Miguel Lizana

Influence of developmental stage on sensitivity to ammonium nitrate of aquatic stages of amphibians

In static renewal experiments, we studied how developmental stage influences the effect of ammonium nitrate on embryonic and larval stages of anuran amphibians. The observed lethal effects caused by ammonium nitrate increased with both concentration and duration of exposure. Significant differences were observed in sensitivity to ammonium nitrate as a function of developmental stage in Discoglossus galganoi, Pelobates cultripes, and Bufo calamita. In D. galganoi and P. cultripes, younger individuals displayed greater acute effects from the chemical fertilizer compared with older individuals. For example, 100% of P. cultripes hatchlings died after 4 d of exposure to a nominal concentration of 225.8 mg N-NO3NH4/L, whereas less than 40% of individuals from older larval stages died when exposed to this concentration. A delay of 4 d in the beginning of the exposure to the chemical was enough to cause significant differences in sensitivity. Bufo calamita showed a higher sensitivity in later larval stages after 12 d of exposure. Hyla meridionalis and B. calamita were less sensitive than the other two species. Peak ammonium nitrate concentrations usually occur when amphibians are breeding and, thus, when the most sensitive aquatic stage is in the water. The developmental stage of the test animals should be considered when evaluating the risk of ammonium nitrate to amphibians.

The absence of species and sex recognition during mate search by male common toads, Bufo bufo

During mate search male Bufo bufo do not discriminate between green frogs and conspecifics, between sexes or between gravid females that differ in body size. We studied mate recognition and the mating behaviour of male European common toads, B. bufo using field-based choice experiments. When given a simultaneous choice between R. perezi and B. bufo both matched in size, male toads did not discriminate between species and amplected a frog or a toad with equal frequency. When a male toad amplected a frog, the frog uttered a release call but the toad did not release the frog and the amplexus lasted from several seconds to a few minutes. Usually frogs fought to release the male toad but test toads were tenacious and usually attempted several times to clasp the frog when the frog slipped away from toad. When given a simultaneous choice between a male and a female of equal size, males did not discriminate between the sexes and attempted to amplex a male or a female with equal frequency. When a test male clasped a stimulus male, the stimulus male uttered a specific release call that caused the test male to release the stimulus male. Male-male amplexus never lasted more than 3 sec, and consequently, the search cost associated with mating with the wrong sex was relatively low. Males did not discriminate between gravid females that differed in body size. Moreover, there was no assortative mating by size. Male-female amplexus was tenacious and prolonged in the three experiments.

The pond network: can structural connectivity reflect on (amphibian) biodiversity patterns?

Landscape connectivity is a very recurrent theme in landscape ecology as it is considered pivotal for the long term conservation of any organism’s populations. Nevertheless, this complex concept is still surrounded by uncertainty and confusion, largely due to the separation between structural and functional connectivity. Amphibians are the most threatened vertebrates around the globe, in Europe mostly due to habitat alteration, and to their particular life cycle. Pond breeding amphibians are considered to be organised in metapopulations, enhancing the importance of landscape connectivity in this group of animals. We sampled the amphibian species present in two pond groups in Central Western Spain. We applied the graph theory framework to these two pond networks in order to determine the importance of each pond for the entire network connectivity. We related the pond importance for connectivity with the species richness present in each pond. We tested if connectivity (partially) determined the presence of the amphibian species sampled using logistic regression. The results show that the structural connectivity of the pond network impacts on the amphibian species richness pattern and that the importance of the pond for the connectivity of the network is an important factor for the presence of some species. Our results, hence, attest the importance of (structural) landscape connectivity determining the pattern of amphibian (functional) colonization in discrete ponds.

