Variation in UV sensitivity among common frog Rana temporaria populations along an altitudinal gradient

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ZOOLOGY Zoology 111 (2008) 309–317 www.elsevier.de/zool

Variation in UV sensitivity among common frog Rana temporaria populations along an altitudinal gradient Olivier Marquis, Claude Miaud Laboratoire d’Ecologie Alpine, Universite´ de Savoie, CNRS UMR 5553, 73 376 Le Bourget du Lac, France Received 30 April 2007; received in revised form 30 August 2007; accepted 1 September 2007

Abstract Solar ultraviolet-B (UV-B) radiation can be harmful for developing amphibians. As the UV-B dose increases with altitude, it has been suggested that high-altitude populations may have an increased tolerance to high levels of UV-B radiation as compared to lowland populations. We tested this hypothesis with the common frog (Rana temporaria) by comparing populations from nine altitudes (from 333 to 2450 m above sea level). Eggs collected in the field were used for laboratory experiments, i.e., exposed to high levels of artificial UV-B radiation. Eggs were reared at 1472 1C and exposed to UV treatments until hatching. Embryonic developmental rates increased strongly and linearly with increasing altitude, suggesting a genetic capacity for faster development in highland than lowland eggs. Body length at hatching varied significantly with UV-B treatments, being lower when eggs developed under direct UV-B exposure. Body length at hatching also increased as the altitude of populations increased, but UV-B exposure times were shorter as altitude of population increased. However, the body length difference between exposed and non-exposed individuals in each population decreased as altitude of populations increased, suggesting a costly effect of UV exposure on growth. Type of UV exposure did not influence the mean rates of embryonic mortality and deformity, but both mortality and deformity rates increased as the altitude of populations increased (while UV-B exposure duration decreased). The effect of UV-B on body length at hatching, mortality, and deformities suggests that the sensitivity to UV-B varied among populations along the altitudinal gradient. These results are discussed in evolutionary terms, specifically the potential of R. temporaria high-altitude populations to develop local genetic adaptation to high levels of UV-B. r 2008 Elsevier GmbH. All rights reserved. Keywords: UV resistance; Altitude; Local adaptation; Amphibians; Embryonic development

Introduction A high level of variability and diversity is usually observed among populations, or demes, through varying ecological conditions (Mayr 1963). Environmental gradients tend to increase the phenotypic plasticity of traits with phenotypes optimised to the local environCorresponding author.

E-mail address: [email protected] (C. Miaud). 0944-2006/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2007.09.003

mental conditions (Sultan and Spencer 2002), and when gene flow is restricted among populations along these gradients, selection tends to favour local adaptation over plasticity (Pigliucci 2001). Differences in mean phenotype among populations can be considered adaptive if they have a genetic basis, and if they confer a fitness advantage in a given environment (Pease et al. 1989). Mountain slopes, which can exhibit considerable ecological variation at relatively short distances, thus offer the opportunity to test hypotheses on the

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determinism of variation, i.e., phenotypic versus genetic, among populations. Spatial and temporal variations in UV-B levels on earth depend on various factors, including the thickness of the ozone layer, latitude, and altitude (Cockell and Blaustein 2001). For example, intensity of UV-B radiation increases by 19% with 1000 m increase in elevation in the Alps (Blumthaler et al. 1992), and species with wide altitudinal distributions often have specific adaptations: an increased concentration of soluble UV-B absorbing compounds in leaves and a reduction of leaf area in plants (Day 1993), a change in pigmentation in Collembola, or in melanisation of the dorsal portion of the carapace in Daphnia from alpine lakes (Hessen 2002). Amphibians are a good model for studying local adaptation as they can experience a wide range of environments and are relatively limited in their movements. Several local adaptations to various stressors have been demonstrated, such as sensitivity to nitrate (Johansson et al. 2001) and tolerance to salinity (Gomez-Mestre and Tejedo 2003). Moreover, local adaptations of life history traits have been identified in populations living at various altitudes (Ficetola and De Bernardi 2006) and latitudes (Merila¨ et al. 2000a) and in different landscapes (Skelly 2004). Many studies have addressed the impact of UV-B radiation on amphibians (Blaustein et al. 1996; Davis et al. 1996; Nagl and Hofer 1997; Corn 1998; Cummins et al. 1999; Ankley et al. 2000; Belden et al. 2000; Merila¨ et al. 2000b; Pahkala et al. 2000; Belden and Blaustein 2002b), but few have focused on variation of UV sensitivity among populations. In the common frog, Rana temporaria, embryos originating from eight populations spanning a 1200 km latitudinal gradient across Sweden did not exhibit clear variation in UV-B sensitivity (Pahkala et al. 2002). In the salamander Ambystoma macrodactylum, larvae from highland populations were less sensitive to UV-B radiation than those from lowland populations (Belden and Blaustein 2002a). R. temporaria is one of the most widespread anurans in Europe. It breeds in a wide range of shallow freshwater habitats from northern Spain to the northern parts of Fennoscandia and from sea level to 2745 m above sea level in the Alps (Gasc et al. 1997). Clutches are deposited in shallow water, and embryos are often directly exposed to solar radiation. Life history traits of the common frog exhibit variation along altitudinal and latitudinal gradient (review in Miaud and Merila¨ 2001). Local adaptation in variation of UV-B sensitivity was tested along the latitudinal gradient (Pahkala et al. 2002), but has not yet been studied along an altitudinal gradient. The aim of this study is to analyse variation in response to UV-B radiation among several R. tempor-

