BACKGROUND
stressful environments  can impose strong ecological and evolutionary pressures at a local level
<cit> .
population persistence depends on the ability of individuals to respond to environmental stress through adaptive, plastic or behavioural mechanisms that maximise fitness
<cit> .
extremes of ph , water availability , and temperature  have been observed to drive adaptive population divergence.
high-latitudes and altitudes experience low temperatures that can result in shorter activity periods and longer periods of freezing
<cit> .
plastic and adaptive responses to low temperature environments have been widely recorded  and can result in cryptic divergence between populations inhabiting different temperature regimes .
temperature is often the major abiotic factor that influences physiological mechanisms in ectotherms
<cit>  and growth slows in response to cold environments
<cit> .
reduced activity periods in low-temperature environments, in combination with low-temperature driven growth-rate reductions, can result in lower sizes at important life-history events such as metamorphosis and reproduction
<cit> .
smaller sizes can translate to lower fitness when weight is positively correlated with survival or reproductive success
<cit> .
assessing the mechanisms that facilitate survival in challenging environments is important for understanding how populations respond to ecological and evolutionary pressures, particularly in a globally changing climate
<cit> .
potential responses to maximise size at important life history stages in low-temperature environments include altering metabolic rate , developmental period , or temperature activity range .
populations that inhabit high-altitude environments experience lower temperatures and shorter activity periods than their low-altitude neighbours and offer an excellent opportunity to assess how survival is facilitated in environments where growth is constrained
<cit> .
amphibians are a particularly good model for studying physiological and behavioural responses to growth constraints, as size at metamorphosis is positively correlated with survival in the subsequent terrestrial life-history stages
<cit> .
variation in metabolic rates between individuals is a common occurrence in nature
<cit> , but the effects on fitness are still relatively unknown
<cit> .
resting metabolic rate, here defined as the energetic cost of self-maintenance
<cit> , has been linked to multiple physiological and behavioural traits including predator avoidance, foraging behaviour, swimming performance and growth
<cit> .
growth imposes a significant physiological cost and can result in a trade-off with other physiological mechanisms
<cit> , especially when resources are limited
<cit> .
as the vast majority of energy expenditure in ectotherms is maintenance costs , small differences in resting metabolic rate can result in large differences in energy available for growth
<cit> .
an increased growth rate can result in a larger size at important life history events and has been linked to a reduced resting metabolic rate in sagebrush lizards at high-altitude
<cit> , sydney rock oysters from growth rate-selected stock
<cit>  and snapping turtles
<cit> .
attempts to assess the physiological trade-offs facilitating higher growth rates in larval common frog  at high latitudes have found no link to reduced metabolic rates
<cit> .
however, as temperature is not linearly related to latitude in sweden
<cit> , where these experiments were conducted, these results may mask the true nature of the temperature-metabolic rate relationship.
therefore, further research in a system with a linear temperature change is required to elucidate the relationship between resting metabolic rate, growth rate and temperature.
another potential response to maximise size at important life-history events, is to increase the time available for growth prior to metamorphosis or reproduction by extending development over multiple growth periods
<cit> .
the concept of delayed development, or diapause, has been commonly observed in insects, often in terms of cohort splitting where different cohorts within a population complete development at different times of the year, or even in different years
<cit> .
in amphibians, the period immediately prior to metamorphic climax is accompanied by a loss of weight
<cit> , but a lower weight decreases the chances of adult survival
<cit> .
therefore, overwintering at a higher weight, but still at the larval stage, and metamorphosing the following year has the potential to increase survival, and has been recorded in a number of temperate amphibian species
<cit> .
however, low winter temperatures at high-altitude can lead to prolonged periods of freezing
<cit> .
therefore, in order to survive, overwintering larval amphibians must be able to respond to freezing temperatures via freeze avoidance  or freeze tolerance .
freeze tolerance depends on the ability to restrict ice formation to extra-cellular areas, which is mediated by accumulation of low molecular weight carbohydrates in the blood
<cit> .
the ability to tolerate freezing has been linked to glucose accumulation in the blood, via release of liver glycogen, in the frogs r. sylvatica, r. lessonae and r. esculenta <cit> , and with glycerol accumulation in hyla versicolor <cit> .
however, all previous studies have focussed on freeze tolerance in adult amphibians and the potential for freeze survival in the larval stage has, to the best of our knowledge, never been studied.
the ability of a tadpole to survive freezing would extend the time available for growth to, and thus size at, metamorphosis in amphibians breeding in temperate climates.
a third alternative response to larval growth constraints would be for adults to adapt behaviourally rather than amphibian larvae adapting physiologically.
adults have the potential to expand the growing season for larvae by breeding earlier in the year
<cit> .
in temperate amphibians, breeding is closely linked to temperature
<cit>  and frequently occurs immediately after winter dormancy .
