Miguel Tejedo

LOCAL ADAPTATION OF AN ANURAN AMPHIBIAN TO OSMOTICALLY STRESSFUL ENVIRONMENTS

Abstract. Water salinity is an intense physiological stress for amphibians. However, some species, such as Bufo calamita, breed in both brackish and freshwater environments. Because selection under environmentally stressful conditions can promote local adaptation of populations, we examined the existence of geographic variation in water salinity tolerance among B. calamita populations from either fresh or brackish water ponds in Southern Spain. Comparisons were made throughout various ontogenetic stages. A combination of field transplant and common garden experiments showed that water salinity decreased survival probability of individuals in all populations, prolonged their larval period, and reduced their mass at metamorphosis. However, significant population X salinity interactions indicated that the population native to brackish water (Saline) had a higher salinity tolerance than the freshwater populations, suggesting local adaptation. Saline individuals transplanted to freshwater environments showed similar survival probabilities, length of larval period, and mass at metamorphosis than those native to freshwater. This indicates that increased tolerance to osmotic stress does not imply a loss of performance in freshwater, at least during the larval and juvenile phases. Despite the adaptive process apparently undergone by Saline, all populations still shared the same upper limit of embryonic stress tolerance (around 10 g/l), defining a window of salinity range within which selection can act. Significant differences in embryonic and larval survival in brackish water among sibships for all populations suggest the existence of a genetic basis for the osmotic tolerance.

Plasticity in Metamorphic Traits of Natterjack Tadpoles: The Interactive Effects of Density and Pond Duration

Plasticity in developmental time and size at metamorphosis were studied in natural and experimental populations of natterjack toad tadpoles (Bufo calamita), a species which breeds in temporary ponds of variable duration. Amount of rainfall directly affected pond hydroperiod and larval density through an increase in the number of spawning females. As both factors directly affected larval period and thus metamorphic success, we performed a factorial field experiment to examine the response of developmental rates to drought regime and its interaction with population density, by controlling pond duration, larval density and food level. Timing of metamorphosis was affected by larval density and food level. However, the response among larvae due to pond duration was not additive. At low larval density, tadpoles raised in short duration ponds exhibited shorter larval periods than in long duration ponds. In constrast, plasticity in developmental rates due to pond duration was less marked at high larval densities and no difference in length of larval period was found. This may be the result of higher competition levels in short duration ponds through an increase in actual larval density because of decreasing water level and lower metamorphic rates. This higher competitive stress was also evident by smaller metamorphic size in short ponds. At low densities tadpoles in longer duration ponds exhibited no additional growth, indicating that tadpoles that remained for a longer period did not achieve larger metamorphic size. This outcome does not support an adaptive explanation of developmental plasticity that merely may be the consequence of expressing a single program of development in different habitats.

Understanding of the impact of chemicals on amphibians: a meta-analytic review

Many studies have assessed the impact of different pollutants on amphibians across a variety of experimental venues (laboratory, mesocosm, and enclosure conditions). Past reviews, using vote-counting methods, have described pollution as one of the major threats faced by amphibians. However, vote-counting methods lack strong statistical power, do not permit one to determine the magnitudes of effects, and do not compare responses among predefined groups. To address these challenges, we conducted a meta-analysis of experimental studies that measured the effects of different chemical pollutants (nitrogenous and phosphorous compounds, pesticides, road deicers, heavy metals, and other wastewater contaminants) at environmentally relevant concentrations on amphibian survival, mass, time to hatching, time to metamorphosis, and frequency of abnormalities. The overall effect size of pollutant exposure was a medium decrease in amphibian survival and mass and a large increase in abnormality frequency. This translates to a 14.3% decrease in survival, a 7.5% decrease in mass, and a 535% increase in abnormality frequency across all studies. In contrast, we found no overall effect of pollutants on time to hatching and time to metamorphosis. We also found that effect sizes differed among experimental venues and among types of pollutants, but we only detected weak differences among amphibian families. These results suggest that variation in sensitivity to contaminants is generally independent of phylogeny. Some publication bias (i.e., selective reporting) was detected, but only for mass and the interaction effect size among stressors. We conclude that the overall impact of pollution on amphibians is moderately to largely negative. This implies that pollutants at environmentally relevant concentrations pose an important threat to amphibians and may play a role in their present global decline.