aria populations living at different altitudes. The main predictions were (1) increasing levels of UV-B radiation with higher altitude induce variation in embryonic traits among populations; (2) if adaptation to UV-B radiation has occurred, populations from higher altitudes should tolerate higher UV-B radiation levels than populations from lower altitudes.

Materials and methods Populations and study sites We collected eggs from nine populations located at altitudes of 333, 438, 860, 1114, 1318, 1968, 2280, 2445 and 2450 m a.s.l. in the western Alps in 2003. These populations were clearly separated (the closest distance between two populations was 7 km), and without aquatic connections such as a drainage system that could allow tadpole migration from one place to another. Breeding time differed between populations according to altitude, starting in March and ending in June. Samples (parts of spawns containing about 200 eggs) were collected from clutches less than 24 h old (stage 1–8, Gosner 1960). From 4 to 20 samples were collected from different clutches in each population according to the size of the population and availability of recently fertilised clutches: 9 clutches at 333 m, 10 clutches at 438 m, 5 clutches at 860 m, 10 clutches at 1114 m, 20 clutches at 1318 m, 9 clutches at 1968 m, 8 clutches at 2280 m, 4 clutches at 2445 m, and 10 clutches at 2450 m. Eggs were stored in the dark at 4 1C (for no more than 24 h) in the laboratory before the start of the experiment.

Experimental design The experimental design was inspired by the experiments described in Pahkala et al. (2000, 2002). Following field measurements at several altitudes and regions of the Alps by Pachard et al. (1999), Reiter et al. (1982) and Blumthaler et al. (1992) we assumed a total daily irradiance of 33 MJ/m2, with UV-A ¼ 1.72 MJ/m2 and UV-B ¼ 4.71 kJ/m2. This equals the mean cumulative UV-A and UV-B dose received per day at an altitude of about 2500 m in the Alps during summer with a daily light period of 8 h of the UV-A tubes (10.00 a.m. to 6.00 p.m.) and 6 h of the UV-B tubes (11.00 a.m. to 5.00 p.m.). Eggs were exposed to a homogenous UV radiation obtained by eight UV-A (Philips Cleo Performance 100 W) and five UV-B (Philips TL 100W/01) tubes covering a surface of 5.7 m2, at 90 cm above the water surface in the experimental tanks. Exposed eggs were kept in plastic vessels (length 210 mm, width 140 mm, depth 35 mm) with three holes

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1995; Licht and Grant 1997; Diamond et al. 2002). Transmission through the filters was confirmed by measurements performed with a UV/visible spectrophotometer (Shimatzu UV-160A). Embryos were exposed from early embryonic development to hatching (i.e. stages 8–25, Gosner 1960, which represents on average 7 days of exposure) and then preserved in 80% ethanol before measuring.