by adults becoming active and breeding at lower temperatures, larvae would have longer to grow and develop prior to winter dormancy.
the longer time available for growth would allow larvae to reach a larger size at metamorphosis and thus have an increased chance of survival as adults
<cit> .
the common frog  is the most widespread amphibian in europe and occurs from zero to  <dig> metres above sea level within its range, and to over a thousand metres on the mountains of scotland
<cit> .
it is an explosive breeder, with communal spawning taking place immediately after winter dormancy
<cit> ; a 5°c temperature threshold is generally considered to initiate activity and spawning
<cit> .
r. temporaria larvae show increased growth rates in response to low temperatures experienced at high-latitudes and altitudes throughout its range
<cit> .
we have previously shown that local adaptation to high-altitude environments occurs even in the face of high gene flow, suggesting that temperature exerts a strong selective pressure
<cit> .
however, there are also reports of r. temporaria overwintering as tadpoles in scotland, although it is currently unclear whether this response is particularly linked to low-temperature environments
<cit> .
the mountains of scotland offer an excellent opportunity to study the responses that facilitate survival in low-temperature environments, as there is continuous habitat along altitudinal gradients, with temperature decreasing linearly by  <dig> °c for every 100 m gain in altitude
<cit> .
individuals from high-altitude sites in scotland experience substantially lower temperatures than their low-altitude counterparts, with an average mean annual temperature reduction of  <dig> °c at high- compared to low-altitude breeding sites
<cit> .
the overall aim of this study was to assess the physiological and behavioural responses of common frogs in scotland that facilitate survival in low-temperature environments.
in particular, this study answers the questions: 1) do high-altitude adults extend the time available for larval growth by breeding at lower temperatures than low-altitude individuals?
; and 2) do tadpoles sampled from high-altitude sites differ physiologically from those from low-altitude sites, in terms of routine metabolic rate and freeze tolerance?
RESULTS
adult spawning behaviour in relation to altitude
temperature on the day of egg mass observation was, on average,  <dig>  ±  <dig> °c  and did not vary predictably with altitude .
likewise, no significant regression with altitude was found for the average temperature in the week prior to egg mass observation .
degree days prior to egg mass collection was highly variable across sites  but also did not show a significant relationship with altitude .
the date of egg mass collection was on average 30 days later at high- compared to low-altitude sites  and julian spawning day showed a significant positive relationship with altitude : individuals spawned 5 days later for every 100 m gain in altitude .
the daily mean temperature at all sites had exceeded the threshold value of 5°c in the week prior to egg mass collection .
observation day temp and week prior temp are mean values and are accompanied by their standard deviation.
*data not available due to logger failure.
larval physiology in relation to altitude
routine metabolic rate
between eight and  <dig> individuals per site were measured for rmr, due to varying levels of mortality .
mean rmr per site varied between  <dig>  ml o <dig> g-1 h- <dig>  and  <dig>  ml o <dig> g-1 h- <dig> , with an overall average of  <dig>  ±  <dig>  ml o <dig> g-1 h- <dig> .
mountain, altitude, and their interaction were found to be significant in predicting rmr.
a tukey’s hsd test showed a significant difference between high- and low-altitude rmr in individuals from three of the mountains: dub , mnt  and lom  .
the difference between high- and low-altitude rmr was not significant for ime  and law .
the direction of the relationship varied between mountains, with individuals from dub, law and mnt showing a trend for lower rmr at high- compared to low-altitude, whereas individuals from ime and lom had higher rmr at high-altitude .
the post hoc power analysis of the anova used in the tukey’s hsd revealed an effect size of  <dig>  giving an achieved power of  <dig>  to determine a significant difference between the means of rmr by altitude within each mountain.
the power to confidently conclude that no significant interactions have been missed , is generally set at  <dig>
<cit> .
shown are the number of individuals per site measured , the mean routine metabolic rate  and the proportion of survivors following freezing .
standard deviations are indicated for rmr, whereas freeze survival is shown as a single measurement per site.
the results of the tukey’s hsd test of significant difference between the means of rmr of individuals from low- and high-altitude sites, by mountain are shown .
a positive difference between the means  shows that individuals from high-altitude have a lower mean rmr than those from low-altitude, and a negative difference between the means shows that individuals from low-altitude have a lower rmr than those from high-altitude.
