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Behavioural adaptations of Rana temporaria to cold climates
Journal of Thermal Biology 49-50 (2015) 82–90
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Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio
Behavioural adaptations of Rana temporaria to cold climates Gerda Ludwig a,n, Ulrich Sinsch b, Bernd Pelster a a b
Institute of Zoology, University of Innsbruck, Technikerstr. 25, A-6020 Innsbruck, Austria Institute of Sciences, Department of Biology, University of Koblenz-Landau, Universitätsstraße 1, D-56070 Koblenz, Germany
art ic l e i nf o
a b s t r a c t
Article history: Received 3 November 2014 Received in revised form 12 February 2015 Accepted 13 February 2015 Available online 14 February 2015
Environmental conditions at the edge of a species’ ecological optimum can exert great ecological or evolutionary pressure at local populations. For ectotherms like amphibians temperature is one of the most important abiotic factors of their environment as it influences directly their metabolism and sets limits to their distribution. Amphibians have evolved three ways to cope with sub-zero temperatures: freeze tolerance, freeze protection, freeze avoidance. The aim of this study was to assess which strategy common frogs at mid and high elevation use to survive and thrive in cold climates. In particular we (1) tested for the presence of physiological freeze protection, (2) evaluated autumnal activity and overwintering behaviour with respect to freeze avoidance and (3) assessed the importance of different high-elevation microhabitats for behavioural thermoregulation. Common frogs did not exhibit any signs of freeze protection when experiencing temperatures around 0 °C. Instead they retreated to open water for protection and overwintering. High elevation common frogs remained active for around the same period of time than their conspecifics at lower elevation. Our results suggest that at mid and high elevation common frogs use freeze avoidance alone to survive temperatures below 0 °C. The availability of warm microhabitats, such as rock or pasture, provides high elevation frogs with the opportunity of behavioural thermoregulation and thus allows them to remain active at temperatures at which common frogs at lower elevation cease activity. & 2015 Elsevier Ltd. All rights reserved.
Keywords: Amphibians Rana temporaria Freeze protection Freeze avoidance Behavioural thermoregulation Microhabitat selection
1. Introduction Environmental conditions at the edge of a species’ ecological optimum can exert great ecological or evolutionary pressure at local population level. In this case adaptive or plastic responses to stressor at individual level facilitate the survival of populations (Hangartner et al., 2012; Dallinger and Höckner, 2013). Exploring the mechanisms that allow a species to persist in challenging environments is crucial for understanding how species deal with ecological and evolutionary pressure, especially in the light of global climate change. For ectotherms ambient temperature is one of the most important physical aspects of their environment. It affects directly physiological processes such as metabolism, growth, development and reproduction, muscle contraction, oxygen transport, and digestion (Carey and Alexander, 2003; Laugen et al., 2003a) and thus influences timing of hibernation and reproduction (e.g. Terhivuo, 1988; Beebee, 1994; Reading, 1998, 2003; Tryjanowski et al., 2003; Carroll et al., 2009) and global species richness patterns (Buckley and Jetz, 2007). Shorter n
Corresponding author. E-mail addresses: [email protected] (G. Ludwig), [email protected] (U. Sinsch), [email protected] (B. Pelster). http://dx.doi.org/10.1016/j.jtherbio.2015.02.006 0306-4565/& 2015 Elsevier Ltd. All rights reserved.