(diameter 20 mm) on each side covered by plastic mesh to permit water circulation and to prevent the tadpoles from escaping. The vessels were placed in tanks (length 77 cm, width 56 cm, depth 21 cm, containing approximately 78 l of dechlorinated tap water) in a temperaturecontrolled room, where air temperature was 1472 1C. Each day, we measured the water temperature in two randomly chosen tanks and in the plastic vessels. No temperature difference was observed between tanks or between plastic vessels. Previous experiments showed that eggs and tadpoles from the different studied populations developed normally at the chosen rearing temperature (Martin 2004). We used 10 tanks, each containing two floating plastic frames with three vessels on each one (Fig. 1), so that the vessels were continuously filled with 25 mm of water even if the level of the water in the tanks fluctuated. Each vessel on a plastic frame contained 50 eggs, all originating from the same clutch. Thus, each tank contained two groups of 150 eggs composed of three groups of 50 eggs (i.e. one per filter type). In each vessel, eggs were manually separated in order to spread them out in a single level. All nine populations, with the number of clutch varying from 4 to 20, were consecutively placed in the tanks for UV exposure. The experiment consisted of a combination of three UV-B treatments (Fig. 1): (1) Mylar: exposure through a Mylar filter eliminating more than 80% of the UV-B (see Pahkala et al. 2000) and shorter wavelengths, (2) Acetate: exposure through a cellulose acetate filter which transmits approximately 100% of UV-B (considered as a control of the filter effect), and (3) Open: direct exposure to UV without a filter. Both filters did not affected UV-A transmission, this wavelength is known beneficial for enzymatic DNA repairing mechanisms against UV-B damages (Grant and Licht Tank 1

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Statistical analysis When larvae reached stage 25 (Gosner 1960), the mortality rate (i.e. proportion of dead tadpoles or undeveloped eggs/tadpole alive), malformation rate (i.e. proportion of abnormal tadpoles among the living tadpoles at the end of the experiment), and body length (from extremity of the head to the tip of the tail) were recorded. Tadpoles in the same vessel cannot be considered as independent samples, so data from individual vessels were treated as a single sample. Deformed or dead tadpoles were excluded from body length statistics. The effects of the treatments (light exposure) on body length were investigated using a mixed-model analysis of variance in which UV-B treatments (i.e., filter type) and altitude of tested populations were treated as fixed effects, and the effect of clutch or tank treated as a random effect. Including these random effects had a negligible effect, so the analysis was reduced to a simple two-way ANOVA. Exposure duration was highly correlated with the altitude of populations (r2 ¼ 0.96, po0.0001), and adding it as a covariate means that the effect of altitude is lost with no improvement in filter as an explanatory variable. Observed variation with altitude of populations thus has to be interpreted taking

Tank 2

Tank n

A

B

C

D

E

F

A

B

C

D

E

F

A

B

C

D

E

F

= OPEN

= ACETATE

= MYLAR

Fig. 1. Schematic presentation of the experimental set-up. Eggs in each tank came from two clutches (symbolised by letters A–F) and were exposed to three different UV treatments (Mylar ¼ mylar filter, Acetate ¼ cellulose acetate filter, Open ¼ without filter). The order of UV-B treatments within each tank was random. The total number of tanks was 10 and the number of tested clutches varied from 4 to 20 per population. Each of the six vessels in a given tank received 50 eggs at the beginning of the experiment.

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Table 1. Two-ways ANOVA for effects of UV-B treatments and altitude of populations on hatching size of nine Rana temporaria populations Hatching size

df

SS

F

p

Fixed effects Altitude Filter

1 2

58.159 11.827

75.646 7.6916

o0.0001 0.0005

SS: sum of squares.

into account variation in UV-B exposure. The effects of UV-B treatment on survival and frequency of deformities were investigated with generalised linear models with binomial errors and the logit link. Analyses were performed using R 2.5.0 software (R Development Core Team, Vienna, Austria).

Results Embryonic development Within each population and within each clutch, all eggs developed in the same rhythm and hatched within the same 24 h interval. Time to hatching varied greatly among populations. The developmental time was, e.g., close to three times longer at 333 m a.s.l. than at 2450 m a.s.l. The developmental time significantly decreased as the altitude of populations increased (Spearman correlation, rs ¼ 0.87, p ¼ 0.0023, N ¼ 9, Fig. 2).

