R. arvalis is a ranid frog that occurs in the western Palaearctic in a wide range of habitats and acidity levels 31. Breeding occurs in early spring, at higher latitudes soon after spring snow melt, hence coinciding with peak acidity in acidified areas.

Eight populations occurring along a ca. 160 km transect in southwestern Sweden were used in this study (Figure 1, Table 1). All study sites are permanent ponds or small lakes in forested areas (Figure 1, Table 1). Breeding pond pH among these sites ranges from pH 4.0 to 7.3. The exact acidification history of these ponds is not currently known, but is likely influenced by a mix of anthropogenic [acid rain, 2832] and natural acidification (Table 1), which may have been counteracted in some locations with artificial liming 33. The two most neutral sites (Figure 1, Table 1) are buffered naturally due to limestone bedrock 34.

To characterize environmental differences among breeding ponds, pH and several abiotic and biotic variables known to change as a result of acidification, or to be ecologically important for amphibians, were measured (Table 1). The abiotic environment was characterized as pH, latitude and altitude, water temperature, pond size and canopy cover. The biotic environment was characterized as predator abundance and tadpole density. These variables were chosen because seasonal time constraints and temperature can have strong effects on larval life history traits in amphibians 35, canopy cover and pond size may affect amphibian growth and development 3637, and community composition typically shifts towards more insect dominated predator communities and reduced amphibian densities as a result of acidification 28. To increase sample size for environmental inferences, habitat variables were also measured in one additional population, which was, however, not included in the experimental work (Nitta, Table 1).

pH was measured annually since 2007, whereas the other environmental variables were quantified in 2009. pH was measured in April 2007 to 2009, and May and June 2009 with an Orion 9109WL electrode (Thermo Scientific, Inc.) attached to a portable field pH-meter (Orion 260, Thermo Scientific, Inc.). At each pond, pH was measured at three different sites (three measurements per site) during one to three visits/year. pH for each pond was calculated based on pH averages of April 2007-2009 (hereafter "embryonic period") and April 2007 and 2008 (no May and June data available for these years), May and June 2009 (hereafter "larval period").

Latitude and altitude were gained from Google Earth http://earth.google.com/ and used as indicators of large-scale climatic variation. Local temperature was measured with iButton data loggers (DS1921G-F50, Maxim Integrated Products, Inc.) in 2009 from April 14 (early breeding season) until June 17 (when tadpoles were approaching metamorphosis). The loggers were set at measuring interval of three hours and placed just below water surface in sealed plastic bottles (one per two or three different sites within a pond), near R. arvalis breeding sites. Due to technical fault, data from only one logger could be retrieved from two of the ponds (Rud and Sätila, Table 1). For each pond, the mean temperature was calculated over the whole recording period and across sites.

To get an estimate of pond size, pond area was estimated by multiplying pond length × pond width (measurements gained from Google Earth). Shoreline depth at each pond was measured in May and June 2009 (to the nearest 0.01 m) at three randomly chosen, approximately equidistant, sites (using a measuring band with a weight attached to assure a straight line). The average of May and June measurements/pond was used as pond depth. Pond size (m³) was then calculated by multiplying pond area (m²) with pond depth (m) and log-transformed for the statistical analyses. The percentage of forest canopy closure was estimated as the proportion of non-visible sky within an angle of 40°- 45° by the same person (S.H.) in 10% categories 38.

Predator and tadpole densities were estimated at each pond in mid May and mid June 2009 with dip net (32 cm diameter, 0.08 cm mesh size) sweeps. Within each pond, five sweeps at a depth of 0.5-1.5 m over a 2-3 m distance along the shoreline were made at three equidistantly distributed sites at each sampling occasion (May and June). All tadpoles (Bufo bufo, R. arvalis and R. temporaria are the only anurans occurring at these ponds) and all invertebrate (aeshnid, libellulid and zygoptera larvae, notonectid bugs and dytiscid beetles) and vertebrate taxa (fishes and adult newts, Triturus vulgaris and T. cristatus) considered as potential predators were placed in a white square plastic box and photographed for later counts of predator and tadpole numbers. All animals were subsequently released. The total number of predators and tadpoles over the five sweeps/site was counted from the photographs. The average numbers over the three sampling sites per pond in May and June were taken as predator and competitor abundance in each pond.