Contrasting patterns of quantitative and neutral genetic variation in locally adapted populations of the natterjack toad, Bufo calamita

Abstract The relative importance of natural selection and genetic drift in determining patterns of phenotypic diversity observed in nature is still unclear. The natterjack toad (Bufo calamita) is one of a few amphibian species capable of breeding in saline ponds, even though water salinity represents a considerable stress for them. Results from two common‐garden experiments showed a pattern of geographic variation in embryonic salinity tolerance among populations from either fresh or brackish environments, consistent with the hypothesis of local adaptation. Full‐sib analysis showed increased variation in survival among sibships within population for all populations as osmotic stress was increased (broad‐sense heritability increased as salinity raised). Nevertheless, toads native to the brackish water environment had the highest overall survival under brackish conditions. Levels of population genetic differentiation for salinity tolerance were higher than those of neutral genetic differentiation, the latter obtained through the analysis of eight microsatellite loci. Microsatellite markers also revealed little population differentiation, lack of an isolation‐by‐distance pattern, and moderate gene flow connecting the populations. Therefore, environmental stress tolerance appears to have evolved in absence of geographic isolation, and consequently we reject the null hypothesis of neutral differentiation.

Developmental Alterations and Osmoregulatory Physiology of a Larval Anuran under Osmotic Stress

Water salinity represents an environmental stress for many species. Amphibians are particularly sensitive because they are generally poor osmoregulators, and most species are completely absent from brackish and saline environments. We experimentally examined the effect of different salinity levels on larvae of the toad Bufo calamita L., a species that occupies freshwater ponds but can also breed in brackish ponds. Two independent experiments are reported here. In both experiments, tadpoles under saline conditions (ranging between 85 and 200 mOsm) showed a slower developmental rate, metamorphosing between 4 and 9 d later than the controls. Bufo calamita tadpoles reared in brackish water increased their osmolality and solute concentration (mainly sodium and chloride), decreased their levels of glucose, and decreased the total protein content, all measured from whole‐animal extracts. Although most larval anurans are strictly ammoniotelic until the completion of metamorphosis, a few species exposed to dehydrating environments have evolved the ability to use urea as an osmolyte during the larval phase. The data presented here reveal that although B. calamita seems to be yet another exception to the rule of larval strict ammoniotelism, the tadpoles are not able to use urea as an osmolyte and rely on sodium‐chloride balance instead. Preliminary immunoassays of thyroid hormone content suggest a possible decrease in hormone levels induced in water salinity conditions that correlate with a decreased developmental rate.

Evolution and plasticity of anuran larval development in response to desiccation. A comparative analysis

Anurans breed in a variety of aquatic habitats with contrasting levels of desiccation risk, which may result in selection for faster development during larval stages. Previous studies suggest that species in ephemeral ponds reduce their developmental times to minimize desiccation risks, although it is not clear how variation in desiccation risk affects developmental strategies in different species. Employing a comparative phylogenetic approach including data from published and unpublished studies encompassing 62 observations across 30 species, we tested if species breeding in ephemeral ponds (High risk) develop faster than those from permanent ponds (Low risk) and/or show increased developmental plasticity in response to drying conditions. Our analyses support shorter developmental times in High risk, primarily by decreasing body mass at metamorphosis. Plasticity in developmental times was small and did not differ between groups. However, accelerated development in High risk species generally resulted in reduced sizes at metamorphosis, while some Low risk species were able compensate this effect by increasing mean growth rates. Taken together, our results suggest that plastic responses in species breeding in ephemeral ponds are constrained by a general trade-off between development and growth rates.

Reaction norms for metamorphic traits in natterjack toads to larval density and pond duration

The evolution of environmentally‐induced changes in phenotype or reaction norm implies both the existence at some time of genetic variation within a population for that plasticity measured by the presence of genotype x environment interaction (G x E), and that phenotypic variation affects fitness. Otherwise, the genetic structure of polygenic traits may restrict the evolution of the reaction norm by the lack of independent evolution of a given trait in different environments or by genetic trade‐offs with other traits that affect fitness. In this paper, we analyze the existence of G x E in metamorphic traits to two environmental factors, larval density and pond duration in a factorial experiment with Bufo calamita tadpoles in semi‐natural conditions and in the laboratory.

Large male mating advantage in natterjack toads, Bufo calamita : sexual selection or energetic constraints ?

