vast anatomical, physiological and ecological changes in the organism. The timing and body condition at which metamorphosis occurs largely determine the likelihood of survival in larval and juvenile amphibians. Growth and development are remarkably decoupled over long periods of the amphibian larval ontogeny. This allows amphibian larvae to grow without advancing in development under benign conditions of high food availability, reduced competition, and abundant water, or else accelerate development at the expense of truncating growth when conditions worsen, as when at risk of pond drying. Such fine-tuning of growth and development relies on the ability of amphibian larvae to sense their environment and is regulated by neuroendocrine pathways, which can activate/repress multiple metabolic cascades, which in turn can involve short and long-term consequences for body condition and even life span. The main objective of this thesis is to understand the physiological mechanisms enabling developmental and growth plasticity in amphibians, and their consequences. Firstly, several simultaneous experiments were conducted to study the physiological alterations inflicted by common potential external stressors on amphibian larvae. In particular, we studied the effect of varying levels of salinity, herbicide, water pH, types of predators, and temperature in spadefoot toad larvae (Pelobates cultripes) evaluating their changes in corticosterone level, metabolic rate, activity of various antioxidant enzymes, lipid peroxidation, and immune state. Most of the studied levels factors caused some physiological imbalances in tadpoles, although high levels of salinity and herbicide caused the most dramatic physiological alterations. Tadpoles showed decreased levels of corticosterone when exposed to native predators, congruent with the common reduction in foraging and metabolic activity in the presence of predators. Interestingly, however, tadpoles did not reduce corticosterone in the presence of invasive predators, indicating a lack of innate recognition. Furthermore, corticosterone and the antioxidant enzyme glutathione reductase were the most sensitive parameters against the studied factors, and hence good candidates for further use in physiological monitoring of natural populations. Pond drying and predators are two of the main risks for amphibian larvae in temporary ponds. Physiological consequences of plastic responses in tadpoles after being exposed to both factors may allow us to understand short-term consequences of the developmental alterations induced, as well as to predict their long-term effects. We crossed the presence/absence of predators with permanent and drop-down water levels in a 2x2 experimental design simulating natural conditions in large outdoor mesocosms to test for induced changes in life-history traits, and also assessed their effects on fat reserves, oxidative stress, and telomere length of the surviving Pelobates cultripes juveniles that survived to both risks. Tadpoles accelerated their development in response to decreased water level, but at the expense of metamorphosing smaller and greatly depleting their fat reserves. Cellular oxidative stress due to developmental acceleration was successfully buffered by increased antioxidant enzyme activity, and telomere length remained unchanged. On the other hand, predators greatly reduced larval density, which relaxed competition and allowed survivors to develop fast, grow bigger and accumulate fat. However, tadpoles showed signs of oxidative stress and experienced telomere shortening. Telomere length is reduced during cell replication, and telomere shortening is known to be associated with senescence and reduced life span. Therefore, regarding body size and fat reserves at metamorphosis, developmental acceleration in response to pond drying compromises short-term survival, although its consequences are reversible in the long run. In turn, oxidative stress and telomere shortening due to rapid growth when tadpoles survived predators are likely reduce their long-term survival. Even within species, organisms do not respond phenotypically to the same extent against environmental inputs. In fact, natural populations often harbor substantial variation in their degree of phenotypic plasticity. Theoretical studies suggest that the evolution of plasticity is limited by costs of maintaining the machinery needed to detect and respond to environmental cues. However, only a few studies have empirically detected maintenance costs of plasticity. In the third chapter of this thesis, we tested for physiological costs of maintaining developmental, growth, and morphological plasticity in Pelobates cultripes larvae in response to both pond drying and predators. For this purpose, we first determined the degree of developmental plasticity of a total of twenty families (sibships) from various populations in response to these two environmental