Five to 11 pairs of males and females in breeding condition were collected from each of the eight populations (168 individuals in total) in spring 2008 (Table 1) and transported to the laboratory at Uppsala University (59°50'N, 17°50'E). The adults were kept in a climate chamber at +2 to 4°C until artificial crosses were made a few days later. Artificial mating prevented any bias due to differences in exposure in the early environment and assured that the offspring in each family were full sibs. The crosses were performed at +16°C according to 1339 with some modifications. Sperm production was stimulated by injecting males with ca. 2 μg/25 g body mass of the fish hormone LHRH [H-7525, Bache Bioscience Inc., 40]. Females were not treated with hormones, as they had already ovulated. Sperm from males was collected by rinsing their cloacae with sterile physiological salt solution into 0.9 l plastic vials containing 10% Amphibian Ringer solution 41. Sperm motility of each male was checked under a microscope. Eggs from the females were subsequently stripped into the sperm solution 39 and treated using standard procedures 13. The fertilized eggs were divided to the treatments two hours after fertilization but before the first cell cleavage.

The embryonic experiment consisted of five to 11 families/population (Table 1) with three pH treatments (embryos: pH 4.0, 4.3 and 7.5) and three replicates/family/treatment. The larval experiment consisted of four to 11 families/population with two pH treatments (larvae: pH 4.3 and 7.5) and nine replicates/family/treatment. These treatments were chosen as they reflect pH levels at our most extreme sites in the wild and are known to reduce embryonic survival and/or cause sub-lethal effects (reduction in growth and developmental rates) in the larvae 2125. During both experiments, the experimental vials were randomly distributed over three blocks (embryos: one replicate per family and treatment per block, larvae: three replicates per family and treatment per block) according to a known temperature gradient within the room. This design resulted in a total of 631 experimental units for the embryonic and a total of 1079 experimental units for the larval experiment.

Embryos and tadpoles were reared in reconstituted soft water [RSW, 42], as described in Räsänen et al. 21. Untreated RSW was used for the neutral (pH 7.5) treatment. RSW in the acid (pH 4.0 and 4.3) treatment was adjusted with 1 M H₂SO₄ over two days before use. To maintain appropriate pH and water quality, water was changed every three days in the embryonic and every two days in the larval part of the experiment. Prior to each water change, pH was measured in randomly selected experimental vials (pH 4.0 and pH 4.3 treatment: three vials; pH 7.5 treatment: one vial) with a Ross Sure-flow electrode (model 8172BN, Thermo scientific, Inc.) and a Thermo Orion 3 Star pH-meter (model 1112000, Thermo scientific, Inc.). pH drift between the water changes was low during the embryonic experiment (mean pH ± S.D. in treatments: pH 4.0 = 4.03 ± 0.05, pH 4.3 = 4.29 ± 0.08, pH 7.5 = 7.63 ± 0.08). It was also relatively low during the first half of the larval experiment (pH 4.3 = 4.49 ± 0.19, pH 7.5 = 7.43 ± 0.13). During the second half of the larval experiment, pH deviated more strongly due to increasing tadpole biomass and food (pH 4.3 treatment = 4.96 ± 0.17, pH 7.5 treatment = 7.14 ± 0.09). However, the low pH treatment always remained clearly acid (below pH = 5.5) and the two treatments never overlapped.

The experiments were conducted in a walk in climate room (+16°C) with a 17L: 7D photoperiod. During the embryonic experiment, 30-50 embryos per replicate were reared in 0.9 l plastic vials containing 0.5 l treatment water. Embryos were reared from fertilization (day = 0) to day 12, i.e. when the larvae had hatched and reached the free-swimming stage upon leaving the egg capsule [ca. Gosner stage 20; 43]. Survival of embryos was recorded during each water change (see below).

For the larval experiment, a randomly selected subset of tadpoles from each family was selected from the neutral embryonic treatment as soon as they had reached Gosner stage 25 (complete gill absorption and initiation of independent feeding, Gosner 1960), which occurred 12 - 15 days after fertilization. We used larvae from the neutral treatment because of high mortality in the acid treatments, which constrained the availability of healthy larvae and might have biased results due to selective mortality. Carry over effects from embryonic to larval stages have been shown to be small 44, suggesting that hatchlings transferred from pH 7.5 (embryonic neutral treatment) to pH 4.3 (larval acid treatment) are little affected by the change in pH. The tadpoles were randomly assigned to the two pH treatments and reared singly in 0.9 l opaque plastic containers containing 0.7 l treatment water. Tadpoles were fed ad libitum with finely chopped and parboiled spinach every second day. The amount of food was increased with increasing age of the tadpoles. When the tadpoles approached metamorphosis (emergence of at least one front leg; Gosner stage 42) the vials were checked daily.