Abstract Variation in the effect of male body size on mating success was studied in natterjack toads during two breeding seasons with different temporal patterns of reproductive activity. Larger males were more likely to mate than small males in the season with a single peak and continuous breeding activity, whereas random mating by size was found when reproduction occurred during several peaks separated by periods of inactivity. Playback tests did not reveal any consistent preference by females for call variables related to male body size other than call intensity. Moreover, direct observation of pair formation resulting from capture of females by non-calling males (searchers and satellites) suggested that most females did not have the opportunity to make any choice. Direct male-male competition for females already in amplexus was negligible as suggested by the low rate of take-over success. Body size advantage occurred indirectly through a positive correlation with chorus attendance. One hypothesis is that smaller males may attend the breeding site less frequently because of energy limitations. Energetic constraints may arise in the long term, affecting the energy that should be allocated to growth, which is higher in small males, or in the short term, affecting the capacity for sustained chorus attendance. Growth rate was not correlated with chorus attendance. However, smaller males lost weight more rapidly in both seasons. This might explain why they had a lower attendance rate than larger males, giving rise to a higher mating success in the latter.

Effects of egg size and density on metamorphic traits in tadpoles of the natterjack toad (Bufo calamita)

-We examined whether intrapopulational variation in egg size was correlated with developmental rates and metamorphic traits of natterjack toads (Bufo calamita) grown at different experimental densities. Mean egg size of siblings was positively correlated with both mean size of hatchlings at the initiation of feeding and mean developmental rates to the feeding stage. However, size at and survival to metamorphosis were not related to ovum or hatchling size. At high densities tadpoles had slower growth rates, longer larval periods, and lower survival, and were smaller at metamorphosis. Length of larval period was inversely related to early tadpole growth rate for all densities. Mass at metamorphosis was positively correlated to tadpole growth rate, except at high densities. Size at metamorphosis was inversely related to length of larval period at low densities, but relationships were inconsistent at higher densities. Egg size in amphibians has been viewed as a resource tradeoff for females with limited energy for reproduction. This notion is derived in part from optimality models predicting that for each environment there is an optimal offspring size (Smith and Fretwell, 1974; Brockelman, 1975). Others have suggested that optimality theory is contradicted by the high degree of intraclutch egg size variability observed in amphibians (Kaplan, 1980; Crump, 1981). Egg size variability may be adaptive in unpredictable environments such as temporary ponds where the intensity of competition may vary annually. Larvae from larger eggs would be favored in competitive environments due to their initial size advantage, while in noncompetitive environments larger and smaller hatchlings would be equally successful. By producing eggs of mixed sizes females may increase their fitness relative to those following a pure strat146 This content downloaded from 207.46.13.57 on Fri, 09 Sep 2016 04:31:37 UTC All use subject to http://about.jstor.org/terms METAMORPHOSIS IN NATTERJACK TOADS egy (Crump, 1984; Kaplan and Cooper, 1984). This reasoning assumes a positive correlation between egg size and larval fitness in competitive environments. Hence, under this view the covariation is environmentally mediated, and the effect of egg size on larval fitness should be related to population density or food availability (Kaplan, 1985; Berven and Chadra, 1988). In temporary ponds, the length of larval period may be an important fitness component because it is inversely related to larval survival. In some amphibians, individuals that grow relatively fast metamorphose earlier (Wilbur and Collins, 1973; Collins, 1979; Smith-Gill and Berven, 1979; Travis, 1981, 1983, 1984) and may be less susceptible to size-selective predators (Heyer et al., 1975; Brodie and Formanowicz, 1983). Further, time to and size at metamorphosis may have strong ecological implications related to long-term fitness since both can affect juvenile survival, age at first reproduction, or level of adult fecundity (Smith, 1987; Semlitsch et al., 1988; Berven, 1990). The relationship between these two traits is controlled largely by the environment (Travis, 1984), and may vary in consistency and direction. A gradual drop in water temperature during a cohorts development may produce a positive covariation between traits (Smith-Gill and Berven, 1979). Likewise, covariation may result from competitive release as fast-growing tadpoles undergo metamorphosis and leave the pond. Remaining tadpoles may then have higher growth rates as a result of an increase in the per capita food ration (Collins, 1979). Negative or random covariations may be the result of an environmental decline in per capita food level that is not counterbalanced by competitive release of metamorphs (Alford and Harris, 1988). It also may be the result of heterogeneous environmental effects (Travis, 1984). The natterjack toad (Bufo calamita) breeds in temporary ponds, and is often the only amphibian species present. Tadpole developmental time is short and may last less than 30 d. However, under crowded conditions individuals may extend their larval development up to 80 d. This implies a greater mortality and total absence of metamorphic success in dry years (Tejedo, 1992a); hence, faster growth rate may be highly adaptive. Much variation in egg size exists within populations, and this variation is related to female body size (Tejedo, 1992a). In this report we examined in the laboratory the relationships between two maternally induced variables (egg size and early hatchling size) and four fitness traits: size at metamorphosis; time of development; growth rate; and survival. The relationship was assessed for larvae of different sibships from a single population, under a range of density treatments. Moreover, growth trajectories and covariation between body size at metamorphosis and developmental time were analyzed across the different densities. MATERIALS AND METHODS A total of 41 egg clutches of B. calamita was sampled from a breeding pond at Sierra Morena, C6rdoba, Spain, during January 1988, ca. 12 h after oviposition, and the eggs were between midand late cleavage (stage 8-9; Gosner, 1960) when they were measured. Average egg size was determined by measuring the diameter of 10 randomly-selected eggs from each clutch. For each egg, the diameter was measured to the nearest 0.02 mm with a dissecting microscope and ocular micrometer. From each clutch, we selected 20 eggs within 0.05 mm of the average diameter. These were allowed to develop to Gosner stage 25 (initial feeding phase), at which time the tadpoles were measured and randomly assigned to four experimental densities consisting of either 1 (D1), 2 (D2), 4 (D4), or 8 (D8) individuals per container. Each group of tadpoles was placed in 15 cm diameter plastic pans that contained 250 ml of dechlorinated tap water. The range of experimental densities was similar to that observed in the pond (5-1000 tadpoles/m2; Tejedo, unpubl. data). Tadpoles were fed and the water changed three times per week. Each pan initially received 30 mg of Purina rabbit chow per feeding. Food rations were increased to 70 mg after 30 d of growth. The ration was kept constant throughout the experiment despite the fact that tadpoles in some treatments died or reached metamorphic climax. Food levels at the lower densities (1-2 tadpoles per pan) allowed unlimited growth. However, tadpoles at higher densities (4 and 8 individuals per pan) normally consumed their food by the second and first day after feeding, respectively. The experiment was conducted in laboratory conditions with natural light. Temperature oscillated around 1416 C. We measured snout-to-tail base and total length of tadpoles 15, 30, and 45 d after the start of the experiment. Both length measures minimized handling of tadpoles and were highly correlated with tadpole weight (total length, r2 = 0.801; snout-to-tail base length, r2 = 0.781, n = 53, P 0.20). Consequently, data from different clutches were used as replicates of the density experiment. Tadpole cohorts were considered to be an experimental unit and analyses were performed on their mean values. All larval fitness variables were logetransformed to meet the assumption of homogeneity of variances among densities. Tadpole survivorship was arcsin-transformed to meet the assumptions of a normal distribution and homogeneity of variances among densities. Heterogeneity in variance among densities was observed for metamorphic size and growth rate and therefore a Kruskal-Wallis test was used (Zar, 1984). Linear regression was used to analyze the relationship between metamorphic size and developmental time. The best linear model was fitted with the aid of the Statgraphic Statistical package (STSC Inc., 1986). Analysis of intraclutch egg size variability was determined by measuring a randomly selected sample of 90-110 eggs from each of 10 clutches collected in the same population during the 1989 breeding season. Coefficients of variation (CV) for egg size were calculated. In addition, skewness (g,) and kurtosis (g2) were computed; these values were compared to the expected parametric values in a normal distribution (Sokal and Rohlf, 1981).