Factors influencing wolf Canis lupus roadkills in Northwest Spain

Roadkill is one of the most prominent causes of wildlife mortality. Much research effort has focussed on collisions with ungulates because of traffic safety. However, studies about large carnivore roadkills are scarce despite vehicles being a main cause of mortality. The absence of studies can be explained in part because of difficulties in obtaining sufficient sample sizes. We collected data from locations of 82 wolf roadkill sites in the Castilla y León Region, northwest Spain. We evaluated different models to characterise collision localities using logistic regressions with corrections for rare events. The best models included traffic and human disturbance parameters. Landscape variables did not improve predictive power. Fencing was a decisive key predictor; roadkill was proportionally higher along fenced highways than on similar major roads that lacked fences. Wolf–vehicle collisions were more common in agricultural areas, although wolf densities were lower in these zones. Both the higher density of important roads and a greater proportion of roaming wolves on the plateau may explain this pattern.

Reproductive biology of Pelobates cultripes (Anura: Pelobatidae) in Central Spain

We studied the mating system of a population of western spadefoot toads Pelobates cultripes for a single season. The breeding season lasted 35 days, during which there were four periods of arrival at the breeding pond. Only males arrived at the breeding site in the first peak; additional males and females arrived in subsequent peaks. Minimum temperature and rainfall influenced activity, and maximum temperature was correlated with recruitment and total number of toads that remained in the pond. The breeding sex ratio was slightly male-biased (1.15:1), whereas average operational sex ratio was more strongly male-biased (average 2.97:1). Males migrating early to the breeding pond were larger than males that migrated late. Males remained in the pond longer than females, and fighting between males was observed

Adaptation to osmotic stress provides protection against ammonium nitrate in Pelophylax perezi embryos.

The negative effects of pollution on amphibians are especially high when animals are additionally stressed by other environmental factors such as water salinity. However, the stress provoked by salinity may vary among populations because of adaptation processes. We tested the combined effect of a common fertilizer, ammonium nitrate (0-90.3 mg N-NO3NH4/L), and water salinity (0-2 per thousand) on embryos of two Pelophylax perezi populations from ponds with different salinity concentrations. Embryos exposed to the fertilizer were up to 17% smaller than controls. Survival rates of embryos exposed to a single stressor were always below 10%. The exposure to both stressors concurrently increased mortality rate (>95%) of embryos from freshwater. Since the fertilizer was lethal only when individuals were stressed by the salinity, it did not cause lethal effects on embryos naturally adapted to saline environments. Our results underscore the importance of testing multiple stressors when analyzing amphibian sensitivity to environmental pollution.

Spatial and temporal segregation allows coexistence in a hybrid zone among two Mediterranean vipers (Vipera aspis and V. latastei)

Mediterranean vipers are ecologically very similar. When in contact, they constitute an exceptional model to study the effects of competitive interactions and niche segregation mechanisms. In High Ebro, distinct methodologies (random visual encounter, road sampling and radio-tracking surveys, captive maintenance, standardized transects and ad hoc field observations) were used to compare ecological traits (diet, micro-habitat, activity, reproductive and demographic) of V. aspis, V. latastei and hybrids among both species and determine competitive advantages of each form. Diet preferences were similar but few differences were found in feeding frequency and annual variation in diet consumption. Males of the three forms compete for areas with similar characteristics but differences were found mainly during summer. Significant differences in average movement rates and home range size were found among males. Fecundity and new-borns fitness were lower in V. aspis than in V. latastei, whereas hybrids had intermediate values. Male and female adult hybrids were more abundant than parental forms, whereas female and juvenile V. latastei and juvenile hybrids had high road-mortality. The three forms seem to be competing intensely for resources but the segregation on the spatial and temporal axes of their niches could be reducing interspecific competition and allowing coexistence. The use of different resources probably confers reproductive and demographic advantages for V. latastei and hybrids, respectively. Moreover, reproductive fitness of hybrids suggests the occurrence of endogenous selection. Comparative thermal and genetic studies are needed to clarify the limiting factors of vipers and to establish a model of the hybrid zone.