*significant at p <  <dig> .
na: no freeze tolerance results are available for dubhigh due to complete tadpole mortality prior to the freeze tolerance experiment.
freeze tolerance
ten individuals per site were tested for freeze tolerance, except for lomhigh  and dublow  , due to variable tadpole mortality prior to the experiment.
between 10%  and 80%  mortality was observed post-freezing across sites .
out of  <dig> tadpoles tested for freeze survival from low-altitude sites,  <dig> survived , compared with 12/ <dig>  tadpoles from high-altitude sites .
altitude and weight, but not their interaction, significantly changed the log likelihood when removed from the glmm and were thus included in the final model.
there was no significant effect on the log likelihood of the model when mountain was removed from the model and thus mountain was not included in the final model .
the results of the glmm using the final model  showed that individuals from low-altitude sites had significantly higher survival than those from high-altitude sites .
discussion
adult spawning behaviour in relation to altitude
no significant relationship was found between spawning date and either the temperatures recorded on the day of, or in the week prior to, egg mass observation that would have suggested that breeding occurs at lower temperatures at high altitude sites.
degree days also did not show a significant relationship with altitude but they were highly variable across sites , suggesting that degree days are not an accurate predictor of spawning activity in r. temporaria.
all sites had exceeded the 5°c temperature threshold generally thought to initiate activity and breeding in r. temporaria <cit>  in the week prior to spawning .
our results therefore support 5°c as the activity threshold for r. temporaria regardless of altitude of breeding site, and demonstrate that high-altitude individuals experience a longer period of low-temperatures and delayed spawning compared to low-altitude individuals.
the date of spawning was 5 days later for every 100 m gain in altitude .
as mean annual temperature decreases by  <dig> °c for every 100 m increase in altitude in this system
<cit> , we can infer that spawning is on average one day earlier for every  <dig> °c increase in mean annual temperature.
however, further information regarding the date of spawning between 223 m and 720 m  is needed to confirm this pattern.
it is interesting to note that for the mountains with the highest high-altitude sites , the average time difference between spawning at low-and high-altitude is only 24 days, compared with 38 days observed difference between spawning at low- and high-altitude in ime and lom .
this could suggest that high-altitude individuals at the highest breeding sites are breeding at a lower temperature.
however, this is not reflected as a significant difference between high- and low-altitude spawning temperatures in these mountains .
the longer period of low temperatures at high- compared to low-altitudes prior to spawning supports the hypothesis that high-altitude individuals experience a shorter annual activity period but does not support the hypothesis that breeding occurs at lower temperatures at high altitudes to provide a longer developmental period, facilitating survival at high-altitude within r. temporaria.
breeding at a lower temperature would allow individuals to spawn earlier in the year, thus providing their offspring with a longer period in which to develop prior to metamorphosis
<cit> , such as has been found for amphibian species at higher latitudes compared to their lower latitude counterparts
<cit>  but, to the best of our knowledge, has not yet been observed within-species.
however, phenological studies that have quantified within-species breeding temperature have typically used local weather station data
<cit>  and so the spatial scale might not be not fine enough to represent local conditions in mountain areas, where temperatures can vary rapidly over short geographical distances
<cit> .
therefore, such studies are liable to miss within-species differences in spawning temperature in relation to altitude.
when we quantified temperature at a local level, our results support the absence of a shift in breeding temperature in low-temperature environments, contributing to knowledge of the fundamental niche of r. temporaria.
however, we did find that r. temporaria can substantially alter the date they breed in response to the environmental conditions experienced, even between geographically close breeding sites, demonstrating a high degree of plasticity in terms of breeding timing.
larval physiology in relation to altitude
routine metabolic rate
there was a significant interaction between mountain and altitude, which complicated interpretation of rmr.
posthoc tests indicated no significant differences in rmr between tadpoles sampled from high and low sites on ime or law.
in regards to the other three mountains, there were significant differences but not always in the same direction: for dub and mnt tadpoles sampled from high elevation showed a decreased rmr relative to those from low elevation, but the largest difference due to elevation was at lom, where tadpoles from high elevation showed a substantial increase in rmr compared to those from low elevation .
however, due to the moderate power of the model , it is possible that some significant relationships were missed.
it has been suggested that a lower resting metabolic rate can allow more energy to be allocated to growth in resource-limited environments
<cit>  and a link between lower rmr and increased growth rate has been found in the southern toad
<cit> , sydney rock oyster
<cit>  and snapping turtle
<cit> .
furthermore, sears
<cit>  found that both increased growth rates and reduced rmr were positively correlated with altitude in sagebrush lizards.
the three mountains in our study system where rmr was lower in individuals from high-altitudes  have been shown to be locally adapted to temperature parameters, with larval period decreasing and growth rate increasing at high-altitude
<cit> .
therefore, the lower rmr of individuals from high-altitude from dub, mnt and law, is in line with the increased growth rates observed at these sites.
lindgren and laurila
<cit>  did not find a link between growth rates and rmr in r. temporaria along a latitudinal gradient in sweden.
therefore, this is the first tentative evidence of reduced rmr being linked to increased growth rate as an adaptation to low-temperatures in an amphibian.