Embryonic survival was recorded by visual inspection at each water change, but eggs were left untouched and only final survival (day 12) was used in the statistical analyses. Any unfertilized eggs were determined in conjunction to first water change (day 3) and were excluded from the analyses of survival. Embryos were considered dead if they did not hatch (i.e. escape the egg capsule) or were abnormal at hatching (e.g., bent spine, truncated body and oedema), as survival of abnormal hatchlings is very low 45. Embryonic survival was therefore estimated as healthy hatched larvae at day 12/total number of fertilized eggs in each experimental unit.

In the larval experiment, survival was monitored at each water change, but only final survival was used in the statistical analyses. At metamorphosis, mass, larval period and average growth rate was estimated for each individual. Individuals were dry blotted and their wet mass measured with an electronic balance (to the nearest 0.1 mg). Larval period was estimated as the number of days from Gosner stage 25 (day 0 of larval experiment) to metamorphosis. Average daily growth rate (mg/day) was defined as the ratio of mass at metamorphosis/larval period.

Because maternal investment can differ among acid and neutral R. arvalis populations 46, and because egg size/initial size mediated maternal effects can affect larval performance of R. arvalis in a pH dependent way 21, average egg size of each parental female, as well as initial size (size at Gosner stage 25) of each experimental tadpole, was measured from digital images. For egg size, 20-40 eggs/female, and for initial size the individual stage 25 tadpole, were placed in a small petri dish, illuminated from below and photographed with a digital camera (Olympus Camedia C-5060 WideZoom with 5.1 megapixels). For photographing, eggs were covered with water and tadpoles placed in a thin layer of water to avoid dehydration whilst preventing movement. Egg size was measured as area (mm²) and initial size of tadpoles as total length from tip of the nose to tip of the tail (to the nearest 0.01 mm) using the public domain program ImageJ, version 1.39 u http://rsbweb.nih.gov/ij/.

Embryonic survival - Low pH severely reduced embryonic survival, as indicated by a significant pH treatment effect (Table 2, Figure 2A). However, populations differed strongly in their responses to pH, as indicated by a highly significant pond pH × pH treatment interaction (Table 2). Contrast results revealed that embryonic survival and pond pH were strongly negatively correlated at pH 4.0 (Figure 2A; b ± S.E. = -1.86 ± 0.41, P < 0.001), not significantly correlated at pH 4.3 (Figure 2A; b = -0.28 ± 0.40, P = 0.484), and significantly positively correlated at pH 7.5 (Figure 2A; b = 1.07 ± 0.54, P = 0.048) - indicating that populations with highest embryonic survival in the acid treatment had slightly reduced survival in the neutral treatment. Embryonic acid tolerance (survival at pH 4.0) was significantly correlated with latitude (Additional file 4), most probably because of the strong spatial correlation between pond pH and latitude (Additional file 4). Using latitude instead of pond pH for the contrast analysis revealed that embryonic survival and latitude were strongly negatively correlated at pH 4.0 (b ± S.E. = -1.81 ± 0.40, P < 0.001) but not significantly correlated at pH 4.3 (b = -0.13 ± 0.40, P = 0.744) or pH 7.5 (b = 0.64 ± 0.53, P = 0.232).

Significant family and family × pH treatment interactions indicated within population maternal effect/genetic variation in embryonic survival and pH tolerance (Table 2). Egg size differed significantly among populations (F_{7, 63} = 3.07, P = 0.008), but was not significantly correlated with pond pH (log b = -0.05 ± 0.04, P = 0.206). There was no significant egg size or pH treatment × egg size interaction in embryonic survival, and its inclusion as a covariate did not qualitatively change the results (not shown). This indicates that family effects in embryonic survival are independent of egg size.

Larval traits - Across all populations and treatments, larval survival was generally very high (ranging from 92.3% in Sätila to 98.8% in Lomsjö at pH 7.5, and from 84.2% in Sätila to 100% in Bergsjö and Rud at pH 4.3). There were no significant pond pH, pH treatment or their interaction effects on larval survival (all P > 0.40) and the data were therefore not further analyzed.

Metamorphic mass and growth rate decreased, and larval period increased at pH 4.3 compared to pH 7.5 (Figure 2B, C and 2D), as indicated by significant pH treatment main effects (Table 3). This indicates sub-lethal acid stress effects on tadpoles. Furthermore, populations have diverged in all three larval traits, as indicated by the significant pond pH main effects (Table 3). Significant linear contrasts further showed that metamorphic mass, larval period and growth rate increased with decreasing breeding pond pH (Table 3, Figure 2B, C and 2D). There was no significant pond pH × pH treatment effect on metamorphic mass or growth rate (Table 3 and 3). In contrast, larval period was more negatively affected by the acid treatment in neutral than in acid origin populations (ca. 2.5-3.5 days vs. 0-2 days delay, respectively; Figure 2C), giving rise to a significant pond pH × pH treatment interaction (Table 3). This suggests that acid origin populations have higher larval acid stress tolerance. The pH treatment dependent phenotypic divergence was also reflected in the relationship between pond pH and larval period: the correlation was stronger at pH 7.5 (log b ± S.E. = -0.09 ± 0.01, P < 0.001) than at pH 4.3 (log b ± S.E. = -0.06 ± 0.01, P < 0.001; Figure 2C).