VARIABLE BREEDING PHENOLOGY AFFECTS THE EXPOSURE OF AMPHIBIAN EMBRYOS TO ULTRAVIOLET RADIATION and OPTICAL CHARACTERISTICS OF NATURAL WATERS PROTECT AMPHIBIANS FROM UV‐B IN THE U.S. PACIFIC NORTHWEST: COMMENT

1Department of Zoology, 3029 Cordley Hall, Oregon State University, Corvallis, Oregon 97331 USA 2United States Geological Survey, Oregon Cooperative Fish and Wildlife Research Unit, Department of Fisheries and Wildlife, Oregon State University, Corvallis, Oregon 97331 USA 3Department of Evolutionary Biology, Donana Biological Station, CSIC, Apartado 1056, Sevilla 41013, Spain 4Department of Biology, 112 Science Place, University of Saskatchewan, Saskatoon, Saskatchewan S7N5E2 5Department of Biology, 2125 Derring Hall, Virginia Polytechnic Institute and State University Blacksburg, VA 24061 USA 6Department of Biology, Pennsylvania State University, 208 Mueller Lab, University Park, PA 16802 USA 7Departamento de Biologia Animal, Ecologia, Parasitologia y Edafologia, Universidad de Salamanca, 37071 Salamanca, Spain 8Natural Science Division, Pepperdine University, Malibu, CA 90263 USA

Alteration of courtship behavior because of water acidification and minor effect of ammonium nitrate in the Iberian newt (Lissotriton boscai)

Although many studies have assessed the effects of water pollution on amphibians, few have focused on the alteration of vital behaviors. Newt species usually display complex courtship behavior that may be altered by sublethal levels of contaminants. We analyzed the effects of ammonium nitrate and water acidification on courtship behavior of the Iberian newt (Lissotriton boscai). We exposed newt couples to three treatments (control, nominal 90 mg N--NO3NH4/L, and nominal pH 4) and recorded the occurrence of each of the three courtship phases (orientation, static display, and spermatophore transfer) as well as the latency to initiate courtship, overall courtship time, male perseverance, and female responsiveness. Major effects were observed in the acid treatment. Low pH inhibited orientation. None of the control couples failed to court, whereas 17% of couples from the low-pH treatment did not perform any courtship movement. Latency to initiate courting was twice as high in couples exposed to acidification as in controls. Low pH also affected the overall time of courtship and perseverance time of males in unsuccessful encounters. Ammonium nitrate did not cause any significant effect. Only the female response index and the duration of the tail-fanning movement (the major display during L. boscai courtship) were slightly reduced by the fertilizer. We report a pollution-mediated alteration of courtship behavior, to our knowledge for the first time, in amphibians. This kind of sublethal effect should be considered when estimating potential impacts of water pollution on natural populations.

Influence of a Combination of Agricultural Chemicals on Embryos of the Endangered Gold-Striped Salamander (Chioglossa lusitanica)

Pollution from agrochemicals may be contributing to the global decline of amphibian populations. Environmentally relevant concentrations of a fertiliser, ammonium nitrate, and a commercial formulation of the herbicide glyphosate Roundup Plus were tested on the embryonic development of Chioglossa lusitanica. This study introduces new data at three different levels. First, we provide previously unknown information about hatchling traits of C. lusitanica. Second, we present the first ecotoxicological study of this endangered species, to which environmental pollution is considered a major threat. Third, we conduct the first experiment with an amphibian species exposed to a mixture of a glyphosate-based herbicide and a nitrogenous fertiliser. Control individuals hatched with an average (±SD) total length of 18.77 (±2.02) mm and at an average Harrison’s developmental stage of 44.58 (±1.24). Mean hatching time among controls was 11.52 (±1.29) weeks. None of the chemicals or their interaction produced lethal effects; however, a significant interaction was found when analysing total length at hatching. Individuals exposed to the herbicide hatched at a larger size than controls, and this effect was especially clear when the fertiliser was added to the water. The absence of pollutant-related mortality or severe sublethal effects is in agreement with most studies indicating a high tolerance of amphibian embryos to agrochemicals. However, further research considering other life stages and additional natural factors (i.e., predators, food availability) is needed to estimate the ecological impact of chemical mixtures on C. lusitanica.