factors. Simultaneously, we assessed among families variation in body mass, fat reserves, metabolic rate, antioxidant enzyme activities, lipid peroxidation, reduced/oxidized glutathione, and immune state under benign control conditions. We tested the existence of costs of plasticity by testing for an association between the degree of plasticity of each family and the molecular markers of physiological stress. We found maintenance costs of plasticity associated to the degree of developmental and growth plasticity induced by predators, in terms of increased glutathione reductase activity and granulocyte to lymphocyte ratio, respectively. Morphological plasticity in response to both pond drying and predators were also linked to levels of antioxidant enzymes, lipid peroxidation, immune state or growth. Also, we detected trade-offs between the developmental responses of larvae to each factor so that genotypes (families) that readily accelerated development in response to pond drying were not effective in delaying metamorphosis in the presence of predators. Such a trade-off suggests possible constrains on the evolution of adaptive plasticity to conflicting environmental stimuli. Environmental heterogeneity can affect the degree of adaptive plasticity, which may also imply short and long-term consequences. In amphibians, the ability to accelerate development commonly results adaptive, since larvae usually inhabit pools in which water availability is seasonal and heterogeneous. Adaptive developmental plasticity is expected to evolve despite its possible costs when environmental heterogeneity precludes a single phenotype to maximize fitness across all conditions. Swedish Rana temporaria island populations show marked differences among populations in developmental rate and their responsiveness to pond drying so that populations with more variable durations in pond hydroperiod tend to show higher levels of developmental plasticity. In the fourth chapter, we study the physiological mechanisms and consequences of divergent developmental plasticity among some of these R. temporaria populations. We exposed larvae from six populations from three different island habitat types differing in their pool drying regime to simulated desiccation to determine their developmental plasticity. Populations from islands with only ephemeral pools showed higher developmental plasticity than populations from islands with permanent or long-lasting ponds. Individuals from islands with ephemeral ponds experience physiological alterations indicative of physiological costs of increased plasticity, such as altered catalase and glutathione reductase activities, and reduced telomere length. Elevated antioxidant activities indicate metabolic costs associated to increased developmental plasticity which may also compromise the health and lifespan of individuals and the viability of those populations, as shortened telomeres suggested. During the course of this thesis, we have also evaluated the suitability of some methodological aspects. (I) We evaluated the performance of three commonly used procedures for corticosterone determination using Xenopus laevis tadpoles: radioimmunoassay (RIA) in whole-body homogenates, enzyme immunoassay (EIA) on whole-body, and EIA on plasma. Each procedure presented advantages and disadvantages regarding sensitivity, the use of radioactivity, sampling size, or handling time. RIA is preferred in small-bodied animals from which blood cannot be obtained. EIA in plasma resulted a good non-radioactive alternative when blood sampling is possible. EIA on whole-body homogenates was the less sensitive procedure, although it may be a non-radioactive useful alternative to assess qualitative changes in corticosterone in small individuals when considerable differences are expected. (II) Immune response in amphibians has been commonly evaluated through indirect methods like phytohemagglutinin (PHA) injections or by direct like cell counts from blood smears. Here, we validated immunological evaluations in amphibians by means of flow cytometry. The immunological state of Pelobates cultripes tadpoles were experimentally altered by exposing them to exogenous corticosterone. Then, leukocyte proportions were quantified through both blood smears and flow cytometry. Both techniques showed similar patterns of leukocyte proportions. Once validated, flow cytometry also allowed quantification of changes in absolute number of leukocytes. The suitability of both techniques attending to accuracy, body size requirements, or the useful in field studies was also discussed. The results obtained in this thesis highlight the key role of physiological mechanisms in amphibian larvae plasticity and in its evolution. Therefore, for a holistic knowledge of ecological and evolutionary process results essential to understand the physiology underlying them., Universidad Pablo de Olavide. Departamento de Biología Molecular e Ingeniería Bioquímica, Postprint