Significant family main effects indicated within population maternal effect/genetic variation in all three larval traits (Table 3; see discussion for the role of genetic and maternal effects in these populations). In addition, a significant pH treatment × family effect in larval period suggested within population maternal effect/genetic variation in pH tolerance for this trait. Populations (F_7,54 = 2.23, P = 0.046) and families within populations (Z = 5.09, P < 0.001) differed significantly in initial size, but initial size was not significantly correlated with pond pH (log b ± S.E = -0.01 ± 0.02, P = 0.426). When initial size was included in the models of larval life-history trait variation as a covariate, it was significantly positively related to metamorphic mass (Additional file 5; log b ± S.E. = 0.27 ± 0.09, F = 8.19, P = 0.004) and growth rate (Additional file 5; log b = 0.42 ± 0.10, F = 17.80, P < 0.001), and significantly negatively related to larval period (Additional file 5; log b = -0.15 ± 0.06, F = 7.36, P = 0.007). These results indicate that initially large larvae grew and developed faster, but these effects were independent of pH treatment (non-significant initial size × pH treatment interaction). Moreover, the results remained qualitatively unchanged when initial size was included as a covariate (Additional file 5), indicating that family or population level responses were independent of initial size.

The analyses comparing effects of all single habitat variables (Table 4) or habitat indices (Additional file 3) found that the main correlates of phenotypic divergence were pH, latitude and predator density. In both treatments, metamorphic mass was most strongly, and negatively, related to pond pH (Table 4), and negatively, but more weakly, to latitude (Table 4). The pattern was different for larval period: larval period was most strongly, and negatively, related to latitude in both treatments (Table 4), and negatively, but more weakly, related to pond pH, and only in the high pH treatment (Table 4). These effects suggest that acidity may select for an increase in metamorphic size and climatic selection for a shorter larval period in the north. In contrast, larval growth was most strongly (positively) related to one of the habitat indices (i.e. habitat2, Additional file 3) and to predator density, and marginally (positively) to pond pH (Table 4). However, these relationships were apparent only at pH 4.3. These results suggest that predators, possibly together with canopy cover and/or pond pH, may be the most important selective factors behind divergence in growth rate.

Adaptive divergence along environmental gradients has been mainly studied in the context of adaptation to metal pollution [e.g., 1051] and climate [e.g., 5253], whereas studies along other stress gradients are rare. Our study adds to these studies, and provides further evidence for adaptation to acidification (zooplankton [e.g., 19], fish [e.g., 20] and amphibians [reviewed in 17].

Local adaptation is expected to arise due to fitness trade-offs among contrasting environments 54. In this vein, the increased embryonic survival of acid origin populations under acid conditions is clearly adaptive. In contrast, we found only marginal evidence for a trade-off between this increased acid tolerance and performance at neutral pH (populations with highest embryonic survival in the acid treatment had slightly reduced survival in the neutral treatment). Field transplant experiments suggest that the patterns for embryonic survival observed here under simple laboratory conditions also hold in the wild 5055. The reasons for this "asymmetric" adaptation are not clear, but could arise if fitness trade-offs become apparent only at extreme conditions (neutral conditions are generally benign) or in other contexts or traits [e.g., 356]. Although the fitness consequences of the observed divergence in larval traits are less clear, large metamorphic size and short larval period are generally assumed to have a positive effect on fitness in amphibians [e.g., 22]. Larval traits show evidence for trade-offs along the acidification gradient (i.e. acid origin populations were large but developed slower, whereas neutral origin populations were smaller but developed faster). Selection seems to favor different local optima along this acidification gradient, which suggests the potential for local adaptation in this system. Although more detailed studies and direct fitness estimates are needed to confirm local adaptation, the patterns observed here for embryonic and larval traits are strongly indicative of adaptive divergence and emphasize the importance of early life-history stages for adaptation [e.g., 111357].