however, the significantly higher rmr at high- than low-altitude in lom , and the non-significant difference between lawhigh and lawlow in terms of rmr, could suggest that local conditions other than altitude are also important in driving divergence in rmr.
for instance, the lowest rmr were observed in individuals from imelow and lomlow, where spawning occurred at the lowest temperatures of all the sites , and this could suggest that low rmr is beneficial in tolerating low temperatures at all altitudes, particularly early in the season when temperatures can fall after spawning.
it has also been speculated that increased growth rate can be a result of increased time spent foraging, facilitated by lower predator presence in low-temperature environments, and is unrelated to rmr
<cit> .
furthermore, rmr is influenced by temperature and there was up to a 3°c difference in temperature whilst measuring rmr of individuals from different sites, as well as a difference between the temperature at which the tadpoles were raised  and the temperature at which rmr was measured .
therefore, further research is needed to assess whether site-specific differences in rmr remain constant through time and whether other mountains with breeding sites of above 900 m also show reduced metabolic rate at high- vs. low-altitude.
freeze tolerance
just over half of the tadpoles that were frozen survived  and altitude was significant in predicting freeze survival: individuals sampled from low-altitude survived freezing significantly better than those from high-altitude .
voituron et al.
<cit>  suggested that r. temporaria adults were freeze intolerant, as 100% mortality was observed after eight hours of complete bodily fluid freezing.
however, pasanen and karhapää
<cit>  found that r. temporaria adults could survive 24 hours in a sub-zero environment but died within three days .
our results suggest that tadpoles of r. temporaria are also capable of surviving short periods of freezing.
this is the first time, to the best of our knowledge, that larval freeze tolerance has been demonstrated in any amphibian.
although the results presented here suggest that tadpoles are capable of surviving being frozen, the finding of a greater survival of low- compared to high-altitude individuals appears counterintuitive, given the longer period of sub-zero temperatures at high- than low-altitude in scotland
<cit>  and the sub-zero average winter temperature of - <dig> °c at high-altitude, compared to  <dig> °c at low-altitude at the breeding sites used in this study
<cit> .
indeed an increase in freeze tolerance with altitude has been found in plants  and insects
<cit> , but freeze tolerance and altitude has not been explicitly linked in herptiles.
however, evolution of freeze tolerance in frogs, lizards and turtles has been linked to ecological pressures relating to winter temperatures experienced
<cit> .
in general, freeze tolerant species are those that terrestrially overwinter in sub-zero temperatures, as opposed to avoiding freezing in deep water bodies
<cit> .
therefore, a higher freeze tolerance would suggest that low-altitude tadpoles are more often exposed to freezing temperatures, whereas high-altitude tadpoles may avoid such exposure altogether by inhabiting deep water bodies or metamorphosing prior to winter.
formation of deep water pools is inhibited at high-altitudes in scotland due to the rocky, exposed landscape
<cit> .
therefore, freeze exposure is more likely avoided in high-altitude tadpoles by metamorphosing within a single active season, facilitated by a faster growth rate in conjunction with a lower rmr .
therefore, our results potentially suggest that of the tadpoles found overwintering in scotland
<cit> , low-altitude individuals are more likely to overwinter as tadpoles than high-altitude individuals.
however, it is possible that long periods of snow cover at high-altitude
<cit>  could insulate tadpoles from freezing temperatures even in shallow water bodies.
therefore, further field research is needed to assess whether fewer, if any, high- than low-altitude individuals overwinter as tadpoles.
CONCLUSIONS
r. temporaria adults do not show behavioural adaptations in terms of breeding at lower temperatures, instead they delay spawning based on the temperature experienced.
however, r. temporaria larvae appear to have the potential to physiologically adapt to low-temperature environments, although these relationships, and the results we obtained for routine metabolic rate and freeze tolerance, are not always straightforward to interpret.
therefore, our results suggest that survival at high-altitude may be facilitated by physiological mechanisms that permit faster growth rates, allowing completion of larval development within a shorter period of time, alleviating the need for adaptations that extend the time available for larval growth.
how individuals respond to environmental temperature at a local level is an important step in relating ecological and evolutionary pressures to phenotypes.