Covariation of Morphology and Jumping Performance in Newly Metamorphosed Water Frogs: Effects of Larval Growth History

Abstract We examined the functional prediction of covariation between relative hind limb morphology and jumping performance for newly metamorphosed frogs from the hybridogenetic Rana esculenta complex. For the two parental species, R. lessonae and R. ridibunda, and three coexisting hybrid hemiclones (GUT1, GUT2, GUT3), we varied larval densities to test the relationship of morphology to performance at both the intraspecific and interspecific levels. Across all genotypes, frogs raised at low density were larger and jumped farther than those raised at high densities. Size-independent maximum jumping performance was repeatable for all hemiclones and species. Comparisons across densities indicated that covariation between morphology and performance matched the biomechanical prediction except for R. ridibunda. Rana ridibunda frogs raised at low larval density jumped relatively farther than those raised at high larval density but, in spite of differing growth environments, did not show differences in relative length of the hind limb. At the intraspecific level, significant performance gradients for hind limb length were found in the hemiclones GUT1 and GUT3 and in R. ridibunda at low larval density. Relative jumping performance of GUT2 and R. lessonae at low larval density and all genotypes at high larval density were not explained by the partial effect of any morphological trait. At the interspecific-interclonal level the biomechanical prediction was confirmed only at the high larval density. These results reveal a lack of congruence of the morphofunctional relationships both at different larval growth conditions within populations and also at intra- and interpecific levels.