In order for trait divergence to be an adaptation to divergent selection, it is necessary to confirm a heritable basis to the phenotypic traits 58. We here used full-sib crosses on wild caught adults and reared embryos and larvae under laboratory common garden conditions, which does not allow to infer the genetic basis. However, several lines of evidence suggest that the observed patterns indeed reflect adaptive divergence. First, quantitative genetic crosses indicate that variation in embryonic acid tolerance is driven by maternal effects 25265059, which are clearly adaptive (embryos of acid origin mothers have higher acid tolerance). Whether these maternal effects have a genetic rather than environmental basis is work in progress. Second, divergence in larval traits is primarily due to direct genetic effects 59. Reciprocal crosses between three population pairs 59, and within-population breeding experiments with half-sib (NCII) designs in a subset of four of the present populations (K. Räsänen and A. Laurila, unpubl. data), find evidence for direct genetic effects, negligible maternal effects and moderate to high heritability (h² up to 0.6) in larval traits. Furthermore, when we statistically controlled for initial larval size - indicative of maternal effects - the among population differences in trait divergence were maintained (Additional file 5). Third, correlations between the extent of phenotypic and environmental differences suggest that variation in trait divergence might reflect variation in the strength of divergent selection [e.g., 30]. Q_ST-F_ST comparisons along this gradient 60 further indicate that the patterns of phenotypic divergence observed here are most likely a result of divergent natural selection rather than neutral processes, such as drift and gene flow 61. We next turn to the discussion of the putative selective pressures along this acidification gradient.

Our phenotype-environment correlations, using several different abiotic and biotic environmental variables, found that trait divergence most strongly correlated with pond pH, latitude and predator density. Although we found a correlation between latitude and embryonic acid tolerance, we have no reason to expect increased selection or environmental influence for higher embryonic acid tolerance at lower latitudes. Moreover, divergence in embryonic acid tolerance is also seen between populations at similar latitudes 13. Instead, we argue that this correlation most likely arose due to spatial autocorrelation between acidity and latitude, and that divergence in embryonic acid tolerance is most obviously driven by pond pH. This is also supported by the parallel patterns between laboratory and transplant experiments 5055.

Complex life-history traits - such as larval growth and development - are likely under simultaneous selection from different sources 62. In this vein, it is not surprising that our phenotype-environment correlations found that divergence in metamorphic mass is most closely related to pond pH, whereas divergence in larval period is most closely related to latitude, and larval growth to predator density, respectively.

Stressful (here acid) environments may also cause various forms of selection on stress tolerance and/or trait means [e.g., 356], such as organismal growth and development rates [e.g., 663]. Several hypotheses relating to larval life-history trait divergence on the acid-neutral axis were discussed in detail in 21 and we do not repeat them all here. For metamorphic mass, we suggest that large size may be more strongly selected for in acid environments to counteract the negative effects of acidic pH. Large size might be selected for due to increased fitness of large juveniles or adults during the terrestrial phase (e.g. either increased overwintering survival or reproductive success [e.g., 2122]).

For larval period, we found that development rate increased at higher latitudes, which is in accordance with countergradient selection 63. However, in contrast to the commonly observed pattern in other Scandinavian Rana populations (e.g. 46, 47), we found no significant relationship between latitude and growth. We propose that in our study system, seasonal time constraints in the north favor faster development, whereas selection by predators (possibly in combination with canopy cover and/or pond pH) may drive divergence in growth rates. However, the relative contribution of climatic (latitude) and pH mediated selection on larval development is difficult to disentangle here as acidity and latitude were closely spatially correlated. Interestingly, populations with higher predator densities had higher growth rates, particularly in the low pH treatment. This could indicate selection for higher growth rates through gape limited predators [e.g., 64], such as libellullid dragonflies, which are one of the most common predators of R. arvalis tadpoles in acidic ponds [65, this study]. To shed light on this hypothesis, direct tests of predator mediated effects on larval performance are under way in our laboratory 59.

A few additional points need to be considered. First, our study provides correlative evidence for different selective agents acting on different fitness traits, and that different larval traits respond in part independently. As larval life-history traits can be phenotypically and genetically highly correlated [e.g., 66], it would be interesting to study to what extent the different traits indeed respond independently to the different selective agents along environmental stress gradients 67. Second, although we find strong correlations between some of our environmental variables and the extent of phenotypic divergence among local populations, there clearly is potential for several other factors to influence the extent of phenotypic divergence. These include variation in the extent of gene flow [e.g., 3068], the amount and type of genetic variation [e.g., 69], as well genetic [e.g., 70] and ecological trade-offs [e.g., 71] among interacting selective forces. Our study was not designed to directly test for effects of individual habitat variables or other constraining factors to adaptation to acidity. Additional studies are therefore needed to better understand the relative importance of different selective or constraining factors to adaptive divergence in this system.