 stressful environments can impose strong ecological and evolutionary pressures at a local level. population persistence depends on the ability of individuals to respond to environmental stress through adaptive, plastic or behavioural mechanisms that maximise fitness. extremes of ph, water availability, and temperature have been observed to drive adaptive population divergence. high-latitudes and altitudes experience low temperatures that can result in shorter activity periods and longer periods of freezing. plastic and adaptive responses to low temperature environments have been widely recorded and can result in cryptic divergence between populations inhabiting different temperature regimes. temperature is often the major abiotic factor that influences physiological mechanisms in ectotherms and growth slows in response to cold environments. reduced activity periods in low-temperature environments, in combination with low-temperature driven growth-rate reductions, can result in lower sizes at important life-history events such as metamorphosis and reproduction. smaller sizes can translate to lower fitness when weight is positively correlated with survival or reproductive success. assessing the mechanisms that facilitate survival in challenging environments is important for understanding how populations respond to ecological and evolutionary pressures, particularly in a globally changing climate. potential responses to maximise size at important life history stages in low-temperature environments include altering metabolic rate, developmental period, or temperature activity range. populations that inhabit high-altitude environments experience lower temperatures and shorter activity periods than their low-altitude neighbours and offer an excellent opportunity to assess how survival is facilitated in environments where growth is constrained. amphibians are a particularly good model for studying physiological and behavioural responses to growth constraints, as size at metamorphosis is positively correlated with survival in the subsequent terrestrial life-history stages. variation in metabolic rates between individuals is a common occurrence in nature, but the effects on fitness are still relatively unknown. resting metabolic rate, here defined as the energetic cost of self-maintenance, has been linked to multiple physiological and behavioural traits including predator avoidance, foraging behaviour, swimming performance and growth. growth imposes a significant physiological cost and can result in a trade-off with other physiological mechanisms, especially when resources are limited. as the vast majority of energy expenditure in ectotherms is maintenance costs, small differences in resting metabolic rate can result in large differences in energy available for growth. an increased growth rate can result in a larger size at important life history events and has been linked to a reduced resting metabolic rate in sagebrush lizards at high-altitude, sydney rock oysters from growth rate-selected stock and snapping turtles. attempts to assess the physiological trade-offs facilitating higher growth rates in larval common frog at high latitudes have found no link to reduced metabolic rates. however, as temperature is not linearly related to latitude in sweden, where these experiments were conducted, these results may mask the true nature of the temperature-metabolic rate relationship. therefore, further research in a system with a linear temperature change is required to elucidate the relationship between resting metabolic rate, growth rate and temperature. another potential response to maximise size at important life-history events, is to increase the time available for growth prior to metamorphosis or reproduction by extending development over multiple growth periods. the concept of delayed development, or diapause, has been commonly observed in insects, often in terms of cohort splitting where different cohorts within a population complete development at different times of the year, or even in different years. in amphibians, the period immediately prior to metamorphic climax is accompanied by a loss of weight, but a lower weight decreases the chances of adult survival. therefore, overwintering at a higher weight, but still at the larval stage, and metamorphosing the following year has the potential to increase survival, and has been recorded in a number of temperate amphibian species. however, low winter temperatures at high-altitude can lead to prolonged periods of freezing. therefore, in order to survive, overwintering larval amphibians must be able to respond to freezing temperatures via freeze avoidance or freeze tolerance. freeze tolerance depends on the ability to restrict ice formation to extra-cellular areas, which is mediated by accumulation of low molecular weight carbohydrates in the blood. the ability to tolerate freezing has been linked to glucose accumulation in the blood, via release of liver glycogen, in the frogs r. sylvatica, r. lessonae and r. esculenta, and with glycerol accumulation in hyla versicolor. however, all previous studies have focussed on freeze tolerance in adult amphibians and the potential for freeze survival in the larval stage has, to the best of our knowledge, never been studied. the ability of a tadpole to survive freezing would extend the time available for growth to, and thus size at, metamorphosis in amphibians breeding in temperate climates. a third alternative response to larval growth constraints would be for adults to adapt behaviourally rather than amphibian larvae adapting physiologically. adults have the potential to expand the growing season for larvae by breeding earlier in the year. in temperate amphibians, breeding is closely linked to temperature and frequently occurs immediately after winter dormancy. by adults becoming active and breeding at lower temperatures, larvae would have longer to grow and develop prior to winter dormancy. the longer time available for growth would allow larvae to reach a larger size at metamorphosis and thus have an increased chance of survival as adults. the common frog is the most widespread amphibian in europe and occurs from zero to metres above sea level within its range, and to over a thousand metres on the mountains of scotland. it is an explosive breeder, with communal spawning taking place immediately after winter dormancy; a 5°c temperature threshold is generally considered to initiate activity and spawning. r. temporaria larvae show increased growth rates in response