growing seasons impose time constraints on growth and development (Laugen et al., 2003b; Muir et al., 2014a), sub-zero temperatures during winter pose the risk of freezing and set geographical limits to the distribution of species. Only two amphibian species, the North American wood frog (Lithobates [Rana] sylvaticus) and the European common frog (Rana temporaria) are able to inhabit regions north of the Arctic Circle. Both species exhibit a wide geographic distribution: L. sylvaticus occurs from Alaska to Labrador, south to New Jersey, northern Georgia and northern Idaho (Chubbs and Phillips, 1998). R. temporaria is found from north of the Arctic Circle in Scandinavia to northern Spain, and in mountainous regions up to an elevation of 2600 m (Gasc et al., 1997). In both species populations are subject to a variety of climatic conditions, which substantially modify their annual activity cycle. For example, at the southern edge of their distribution, common frogs do not hibernate and breeding is initiated at the beginning of winter (Bea et al., 1986). In lowland populations in Switzerland and France common frogs can be found breeding as early as February (e.g. Ryser, 1989; Augert and Joly, 1993). With increasing latitude or altitude breeding starts later (e.g. Elmberg, 1990; Elmberg and Lundberg, 1991) and can occur as late as May in northern or high alpine populations. In general, there are three ways amphibians can cope with temperatures below zero (Pinder et al., 1992): (1) tolerate freezing
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of extra-cellular body fluids (freeze tolerance) (Storey and Storey, 1984); (2) production of cryoprotectants to prevent cells from freezing (freeze protection) (Storey and Storey, 1988) and (3) choosing hibernation sites where temperatures do not drop below zero (freeze avoidance) (Boutilier et al., 2000; Sinsch and Leskovar, 2011). The behavioural and physiological adaptations of L. sylvaticus to sub-zero temperatures are well studied. Wood frogs cease activity when temperatures drop below 4.4 °C (Bellis, 1962). Their ability to tolerate extra-cellular freezing and to prevent intra-cellular freezing by using glucose as cryoptrotectant (e.g. Storey and Storey, 1984, 1988; Layne and Lee, 1987; Costanzo et al., 1992), allows for terrestrial hibernation underneath leaf litter and snow, where temperatures drop as low as 7 °C (Schmid, 1982; MacArthur and Dandy, 1982). In contrast, our understanding of how R. temporaria deals with low temperatures is less clear. So far studies in high elevation or northern populations have focused on summer microhabitat use and diel activity (Vences et al., 2000), age structure (Ryser, 1996; Patrelle et al., 2012), breeding behavior (Elmberg, 1990; Elmberg and Lundberg, 1991) and larval development (e.g. Aebli, 1966; Angelier and Angelier, 1968; Brand and Grossenbacher, 1979; Laugen et al., 2003a; Muir et al., 2014a). With respect to the hibernation ecology of R. temporaria, the occurrence of both, aquatic and terrestrial hibernation has been reported (Viitanen, 1965; Koskela and Pasanen, 1974; Hagström, 1982; Elmberg, 1990; Pasanen and Sorjonen, 1994). Voituron et al. (2009) found 100% mortality in adult common frogs after eight hours of complete freezing of body fluids. Pasanen and Karhapää (1997) observed that adult R. temporaria survived 24 h exposure to sub-zero temperatures, but died within three days after exposure. Muir et al. (2014a) found that 82% of R. temporaria larvae from low-elevation sites survived short periods of freezing. From high elevation sites, only 32% survived. However, data on physiological and behavioural responses to low temperatures of wild adult common frogs are lacking. This gap of knowledge in the general biology of R. temporaria limits our ability to link local responses to phenotypes and thus assess the impact of environmental change on this species. Therefore the objectives of the present study were to compare mid- and high-elevation adult common frogs in terms of their physiological and behavioural adaptations to cold climates. In particular we (1) tested for the presence of physiological freeze protection, (2) evaluated autumnal activity and overwintering behaviour with respect to freeze avoidance and (3) assessed the importance of different high-elevation microhabitats for behavioural thermoregulation.
2. Material and methods 2.1. Study areas The study was conducted in two common frog populations at different elevations. The high elevation population is located in an alpine basin in the Fotsch Valley in the northern Stubai Alps, Austria (11°12′53″E, 47°08′59″N) at an elevation of 2300 m. A detailed description of this site is found it Ludwig et al. (2013). Based on annual egg mass counts during the breeding periods between 2011 and 2014 population size is estimated to be around 700 adults. Reproductive activity usually starts in mid to late May. The subalpine population is located in a small wetland area in the northeast of the city of Innsbruck, Austria (11°24′58″E, 47°17′11″N) at an elevation of 650 m. Several small ponds are surrounded by marsh area and mixed forest. Based on egg mass counts in spring 2013 and 2014 the population size is estimated to be around 200 adults. Breeding usually starts in mid-March.