Memoria presentada por la Licenciada en Biología M. Rosa Arribas Ramos para optar al título de Doctora por la Universidad Pablo de Olavide, [ES] La estructura y dinámica de las comunidades ecológicas están determinadas y reguladas por una serie de procesos clave que influyen en la distribución y la abundancia de las especies (p. ej. selección, deriva, especiación y dispersión), y por interacciones ecológicas como la competencia y la depredación. La importancia relativa de la competencia y la depredación dependerá en gran medida del grupo local de especies, el tipo de depredadores y el nivel de segregación de nicho. Los anfibios constituyen un sistema idóneo para abordar estas cuestiones, ya que son especies clave en las redes tróficas, con un papel importante en diversos procesos ecológicos. Además, el efecto de las larvas de anfibios en los sistemas acuáticos, así como su nicho trófico, dependen de sus complejas interacciones con competidores y depredadores, ya que responden a ellos alterando su morfología y comportamiento. Ante una situación generalizada de dramáticos declives en las poblaciones de anfibios a nivel global, es fundamental caracterizar el papel funcional de los anfibios en los sistemas naturales para poder comprender las posibles consecuencias de su declive y extinción local. El principal objetivo de la presente tesis es conocer el potencial regulador de las larvas de anfibios según las condiciones ambientales, centrándonos en investigar su papel sobre otros niveles tróficos y sus interacciones con la vegetación acuática, así como comparar sus efectos en sistemas mediterráneos y sistemas tropicales. Gracias a experimentos en mesocosmos, que simulan las características ambientales en condiciones controladas, evalué el efecto de una comunidad de larvas de anfibios del Parque Nacional de Doñana sobre diversos parámetros físico-químicos y biológicos característicos del medio. Se detectó un importante efecto de las larvas de anfibios sobre estos parámetros, mayoritariamente cuando se encontraban en alta densidad, causando una marcada reducción de la biomasa de plantas, aumentando la turbidez del agua y los nutrientes tales como el amonio. Las larvas de anfibios afectaron asimismo la abundancia de algas, inferida indirectamente estimando la concentración de clorofila del agua, y redujeron la diversidad de zooplancton. Sin embargo, se observó que estos efectos no eran consecuencia general de todas las especies de anfibios, sino que se debían fundamentalmente a las larvas del sapo de espuelas (Pelobates cultripes). Las larvas de esta especie son desproporcionalmente grandes comparadas con las larvas de las otras especies con las que coexiste, y consume una gran cantidad de biomasa de macrófitos. Esta especie por sí sola alteró la composición del zooplancton, que en presencia de las larvas de P. cultripes se caracterizó por una mayor proporción relativa de copépodos respecto a cladóceros. A este sesgo en la comunidad de zooplancton pueden contribuir también las especies de urodelos, (tritones y salamandras), pues son eficientes carnívoros que pueden alterar considerablemente la composición de las comunidades de zooplancton. Además de la densidad, también se manipuló experimentalmente la presencia de depredadores nativos e invasores, que se introdujeron libres en el medio o mantenidos en jaulas para distinguir los efectos indirectos no derivados de su depredación. Los efectos directos del depredador invasor, el cangrejo rojo americano, fueron los más perjudiciales, tanto en la reducción exacerbada de la supervivencia de anfibios como en las características fisicoquímicas del medio (p. ej. gran aumento de la turbidez y los nutrientes o reducción casi total de la biomasa de plantas). Mediante el uso de isótopos estables se evaluó la plasticidad trófica en la comunidad de larvas de anfibios derivada de los cambios que se habían detectado en las condiciones ambientales. Observamos variaciones en los valores isotópicos de carbono (δ13C) y nitrógeno (δ15N) entre especies y según los tratamientos a los que fueron expuestos. La alta densidad de larvas, la presencia de P. cultripes y el cangrejo rojo americano produjeron una disminución en el δ13C de los anfibios, relacionado con un aumento en su dieta de recursos con menor δ13C, como detritos o zooplancton. Por tanto, la alimentación oportunista de los anfibios está ampliamente condicionada por la competencia, mientras que los efectos indirectos de los depredadores son leves en el sistema templado estudiado. Sin embargo, en sistemas neotropicales se observa que los depredadores tienen un mayor papel en la estructura de las comunidades de anfibios, siendo responsables de una alteración de la posición trófica relativa de las diferentes especies estudiadas en la zona central de Panamá, así como de cambios en su crecimiento y en su supervivencia. Asimismo, la composición