16

Body length at hatching varied significantly with UV-B treatment and altitude of populations (Table 1). In eight of the nine populations, eggs exposed to direct UV-B irradiation resulted in smaller tadpoles at hatching than eggs protected from UV-B exposure (Table 2, Fig. 3). In the last population (2450 m altitude), tadpole length at hatching was similar whatever the UV treatments (Fig. 3). The effect of UV-B on body length decreased with increasing altitude of populations, i.e. the difference between exposed and non-exposed individuals (calculated as the difference between mean length under Mylar and mean length under no filter in each population) significantly decreased with altitude of populations (even when taking into account the difference of exposure duration between populations, rs ¼ 0.32, p ¼ 0.003, Fig. 4). Body length at hatching also varied among populations, increasing with the altitude of the origin of the population (Fig. 3). However, exposure duration decreased during the experiments as the altitude of populations increased, and thus a potential effect of population cannot be distinguished from the effect of exposure duration.

Mortality and deformity rates The mean mortality rate varied from 0.008 (population at 1318 m a.s.l.) to 0.043 (population at 860 m a.s.l.). There was no effect of UV-B treatment on embryonic mortality (Table 3; p ¼ 0.05; however, this estimated p-value for the effect of the filter should be treated with caution because the residual deviance was more than three times larger than the residual degrees of freedom). On the other hand, the mortality rate 15.5

14 12 10 8 6 4 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 Altitude of populations (m)

Fig. 2. Embryonic development times of nine Rana temporaria populations at 1472 1C in the laboratory. Each dot represents the time to hatching of a population (tadpole development from stage 8–25, see Materials and methods). There is no confidence interval because all the eggs of the same clutch and the same population hatched within the same 24 h interval (rs ¼ 0.87, p ¼ 0.0023, N ¼ 9).

Body length at stage 25 (mm)

Embryonic developmental time (days)

18

Body length at hatching

15.0 14.5 14.0 13.5 13.0 12.5

Open Mylar Acetate

12.0 11.5 0

500

1000

1500 2000 Altitude (m)

2500

3000

Fig. 3. Hatching size in nine Rana temporaria populations subject to three different UV-B treatments in the laboratory: Mylar ¼ mylar filter, Acetate ¼ cellulose acetate filter, Openwithout filter.

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Table 2. Summary of mean body length, deformity and mortality rates of tadpoles (stage 25) of the common frog Rana temporaria from nine populations for three UV treatments Treatments

Mean body length (mm) (7S.D.)

Mean deformity rate (7S.D.)

Mean mortality rate (7S.D.)

Mylar filter (N ¼ 85) Acetate filter (N ¼ 87) Open (N ¼ 85)

13.8371.19 13.5371.29 13.2971.24

0.02670.031 0.02970.043 0.02270.034

0.03070.067 0.02970.056 0.02370.062

Open: direct exposure to UV-A and UV-B tubes (exposure similar to cumulative doses received per day at an altitude of 2500 m in the French Alps). Mylar filter: protects from most of the UV-B radiation. Acetate filter: transmits approximately 100% of UV-B radiation. N ¼ number of clutch tested (50 tadpoles per clutch at the beginning of the experiment).

increased as the altitude of populations increased independently of the UV treatment (Table 3). The mean deformity rate varied from 0 (population at 860 m a.s.l) to 0.043 (population at 2450 m a.s.l). The deformity rate was not influenced by the type of UV exposure (i.e., filter type), but increased with the altitude of populations (Table 3).

Discussion

Difference in body length/exposure duration

Living at high altitudes often incurs low temperatures, high doses of UV-B, as well as scarce resources of food and water. These conditions may produce specialised high-altitude phenotypes, such as melanism or thermoregulation of ectotherms (Lawson and King 1996), or morphological adaptation to prevent desiccation of plants (Somersalo et al. 1998). Amphibians in highland populations also exhibited particular life history traits and morphology (reviews in Miaud et al. 1999; Miaud and Merila¨ 2001; Su-Ju et al. 2003; Andreone et al. 0.20