to low temperatures experienced at high-latitudes and altitudes throughout its range. we have previously shown that local adaptation to high-altitude environments occurs even in the face of high gene flow, suggesting that temperature exerts a strong selective pressure. however, there are also reports of r. temporaria overwintering as tadpoles in scotland, although it is currently unclear whether this response is particularly linked to low-temperature environments. the mountains of scotland offer an excellent opportunity to study the responses that facilitate survival in low-temperature environments, as there is continuous habitat along altitudinal gradients, with temperature decreasing linearly by °c for every 100 m gain in altitude. individuals from high-altitude sites in scotland experience substantially lower temperatures than their low-altitude counterparts, with an average mean annual temperature reduction of °c at high- compared to low-altitude breeding sites. the overall aim of this study was to assess the physiological and behavioural responses of common frogs in scotland that facilitate survival in low-temperature environments. in particular, this study answers the questions: 1) do high-altitude adults extend the time available for larval growth by breeding at lower temperatures than low-altitude individuals?; and 2) do tadpoles sampled from high-altitude sites differ physiologically from those from low-altitude sites, in terms of routine metabolic rate and freeze tolerance?  adult spawning behaviour in relation to altitude temperature on the day of egg mass observation was, on average,  ±  °c and did not vary predictably with altitude. likewise, no significant regression with altitude was found for the average temperature in the week prior to egg mass observation. degree days prior to egg mass collection was highly variable across sites but also did not show a significant relationship with altitude. the date of egg mass collection was on average 30 days later at high- compared to low-altitude sites and julian spawning day showed a significant positive relationship with altitude: individuals spawned 5 days later for every 100 m gain in altitude. the daily mean temperature at all sites had exceeded the threshold value of 5°c in the week prior to egg mass collection. observation day temp and week prior temp are mean values and are accompanied by their standard deviation. *data not available due to logger failure. larval physiology in relation to altitude routine metabolic rate between eight and individuals per site were measured for rmr, due to varying levels of mortality. mean rmr per site varied between  ml o g-1 h- and  ml o g-1 h-, with an overall average of  ±   ml o g-1 h-. mountain, altitude, and their interaction were found to be significant in predicting rmr. a tukey’s hsd test showed a significant difference between high- and low-altitude rmr in individuals from three of the mountains: dub, mnt and lom. the difference between high- and low-altitude rmr was not significant for ime and law. the direction of the relationship varied between mountains, with individuals from dub, law and mnt showing a trend for lower rmr at high- compared to low-altitude, whereas individuals from ime and lom had higher rmr at high-altitude. the post hoc power analysis of the anova used in the tukey’s hsd revealed an effect size of giving an achieved power of to determine a significant difference between the means of rmr by altitude within each mountain. the power to confidently conclude that no significant interactions have been missed, is generally set at. shown are the number of individuals per site measured, the mean routine metabolic rate and the proportion of survivors following freezing. standard deviations are indicated for rmr, whereas freeze survival is shown as a single measurement per site. the results of the tukey’s hsd test of significant difference between the means of rmr of individuals from low- and high-altitude sites, by mountain are shown. a positive difference between the means shows that individuals from high-altitude have a lower mean rmr than those from low-altitude, and a negative difference between the means shows that individuals from low-altitude have a lower rmr than those from high-altitude. *significant at p < . na: no freeze tolerance results are available for dubhigh due to complete tadpole mortality prior to the freeze tolerance experiment. freeze tolerance ten individuals per site were tested for freeze tolerance, except for lomhigh and dublow, due to variable tadpole mortality prior to the experiment. between 10% and 80% mortality was observed post-freezing across sites. out of tadpoles tested for freeze survival from low-altitude sites, survived, compared with 12/ tadpoles from high-altitude sites. altitude and weight, but not their interaction, significantly changed the log likelihood when removed from the glmm and were thus included in the final model. there was no significant effect on the log likelihood of the model when mountain was removed from the model and thus mountain was not included in the final model. the results of the glmm using the final model showed that individuals from low-altitude sites had significantly higher survival than those from high-altitude sites. discussion adult spawning behaviour in relation to altitude no significant relationship was found between spawning date and either the temperatures recorded on the day of, or in the week prior to, egg mass observation that would have suggested that breeding occurs at lower temperatures at high altitude sites. degree days also did not show a significant relationship with altitude but they were highly variable across sites, suggesting that degree days are not an accurate predictor of spawning activity in r. temporaria. all sites had exceeded the 5°c temperature threshold generally thought to initiate activity and breeding in r. temporaria in the week prior to spawning. our results therefore support 5°c as the activity threshold for r. temporaria regardless of altitude of breeding site, and demonstrate that high-altitude individuals experience a longer period of low-temperatures and delayed spawning compared to low-altitude individuals. the date of spawning was 5 days later for every 100 m gain in altitude. as mean annual temperature decreases by °c for every 100 m increase in altitude in this system, we can infer that spawning is on average one day earlier for every °c increase in mean annual temperature. however, further information regarding