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2.2. Experiments to test for freeze protection In 2013 17 adult common (16 males, 1 female) frogs were caught in breeding ponds at the beginning of spawning at the subalpine site and their blood glucose levels measured. At the alpine site 23 adults (21 males, 2 females) were caught on snow or ice-slush right after emergence from overwintering and tested for elevated blood glucose levels. Blood was drawn into a heparinized capillary tube from the Vena angularis as described by Nöller (1958) and transferred to a blood glucose meter (Accu-Chek Performa Nano, Roche Diagnostics GmbH, Germany, measuring range: 10–600 mg/dl; measuring precision according to EN ISO 15197:2003 standard: 95% of the individual glucose results shall fall within 7 15 mg/dL of the results of the manufacturer's measurement procedure at glucose concentrations of o75 mg/dL). Sampling was done in situ and did not last longer than 1 min. In addition we recorded air and water temperature using a digital thermometer (RS Digital Thermometer AH-50A Typ K, RS Components, Austria; measuring range: 50 °C to 200 °C; measurement precision: 0.1 °C; measuring error: þ/ 1.5 °C). To test for seasonal changes, we repeated blood glucose measurements in the alpine population in June (N ¼29, 19 males, 29 females) and late August (N ¼ 19, all males). These individuals were all caught in ponds. Blood glucose levels were compared using One-Way ANOVA. Significance level was set to alpha ¼0.05. 2.3. Experiments to test for freeze avoidance Over a period of three years (2011–2013) a total of 46 adult R. temporaria (mass 440 g; 31 males, 15 females) in both populations were marked with radio transmitters (BD-2H, 3.4 g and BD-2TH, 2.9 g, Holohil Systems Ltd., Canada) to monitor their movements. In 2011 transmitters were attached externally. The method of external attachment is described in Ludwig et al. (2013). In 2012 and 2013 transmitters were implanted into the body cavity. For surgery, frogs caught in the field were brought back to the lab and anaesthetized by submersion in a buffered solution of MS-222 (4 mg/L), through a small incision transmitters were implanted into the body cavity. The incision was closed using absorbable sutures (Novosyn HS 15, Braun, Spain) and frogs were rinsed under fresh water until fully recovered. After surgery frogs were kept in a tempered climate chamber (18 °C, L:D¼13:11) for three days to facilitate wound healing and then released at the site of capture. Marked frogs were located at least once a week. At each relocation we recorded the frog's position using GPS (Garmin, eTrex). In addition, we measured the line-of-sight distance each individual had moved between two observations. An animal moving more than 5 m between relocations was classified as active. When an individual remained sedentary for two or more consecutive relocations we assumed it had reached its hibernation site. At each site one data logger (TG-4100 Tinytag Aquatic 2, Gemini Data Loggers Ltd., UK) was placed on the ground and one in the stream to record air and water temperature during fall, winter and spring. Every year data loggers were placed in the same spots on the ground and in streams. Data loggers recording air temperature were placed directly on the ground shaded by a rock (alpine site) or shrubs (subalpine site). Temperatures were recorded in 2 h intervals from the beginning of September until the end of March in the subalpine population and the end of June in the alpine population. We used the telemetry data to calculate the autumnal activity period for each marked frog, defined as the number of days from September 16 (by this date all frogs had been released back into the field) until it reached its hibernation site. For each year in each population we calculated daily average, minimum and maximum temperature between September 16 and December 10. As activity patterns and hibernation behaviour in the alpine population were
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not sex-biased (Ludwig et al., 2013), male and female data were pooled for further analysis. We tested for differences in length of the activity period, and daily mean and daily maximum temperature between years within populations and between populations using One-Way ANOVA and Kruskal–Wallis Test. Significance level was set to alpha ¼0.05. Every spring we recorded the date of emergence from hibernation (high elevation population only) and appearance of first egg masses (both populations). 2.4. Microhabitat potential for behavioural thermoregulation To assess the importance of behavioural thermoregulation based on microhabitat selection, we conducted a series of solar exposure experiments during the 2014 breeding period in the alpine population. For this purpose, 20 adult common frogs were caught in and around breeding ponds and each frog's core, dorsal and ventral temperature was measured in situ using a thermistor (RS Digital Thermometer AH-50A Typ K, RS Components, Austria; measuring range: 50 °C to 200 °C; measurement precision: 0.1 °C; measuring error: þ/ 1.5 °C). Core temperature was measured by inserting the thermistor into the cloaca. After measurements were completed each animal was anaesthetized by submersion in a buffered solution of MS-222 (4 mg/L). When the amphibian failed to respond to a light pinch on a toe-tip, it was placed on snow, rock, pasture or underneath dwarf shrubs. After a 10 min acclimatisation period (Lillywhite, 1975; Sinsch, 1983) we again recorded air and substrate surface temperature as well as the animal's core, ventral and dorsal temperature. Then the animal was moved to the next substrate type and again left to acclimatize for 10 min before temperature measurements were repeated. This procedure was repeated until each individual had passed all four substrate types. The sequence of substrates tested, snow, rock, pasture and shrubs, was the same for all individuals. We tested for differences in substrate, air, core, dorsal and ventral temperature using One-Way ANOVA. To achieve normal distribution, all data sets were ln transformed. In addition we also tested for differences in frogs’ core, dorsal and ventral temperature between substrates using One-Way ANOVA on Ranks. Significance level was set to alpha ¼0.05. All statistical analyses were performed using the software package SigmaPlot 12.5 (Systat Software Inc., USA).