de la comunidad tiene también un gran efecto en el nicho trófico de las larvas de anfibios ya que distinguimos cambios en el δ15N de algunas especies. Dado que el conjunto de nuestros resultados ponen de manifiesto que las larvas de anfibios anuros de sistemas acuáticos temporales son importantes consumidores primarios, no sólo de fitoplancton o perifiton, sino también de plantas vasculares acuáticas, con esta tesis se pretende además averiguar las preferencias de diferentes larvas de anfibios por varias especies de macrófitos y carófitos. El sapo de espuelas es el mayor consumidor de macrófitos, mostrando especial preferencia por los del género Callitriche. La herbivoría por parte de larvas de anfibios puede tener un importante papel en estos ecosistemas, afectando a la composición de las comunidades de plantas, su fenología reproductora o la dispersión de sus semillas. De hecho, el papel de las larvas de anfibios en la dispersión directa o indirecta –por depredadores de anfibios- de, semillas de plantas acuáticas ha sido ignorado hasta el momento, por lo que nuestros datos son las primeras observaciones realizadas en este sentido. Además de las semillas ingeridas directamente de las plantas, los renacuajos pueden consumir también las presentes en el banco de semillas del sedimento junto a los detritos, que es otro componente habitual de su dieta. Se observó que entre un 9 y un 32% de los renacuajos, dependiendo de la especie, transportaban semillas o esporas de la vegetación de las lagunas. De ellos, P. cultripes fue la especie con mayor número de semillas. La especie de planta preferida para el consumo (Callitriche) fue también de la que encontramos más semillas en las heces de los renacuajos y su porcentaje de germinación fue entre el 40 y el 88 % en los renacuajos de las diferentes especies. Los resultados de la presente tesis revelan el importante papel que ejercen las larvas de anfibios sobre la compleja estructura de la comunidad de los sistemas acuáticos. Por ello es esencial ampliar nuestro conocimiento respecto a su papel funcional, con el objetivo de anticipar y entender las consecuencias de sus acusados y previsibles declives poblacionales.

Programa de Doctorado en Estudios Medioambientales, The structure and dynamics of ecological communities is determined and regulated by several key processes that influence in the species distribution and abundance (e.g., selection, drift, speciation, and dispersal), and by ecological interactions as competition and predation. The relative importance of competition and predation will depend largely on the local assemblage of species, the type of predators, or the degree of niche segregation. Amphibians constitute an ideal system to address these questions, as they are key species in the trophic webs with a profound role in several ecological processes. Moreover, the effect of amphibian larvae in the aquatic systems and their trophic niche largely depend on their complex ecological interactions with competitors and predators, as they respond to them altering their morphology and behavior. As amphibians continue showing generalized dramatic and global population declines, it is essential to fully characterize the functional role of amphibians in the natural systems to understand the possible consequences of their local declines and extinction. The main objective of this thesis is to know the regulatory potential of amphibian larvae according to the environmental conditions, focusing on the research about their role over other trophic levels, and their interactions with the aquatic vegetation, comparing their effects in Mediterranean and Neotropical systems. By means of experiments with mesocosms, which simulate the environmental conditions in controlled conditions, I evaluated the effect of a guild of amphibian larvae from Doñana National Park on several biological and physicochemical parameters of the system. We detected an important effect of amphibian larvae on these parameters, mostly when at high larval density, causing a marked reduction of plants biomass, increasing water turbidity and several nutrients like the ammonia. Amphibian larvae also affected algae abundance, indirectly inferred by estimating chlorophyll concentration in the water, and they reduced zooplankton diversity. However, these changes were not produced as a consequence of all amphibian species, but they were caused mainly to the larvae of the western spadefoot toad (Pelobates cultripes). The larvae of this species are disproportionally larger than the other amphibian larvae of the area, and they consume a large amount of macrophyte biomass. This species by itself altered the zooplankton composition, as the presence of tadpoles of P. cultripes increased the relative proportion of copepods in respect to cladocerans. Urodeles (newts and salamanders) can also contribute to this bias in the zooplankton community, as they are efficient carnivorous