0.15

0.10

2004). Latitude and altitude are sources of a natural UV-B variation (Cockell and Blaustein 2001), and geographic variation in UV tolerance among amphibian populations has often been mentioned (Blaustein et al. 1994; Adams et al. 2001), but few studies have demonstrate it along a UV-gradient. The present study tested the UV-tolerance variation of nine populations along an altitudinal gradient in the Alps. A phylogeographic study based on variation of the cytochrome b (DNAmt) gene across the distribution of the common frog in Europe showed that these populations in the western Alps belong to the same haplotype (Pidancier et al. 2003). In the common frog, embryonic developmental rates assessed in the laboratory increased strongly and linearly with increasing altitude, being several times faster in high-altitude populations than in low-altitude populations, confirming previous results obtained with fewer populations (Martin and Miaud 1999). Such differences among populations of this species were also observed in Sweden, but without a linear relationship with latitude (Pahkala et al. 2002). On the other hand, tadpole development rates increased with latitude in a range of temperatures providing strong evidence for seasonality as a selective agent on tadpole development (Merila¨ et al. 2000a; Laurila et al. 2001, 2002; Laugen et al. 2003). When measured in the wild, common frog embryonic development was longer in populations at Table 3. Generalised linear models for effects of UV-B treatments and altitude of populations on egg mortality and deformity rates of nine Rana temporaria populations

0.05

0.00

Deviance

p

Embryonic mortality Fixed effects Filter 2 Altitude 1 Residual 253

5.95 112.93 775.60

0.05 o0.00001

Deformity rate Fixed effects Filter Altitude Residual

4.12 15.06 473.54

0.13 o0.001

df -0.05 0

500

1000

1500 2000 Altitude (m)

2500

3000

Fig. 4. The difference in mean body length at hatching (mean7S.E. in mm) between UV-exposed and non-exposed embryos of the common frog Rana temporaria originating from nine populations along an altitudinal gradient was divided by the number of days of exposure to take into account development rate differences between populations. The quotient obtained decreased as the altitude of the population increased.

2 1 254

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high than in low latitudes because water temperature increased from high to lowland (Martin 2004). In our common garden experiments (this study) with constant water temperature (14 1C), embryonic development was quicker in clutches originated from high-altitude populations than clutches from low-altitude populations. Even if a maternal effect cannot be exclude from the difference observed in this common environment, the existence of faster developing genotypes expressed in highland populations would be another example of countergradient selection in the common frog. Exposed to the total irradiance observed at high altitudes, the eggs of the nine sampled populations of the common frog exhibited low mortality and deformity rates, confirming that this species is resistant to UV radiation (Cummins et al. 1999; Hofer and Mokri 2000; Pahkala et al. 2000, 2001, 2002; Ha¨kkinen et al. 2001). Our experiments did not show mortality and deformity differences between exposed and non-exposed eggs within each population. But, mortality and deformity rates were generally higher in highland populations than in lowland populations (while eggs from highland populations were submitted to shorter periods of UV exposure). Several studies have found no effects (e.g., Blaustein et al. 1996; Ovaska et al. 1997) or negative effects (e.g., Anzalone et al. 1998; Corn 1998; Lizana and Pedraza 1998) of UV-B exposure on amphibian egg hatchability. Answering the question how mortality and deformity rates of UV-B exposed R. temporaria eggs differ among populations will need more studies with various ecologically relevant UV exposures. The experimental UV exposure reduced the body length of tadpoles at hatching, as previously observed in this species (Pahkala et al. 2000). Body length decrease was attributed to the energetic cost of the UV resistance mechanisms in the red-legged frog Rana aurora and the salamander A. macrodactylum (Belden et al. 2000; Belden and Blaustein 2002b) or to a decreased metabolic performance in the common toad Bufo bufo (Formicki et al. 2003). The altitudinal gradient can lead to other selective pressures than those involved in UV tolerance. The solubility of oxygen in water at high altitudes significantly differs from that at low altitudes. UV damage of sub-cellular components and the activation of some UV damage defences are connected with energy cost, which may in turn influence oxygen consumption (Iizawa et al. 1994). Consequently, if high-altitude populations have developed an enhanced oxygen carrying capacity compared to lowland populations, the phenotypic variation observed among populations exposed to UV in the laboratory at 300 m a.s.l. would reflect other physiological capacities and not necessarily UV tolerance. However, previous experiments to compare hatching success, developmental duration and size at