3. Results 3.1. Freeze protection In the high elevation population blood glucose concentration did not differ significantly immediately after emergence from hibernation in May, during spawning (June) and in fall (August) (One-way ANOVA F2,67 ¼1.879, p ¼0.161). All frogs caught right after emergence were found on snow or in ice-slush and were the only ones experiencing substrate surface temperature around 0 °C (mean¼ 2 °C, range: 0.3 to 5.2 °C). Their mean blood glucose level was 37.1 mg/dl (SD ¼16.1). In June and late August mean blood glucose levels were 42.9 mg/dl (SD ¼18.5) and 32.4 mg/dl (SD ¼6.9), respectively. Corresponding mean water temperatures were 9.6 °C and 12.3 °C. In the subalpine population mean blood glucose level during spawning was 46.3 mg/dl (SD ¼ 13.6). It differed marginally (p ¼0.043) from the fall blood glucose level in the high elevation population (Fig. 1). 3.2. Freeze avoidance Out of the 46 frogs originally marked, we were able to follow 25
Fig. 1. Box plots comparing blood glucose concentration of adult common frogs at different times of the year in the alpine and subalpine population. Solid lines inside the box show medians. Lower and upper boundary of the box indicates the 25th and 75th percentile. Whiskers indicate the 90th and 10th percentiles.
individuals to their hibernation sites. The transmitter signal of 14 animals was lost during the course of the study. 2 frogs were found dead. 1 animal was trapped and killed in ice when ponds started to freeze over. 3 individuals were preyed upon by an unknown predator and their transmitters located in a deserted marmot cave. From one individual the transmitter had to be removed after the antenna got tangled up in grass. Hibernation site selection did not vary between populations. In the alpine population 11 individuals hibernated in streams and a slow flowing spring, three individuals hibernated in ponds. In the subalpine population 10 animals hibernated in the stream, one individual retreated to a pond. Terrestrial hibernation did not occur in our study. The length of autumnal activity periods did not differ significantly between years and between populations (One-Way ANOVA F3,22 ¼ 2.8, p ¼0.064, Table 1). In the alpine population the first individual began hibernation on October 4, the last one on November 28. In 2013 early build-up of snow led to a sudden end of the active period on November 7 (Julian day 307; Fig. 2). Subalpine frogs retreated to the stream between October 18 and November 27 (Fig. 3). Mean daily temperature and daily maximum temperature did not differ significantly between years within populations, but did differ between low and high elevation population (Kruskal–Wallis Test, H ¼76.87, p o0.05, Table 1). In 2012 high elevation frogs emerged from streams on May 25 and started spawning on May 27. In 2014 emergence started on May 20, spawning on May 25. Due to the permanent ice and snow cover during winter, stream temperature was not influenced by fluctuations of ambient temperature until ice break in spring. Stream temperature fluctuated between 0.3 °C and 1.0 °C in 2012, and 1.0–3.2 °C in 2014. Corresponding spring air temperature on the ground varied between 1 °C and 1 °C as long as data loggers were covered by snow (Julian day 1–151 in 2012, Julian day 1–144 in 2014) (Fig. 4). The dense vegetation and the lack of snow on the ground did not permit us to observe emergence from hibernation at the subalpine site. Therefore only the onset of spawning was recorded each year. In 2013 spawning started on March 13, in 2014 on March 11. Although the low elevation stream did not freeze over during winter, water temperature remained fairly constant and fluctuated around 5 °C in both years. Due to the lack of a permanent snow cover air temperature near the ground fluctuated during each study period and varied between study periods. In