that can considerably alter the zooplankton community composition. Apart from density, we also experimentally manipulated the presence of native and invasive predators, which were introduced either freely roaming or caged in the mesocosms to distinguish their non-consumptive predator effects. The direct consumptive effects of the invasive predator, the red swamp crayfish, were very harmful in the great survival reduction of amphibians as well as in the physicochemical characteristics of the system (e.g., marked increase in water turbidity and nutrients, pronounced reduction of the plants biomass). Using stable isotopes, we evaluated the trophic plasticity of the amphibian larvae community derived of the changes in the environmental conditions detected in the former experiment. We observed variations in the stable isotopic values for carbon (¿13C) and nitrogen (¿15N) among species and according to the treatments the amphibians were exposed to. High larval density, presence of P. cultripes and presence of red swamp crayfish produced a decrease in the ¿13C of amphibians, related to an increase in carbon-depleted resources in their diet such as detritus or zooplankton. Therefore, the opportunistic feeding of amphibians is widely conditioned by competition, whereas the indirect predator effects are negligible in this temperate system. However, in neotropical systems predators have a greater structuring role in the amphibian community, altering the relative trophic position of the different studied species in central Panama, and changing their growth rate and survival. Furthermore, the community composition also has a great effect in the trophic niche of the amphibian larvae as we distinguished changed in the ¿15N of some of the amphibian species. Since our results bring to light that anuran larvae of temporary aquatic systems are important primary consumers, and not only of phytoplankton or periphyton but also of aquatic macrophytes, this thesis also try to figure out the feeding preferences of different amphibian larvae regarding several species of macrophytes and charophytes. The western spadefoot toad is the greatest macrophyte consumer, showing special preference for those from the genus Callitriche. Herbivory by amphibian larvae can have an important role in these ecosystems, affecting the community composition of plants, their reproductive phenology or their seed dispersal. In fact, the role of amphibian larvae in the direct or indirect ¿through seed predators- seed dispersal of aquatic plants has been ignored until the present study, and therefore our data are the first observations concerning this. Apart from the seeds directly ingested from the plants, tadpoles can also consume the seeds buried in the seed bank while feeding on detritus, as it is a common resource for tadpoles. We observed that between 9 and 32% of the tadpoles, depending on the species, transported seeds or spores from the pond vegetation. Among them, P. cultripes was the species with greatest number of seeds. The preferred plant in the feeding experiment (Callitriche) was also the one from which we found in greatest number in the tadpole feces, whose germination rate was between 40 and 88% depending on the tadpole species. The results of this thesis reveal the important role of amphibian larvae on the complex community structure of aquatic systems. Thus, it is essential to widen our knowledge in respect to their functional role aiming to anticipate and understand the consequences of their pronounced and predictable population declines., Universidad Pablo de Olavide. Departamento de Sistemas Físicos, Químicos y Naturales

[ES] En esta tesis se analizan los requerimientos de hábitat de las especies de anfibios en el Parque Nacional de Doñana, prestando una atención especial a los aspectos metodológicos que pueden sesgar los resultados inferidos. En particular, se ha tenido en cuenta la dinámica temporal del sistema y la fiabilidad de las ausencias, una consecuencia directa de la detectabilidad imperfecta de la especie en el área. También se ha evaluado la utilidad de la teledetección en el estudio de la variación espacial y temporal de los hábitats reproductivos de los anfibios y en la elaboración de modelos estadísticos de distribución de las especies., [EN] In this thesis there are analyzed the requirements of habitat of the species of amphibians in Doñana's National Park, giving a special attention to the methodological aspects that can slant the inferred results. Especially, there has been born in mind the temporary dynamics of the system and the reliability of the absences, a direct consequence of the imperfect detectabilidad of the species in the area. Also the usefulness of the teledetección has been evaluated in the study of the spatial and temporary variation of the reproductive habitats of the amphibians and in the production of statistical models of distribution of the species.