hatching (Martin and Miaud 1999; Martin 2004) of eggs from populations above 2000 m reared in the laboratory at 300 m and in the field yielded no significant differences. We thus assumed that differences observed between UV protected and exposed eggs mainly result from a variation of UV tolerance among populations. This study is the first showing a variation of responses to UV exposure among populations of a frog species along an altitudinal gradient. In the common frog, UV sensitivity decreases with increasing altitude. These results, based on a straightforward experimental design, indicate genetic divergence along an environmental gradient (Pahkala et al. 2001), and imply local adaptation (Schlichting and Pigliucci 1998). However, a parental effect cannot be excluded because the tested embryos were coming from parents living in the field. High-altitude populations of the common frog are exposed to adverse conditions of lighting and temperature during the short favourable period of activity (summer), and are often of relatively small effective size, which can heighten the effects of selective pressures (Miaud and Merila¨ 2001). Gene flow between populations depends on landscape permeability, and populations at high altitudes are often separated by impassable topography (Martin 2004). These conditions tend to favour local adaptation over plasticity (Pigliucci 2001). On the other hand, populations at lower altitudes exhibit more exchanges (with more favourable field topography and larger population sizes, Martin 2004). In this case, plasticity rather than local adaptation should be promoted (Sultan and Spencer 2002). The phenotypes of populations below 1000 m (Fig. 3, and ANOVA on residuals of regression, F ¼ 13.97, p ¼ 0.0096) were more variable than those at higher altitudes. This argues for the maintenance of phenotypic plasticity at low altitudes and of local adaptation at high altitudes. Artificial crossing experiments between individuals of these populations and/or transplantations between populations will have to be conducted to determine the importance of environmental versus genetic influences. Local adaptations are based on singular complex sets of genes (Kawecki and Ebert 2004), which can lead to resistance to UV radiation. This resistance may be due to photo-protective compounds (i.e. UV-absorbing compounds) produced to prevent DNA damage induced by UV-B, or to the activation of enzymatic DNA repair systems. Many photo-protective compounds are known in plants, bacteria and microorganisms. Pigments such as melanin and carotenoids are produced by plants, micro-crustaceans and some cyanobacteria under intense solar radiation (Sommaruga 2001). Mycosporinelike amino acids (MAAs), scytonemin and flavonoids are UV-absorbing compounds found in terrestrial or aquatic plants and bacteria as protection against UV-B radiation (Sommaruga 2001; Rozema et al. 2002).

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In amphibians, melanin is also thought to have an important role in the protection of embryos and larvae against UV-B (review in Blaustein and Belden 2003), and one UV-B absorbing substance (UVAS) was identified in R. temporaria tadpoles exposed to UV-B (Hofer and Mokri 2000). The jelly coat surrounding the eggs of amphibians and heat shock proteins may also play a role in UV protection but no studies have yet clearly demonstrated this (Blaustein and Belden 2003; Rasanen et al. 2003; Marquis et al. 2008). On the other hand, photolyase is a major DNA repair system against damage induced by UV-B radiation in numerous organisms (Pang and Hays 1991; Friedberg et al. 1995). In amphibians, a strong correlation was demonstrated between photolyase activity and resistance to UV-B exposure (Cockell and Blaustein 2001), and photolyase production was induced by UV-B exposure in the wood frog Rana sylvatica embryos (Smith et al. 2000). The variety of mechanisms which provide amphibians with protection against UV radiation implies that many candidate genes are under selection, and thus argues for the existence of local adaptation. In conclusion, our results indicate that common frog embryos exhibit a higher developmental rate when they originate from highland populations, and that they are rather insensitive to UV-B exposure in terms of hatchability and deformity rate. However, variation in length at hatching among populations argues for the existence of a trade-off between UV-B protection or damage repair and early growth. Nevertheless, the developmental speed variation among populations varying in altitude is a confounding effect with regard to other potential phenotypic variations. Further studies taking into account this embryonic effect are required to identify the existence and mechanisms of other local adaptations. Which one is involved in the UV-B resistance of high-altitude common frog populations is still unknown. Finally, as previously noted by Pahkala et al. (2002), one should interpret the results of experiments on embryos with caution because not all the potential negative effects of UV-B irradiation on development are expressed by the hatching stage: a higher rate of developmental anomalies, delayed development and reduced size were observed at metamorphosis when eggs had previously been exposed to increased levels of UV-B (Pahkala et al. 2001).

Acknowledgements The authors are grateful to Annie Millery and many students for their help during field and laboratory work, and B. Anhold for his help with the statistical analysis. Comments from three anonymous reviewers also greatly improved this manuscript.

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