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Table 1 Mean, minimum and maximum values for length of autumnal activity period (d), mean daily temperature (°C) and mean daily maximum temperature (°C) in the alpine population (2300 m asl) and subalpine population (650 m asl). High elevation population 2011
Length of activity period (d) Mean temperature (°C) Mean maximum temperature (°C)
Low elevation population 2013
2012
2013
Mean (SD)
Range
Mean (SD)
Range
Mean (SD)
Range
Mean (SD)
Range
56.9 (9.8) 1.9 (4.7) 5.3 (5.1)
41–76 6.4–11.6 2.7–15.7
40.7 (13.2) 2.6 (2.8) 7.1 (8.9)
18–53 0.2–10.0 0.2–30.0
54.0 (14.4) 7.3 (5.3) 10.5 (6.6)
40–74 3.4–21.1 3.1–24.2
56.4 (9.8) 7.0 (6.6) 9.4 (5.7)
38–62 3.6–16.3 1.3–19.2
2013 sub-zero temperatures occurred frequently. In contrast, in 2014 temperatures did not drop below 0 °C except for the beginning of January (Fig. 5). 3.3. Microhabitat potential for behavioural thermoregulation The surface temperature of pasture was the highest of all four substrates (mean ¼26.4 °C, SD ¼8.9) reaching maxima of 39.4 °C and being significantly higher than corresponding mean air temperature (One-Way ANOVA F2,56 ¼9.99, p o0.001). Also rock surface temperature was significantly above air temperature (OneWay ANOVA F2,56 ¼6.36, p ¼0.003). Shrubs were the thermally most balanced microhabitat. Mean substrate and air temperature did not differ significantly (One-Way ANOVA F2,56 ¼1.42,
p¼ 0.250). The snow's surface was the coldest and significantly lower than air temperature (One-Way ANOVA F2,56 ¼106.06, po 0.001; Fig. 6). Exclusively on snow the frogs’ core, dorsal and ventral temperature differed significantly (One-Way ANOVA F2,56 ¼ 40.8, po0.001), with dorsal temperature being the highest and ventral temperature being the lowest. On rock, pasture and underneath shrubs there was no significant differences between core, dorsal and ventral temperature (One-Way ANOVA, rock: F2,56 ¼ 0.95, p ¼0.40; grass: F2,56 ¼ 0.10, p ¼0.91; shrubs: F2,56 ¼0.02, p¼ 0.98; Fig. 6). When comparing substrate types, core, ventral and dorsal temperature differed significantly between pasture and snow and pasture and shrubs as well as between rock and snow. Between rock and shrubs only core and dorsal temperature differed
Fig. 2. Daily mean (solid line), maximum (upper dashed line) and minimum (lower dashed line) fall air temperature at ground level in the alpine population in 2011 (a) and 2013 (b). Date is shown in Julian days (1 ¼January 1; 259 ¼ September 16; 344¼ December 10). Symbols and numbers indicate when how many frogs retreated into hibernation.
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Fig. 3. Daily mean (solid line), maximum (upper dashed line) and minimum (lower dashed line) fall air temperature at ground level in the subalpine population in 2012 (a) and 2013 (b). Date is shown in Julian days (1 ¼ January 1; 259 ¼September 16; 344¼December 10). Symbols and numbers indicate when how many frogs retreated into hibernation.
Fig. 4. Mean daily stream temperature (dashed lines) and air temperature at ground level (solid lines) at the alpine site between January 1 and May 31 in 2012 (thin lines) and 2014 (bold lines). Date is shown in Julian days (1 ¼ January 1). Triangles indicate the emergence from hibernation, circles indicate the onset of spawning (open symbols ¼ 2012, closed symbols ¼ 2014).