the date of spawning between 223 m and 720 m is needed to confirm this pattern. it is interesting to note that for the mountains with the highest high-altitude sites, the average time difference between spawning at low-and high-altitude is only 24 days, compared with 38 days observed difference between spawning at low- and high-altitude in ime and lom. this could suggest that high-altitude individuals at the highest breeding sites are breeding at a lower temperature. however, this is not reflected as a significant difference between high- and low-altitude spawning temperatures in these mountains. the longer period of low temperatures at high- compared to low-altitudes prior to spawning supports the hypothesis that high-altitude individuals experience a shorter annual activity period but does not support the hypothesis that breeding occurs at lower temperatures at high altitudes to provide a longer developmental period, facilitating survival at high-altitude within r. temporaria. breeding at a lower temperature would allow individuals to spawn earlier in the year, thus providing their offspring with a longer period in which to develop prior to metamorphosis, such as has been found for amphibian species at higher latitudes compared to their lower latitude counterparts but, to the best of our knowledge, has not yet been observed within-species. however, phenological studies that have quantified within-species breeding temperature have typically used local weather station data and so the spatial scale might not be not fine enough to represent local conditions in mountain areas, where temperatures can vary rapidly over short geographical distances. therefore, such studies are liable to miss within-species differences in spawning temperature in relation to altitude. when we quantified temperature at a local level, our results support the absence of a shift in breeding temperature in low-temperature environments, contributing to knowledge of the fundamental niche of r. temporaria. however, we did find that r. temporaria can substantially alter the date they breed in response to the environmental conditions experienced, even between geographically close breeding sites, demonstrating a high degree of plasticity in terms of breeding timing. larval physiology in relation to altitude routine metabolic rate there was a significant interaction between mountain and altitude, which complicated interpretation of rmr. posthoc tests indicated no significant differences in rmr between tadpoles sampled from high and low sites on ime or law. in regards to the other three mountains, there were significant differences but not always in the same direction: for dub and mnt tadpoles sampled from high elevation showed a decreased rmr relative to those from low elevation, but the largest difference due to elevation was at lom, where tadpoles from high elevation showed a substantial increase in rmr compared to those from low elevation. however, due to the moderate power of the model, it is possible that some significant relationships were missed. it has been suggested that a lower resting metabolic rate can allow more energy to be allocated to growth in resource-limited environments and a link between lower rmr and increased growth rate has been found in the southern toad, sydney rock oyster and snapping turtle. furthermore, sears found that both increased growth rates and reduced rmr were positively correlated with altitude in sagebrush lizards. the three mountains in our study system where rmr was lower in individuals from high-altitudes have been shown to be locally adapted to temperature parameters, with larval period decreasing and growth rate increasing at high-altitude. therefore, the lower rmr of individuals from high-altitude from dub, mnt and law, is in line with the increased growth rates observed at these sites. lindgren and laurila did not find a link between growth rates and rmr in r. temporaria along a latitudinal gradient in sweden. therefore, this is the first tentative evidence of reduced rmr being linked to increased growth rate as an adaptation to low-temperatures in an amphibian. however, the significantly higher rmr at high- than low-altitude in lom, and the non-significant difference between lawhigh and lawlow in terms of rmr, could suggest that local conditions other than altitude are also important in driving divergence in rmr. for instance, the lowest rmr were observed in individuals from imelow and lomlow, where spawning occurred at the lowest temperatures of all the sites, and this could suggest that low rmr is beneficial in tolerating low temperatures at all altitudes, particularly early in the season when temperatures can fall after spawning. it has also been speculated that increased growth rate can be a result of increased time spent foraging, facilitated by lower predator presence in low-temperature environments, and is unrelated to rmr. furthermore, rmr is influenced by temperature and there was up to a 3°c difference in temperature whilst measuring rmr of individuals from different sites, as well as a difference between the temperature at which the tadpoles were raised and the temperature at which rmr was measured. therefore, further research is needed to assess whether site-specific differences in rmr remain constant through time and whether other mountains with breeding sites of above 900 m also show reduced metabolic rate at high- vs. low-altitude. freeze tolerance just over half of the tadpoles that were frozen survived and altitude was significant in predicting freeze survival: individuals sampled from low-altitude survived freezing significantly better than those from high-altitude. voituron et al. suggested that r. temporaria adults were freeze intolerant, as 100% mortality was observed after eight hours of complete bodily fluid freezing. however, pasanen and karhapää found that r. temporaria adults could survive 24 hours in a sub-zero environment but died within three days. our results suggest that tadpoles of r. temporaria are also capable of surviving short periods of freezing. this is the first time, to the best of our knowledge, that larval freeze tolerance has been demonstrated in any amphibian. although the results presented here suggest that tadpoles are capable of surviving being frozen, the finding of a greater survival of low- compared to high-altitude individuals appears counterintuitive, given the longer period of sub-zero temperatures at high- than low-altitude in scotland and the sub-zero average winter temperature of - °c at high-altitude, compared to °c at low-altitude at the breeding sites used in this study. indeed an increase in freeze tolerance with altitude has been found in plants and insects, but freeze tolerance and altitude has not been explicitly linked in herptiles. however, evolution of freeze tolerance in frogs, lizards and turtles has been linked to ecological pressures relating to winter temperatures experienced. in general, freeze tolerant species are those that terrestrially overwinter in sub-zero temperatures, as opposed to avoiding freezing in deep water bodies. therefore, a higher freeze tolerance would suggest that low-altitude tadpoles are more often exposed to freezing temperatures, whereas high-altitude tadpoles may avoid such exposure altogether by inhabiting deep water bodies or metamorphosing prior to winter. formation of deep water pools is inhibited at high-altitudes in scotland due to the rocky, exposed landscape. therefore, freeze exposure is more likely avoided in high-altitude tadpoles by metamorphosing within a single active season, facilitated by a faster growth rate in conjunction with a lower rmr. therefore, our results potentially suggest that of the tadpoles found overwintering in scotland, low-altitude individuals are more likely to overwinter as tadpoles than high-altitude individuals. however, it is possible that long periods of snow cover at high-altitude could insulate tadpoles from freezing temperatures even in shallow water bodies. therefore, further field research is needed to assess whether fewer, if any, high- than low-altitude individuals overwinter as tadpoles.  r. temporaria adults do not show behavioural adaptations in terms of breeding at lower temperatures, instead they delay spawning based on the temperature experienced. however, r. temporaria larvae appear to have the potential to physiologically adapt to low-temperature environments, although these relationships, and the results we obtained for routine metabolic rate and freeze tolerance, are not always straightforward to interpret. therefore, our results suggest that survival at high-altitude may be facilitated by physiological mechanisms that permit faster growth rates, allowing completion of larval development within a shorter period of time, alleviating the need for adaptations that extend the time available for larval growth. how individuals respond to environmental temperature at a local level is an important step in relating ecological and evolutionary pressures to phenotypes. 
therefore, survival at high-altitude may be facilitated by physiological mechanisms that permit faster growth rates, allowing completion of larval development within a shorter time period, alleviating the need for adaptations that extend the time available for larval growth. we found that breeding did not occur below 5°c at any site and there was no significant relationship between breeding temperature and altitude, leading to a delay in spawning of five days for every 100 m increase in altitude. the aim of this study was to assess the behavioural and physiological responses that facilitate survival in low-temperature environments.; and 2) do tadpoles sampled from high-altitude sites differ physiologically from those from low-altitude sites, in terms of routine metabolic rate and freeze tolerance? the relationship between altitude and rmr varied by mountain but was lower for individuals sampled from high- than low-altitude sites within the three mountains with the highest high-altitude sites. in particular, we asked: 1) do high-altitude common frog adults extend the time available for larval growth by breeding at lower temperatures than low-altitude individuals? our results suggest that adults at high-altitude do not show behavioural adaptations in terms of breeding at lower temperatures. ectotherms are particularly sensitive to low-temperature environments, which can result in a reduced activity period, slowed physiological processes and increased exposure to sub-zero temperatures. extreme environments can impose strong ecological and evolutionary pressures at a local level.
 extreme environments can impose strong ecological and evolutionary pressures at a local level. ectotherms are particularly sensitive to low-temperature environments, which can result in a reduced activity period, slowed physiological processes and increased exposure to sub-zero temperatures. the aim of this study was to assess the behavioural and physiological responses that facilitate survival in low-temperature environments. in particular, we asked: 1) do high-altitude common frog adults extend the time available for larval growth by breeding at lower temperatures than low-altitude individuals?; and 2) do tadpoles sampled from high-altitude sites differ physiologically from those from low-altitude sites, in terms of routine metabolic rate and freeze tolerance? breeding date was assessed as the first day of spawn observation and local temperature recorded for five, paired high- and low-altitude r. temporaria breeding sites in scotland. spawn was collected and tadpoles raised in a common laboratory environment, where rmr was measured as oxygen consumed using a closed respiratory tube system. freeze tolerance was measured as survival following slow cooling to the point when all container water had frozen.  we found that breeding did not occur below 5°c at any site and there was no significant relationship between breeding temperature and altitude, leading to a delay in spawning of five days for every 100 m increase in altitude. the relationship between altitude and rmr varied by mountain but was lower for individuals sampled from high- than low-altitude sites within the three mountains with the highest high-altitude sites. in contrast, individuals sampled from low-altitudes survived freezing significantly better than those from high-altitudes, across all mountains.  our results suggest that adults at high-altitude do not show behavioural adaptations in terms of breeding at lower temperatures. however, tadpoles appear to have the potential to adapt physiologically to surviving at high-altitude via reduced rmr but without an increase in freeze tolerance. therefore, survival at high-altitude may be facilitated by physiological mechanisms that permit faster growth rates, allowing completion of larval development within a shorter time period, alleviating the need for adaptations that extend the time available for larval growth. routine metabolic ratefreeze tolerancespawning temperaturealtitudescotland 