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Fig. 5. Mean daily stream temperature (dashed lines) and air temperature at ground level (solid lines) at the subalpine site between January 1 and March 31 in 2013 (thin lines) and 2014 (bold lines). Date is shown in Julian days (1 ¼ January 1). Circles indicate the onset of spawning (open symbols ¼ 2013, closed symbols ¼2014).
significantly. Between shrubs and snow only ventral temperature differed significantly (One-way ANOVA on Ranks H3 ¼41.45; p o0.05). No significant difference in core, dorsal and ventral temperature was found between rock and pasture.
4. Discussion At first glance the presence of a large and stable common frog population at a high elevation study site is surprising considering the harsh local climate. Under such extreme environmental conditions amphibians are required to develop a series of adaptations to grow, accumulate energy reserves and reproduce despite overall low temperatures and short growing seasons. 4.1. Freeze protection and freeze avoidance The absence of seasonal blood glucose variation indicates that even high altitude common frogs do not mobilize glycogen as a cryoprotectant. Blood glucose concentrations of emerging high altitude common frogs experiencing temperatures around or below zero were not elevated compared to those of conspecifics tested in summer and fall (Fig. 1) and corresponded to blood glucose levels measured in wood frogs kept at 23 °C (52.02 mg/dl; Storey and Storey, 1984). In comparison, blood glucose concentration in frozen wood frogs reached up to 333.3 mg/dl (Storey and Storey, 1984). Although we were not able to measure blood glucose levels directly during hibernation and therefore cannot exclude the possibility of a change in blood glucose levels between hibernation and emergence, the results of our telemetry study show that common frogs retreat to frost-free hibernation sites during winter months where there is no need for freeze protection. In contrast, emerging frogs can be found on snow or in slush were the surrounding temperatures are around or below 0 °C. If these frogs do not exhibit elevated blood glucose levels it is unlikely that blood glucose levels of frogs exposed to constant water temperature above 0 °C are elevated. These findings suggest that common frogs in subalpine and alpine regions use behavioural
adaptations alone to avoid freezing and to thrive in cold climates. At the first glance air temperature at ground level during winter seem surprisingly mild, especially at the alpine study site, and may raise questions of how representative these data are. Based on the annual climate report of the Austrian Institute for Meteorology and Geodynamics, winter 2011/12 and 2012/2013 were typical winters. Winter of 2013/2014, however, was one of the warmest years in the past 246 years (ZAMG, 2015; online information) The mild subzero temperatures at ground level in the alpine population, can be explained by the fact that dataloggers were snowed in for most of the winter in 2011/12 (Julian day 344 in Fig. 2a to Julian day 150 in Fig. 4) in and again between Julian day 284 and 294 (Fig. 2b) and from Julian day 307 (Fig. 2b) to Julian day 141 (Fig. 4) in 2013/2014. Depending on the overall weather conditions and the presence or absence of frozen soil at the onset of snowfall, air temperature at the snow- soil interface usually fluctuates around 0 °C (Ishikawa, 2003; Zhang, 2005; Rödder and Kneissel, 2012). Due to the lack of a permanent snow cover in the subalpine study area air temperature shows great daily fluctuations and has overall lower temperature minima than alpine air temperature (Fig. 3(a) and (b), Fig. 5). The mild winter of 2013/2014 is reflected in the overall warmer air temperature at the subalpine site (Fig. 5). However, because of the insulation effect of the snow cover (Ishikawa, 2003; Zhang, 2005; Rödder and Kneissel, 2012), the overall milder weather conditions during the winter months in 2013/2014 are not as clearly visible in the data from the alpine site. The lack of freeze protection in subalpine and alpine adult common frogs agrees with the altitudinal variation in freeze protection found in common frog larvae (Muir et al., 2014a). Larvae from breeding sites at 703 m asl. exhibit a lower level of freeze protection than larvae from breeding sites between 77–222 m asl. Muir et al. (2014a) suggested that due to the lack of an insulating snow and ice cover during winter, complete freezing of ponds occurs more frequently at low altitudes. Therefore freeze protection may be selected for at low elevation. A similar evolutionary divergence seems possible for adult common frogs. Whereas
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Fig. 6. Box plots comparing surface temperature of snow (a), rock (b), pasture (c) and shrubs (d) with the core, dorsal and ventral temperatures of common frogs. Solid lines inside the box show medians, dashed lines means. Lower and upper boundary of the box indicates the 25th and 75th percentile. Whiskers indicate the 90th and 10th percentiles.
subalpine and alpine individuals hibernating in streams never experience freezing, freeze protection may be present in common frogs exposed to milder climatic conditions during winter, that allow for terrestrial hibernation, but also bear the risk of occasionally occurring sub-zero temperatures. Further field studies are needed to test this hypothesis. 4.2. Microhabitat potential for behavioural thermoregulation Although mean ambient fall temperature at 2300 m asl. was on average 5 °C lower than at 650 m asl., autumnal activity periods in both populations were of similar length (Table 1). In fall timely migration to hibernation streams ensures that high-elevation common frogs can retreat into the water, whenever temperatures drop below zero, e.g. during night or episodic snow fall. Comparison of the 2013 maximum daily temperatures from the alpine site and the 2013 mean daily temperature from the subalpine site show that maximum values at high elevation are similar or above mean values at low elevation. This suggests that during the day warm microhabitats along the streams, e.g. rocks or pasture, provide high elevation animals with the opportunity for behavioural thermoregulation. The results from the solar exposure
experiments support this hypothesis and show that especially pasture and rock microhabitats allow frogs to raise their body temperature significantly (Fig. 6). Despite overall lower temperature these behavioural traits potentially allow high elevation common frogs to remain active for a similar period of time as their conspecifics at lower elevation and thus give them more time to build up energy stores for overwintering. Also in spring the availability of open water ensures the survival of common frogs at high altitude. At the high elevation site, the break-up of ice over hibernation streams coincides with the ice break on breeding ponds, so that frogs emerging from streams have access to open water during nocturnal temperature drops or cold spells. Since migration from hibernation sites to breeding ponds occurs before snowmelt on land is completed, animals must cross extended patches of snow. To minimise the risk of predation posed by a dark-coloured frog on white background, fast advancement is advantageous. However, in order to move quickly it is crucial for ectotherms to maintain a certain body temperature (Lillywhite, 1970; Vences et al., 2002). We observed two ways in which common frogs accomplish this goal while moving across snow: (1) during resting periods on snow common frogs show an
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atypical upright posture with their front legs completely stretched. This reduces the area of body surface in contact with snow and minimises heat loss through conduction. Our conclusion is supported by the results of the solar exposure experiments. Only on snow dorsal temperature was significantly higher than ventral temperature (Fig. 6). (2) Migrating high elevation common frogs use snow-free spots (e.g. pasture, wind or sun exposed places or small boulders) as heat islands to warm up. The thermal gain by this behaviour agrees with the results from our experiments. Surface temperatures of both pasture and rock were significantly higher than corresponding air temperature and in both cases frogs’ core temperature was significantly higher than air temperature (Fig. 6). In a laboratory-based study Sinsch (1984) found evidence of thermo-regulative behaviour in lowland frogs (60 m asl.). These frogs showed a shift in activity pattern from nocturnal at warmer temperatures to diurnal at colder temperatures. In addition they remained mostly in the terrestrial part of the experimental setup at lower temperatures, whereas the time spent in water increased with increasing temperature. These results suggest that thermoregulative behaviour is also present in lowland common frogs. However, we found that common frogs from the subalpine population did not use thermo-regulative behaviour to extend their autumnal activity period. Instead they ceased activity at temperatures at which their high elevation conspecifics remained active. Since emergence from overwintering occurs earlier and growing seasons are longer at lower elevations, low elevation animals do not need to remain active in fall for a longer time to accumulate sufficient energy reserves for overwintering. Our finding of intraspecific variation in cold tolerance based on behavioural thermoregulation disagrees with the pattern found in L. sylvaticus. In a laboratory based study, Manis and Claussen (1986) found no evidence of intraspecific variation in cold tolerance in individuals along a latitudinal gradient ranging from 37 °34′ N to 45°31′N. They concluded that since wood frogs encounter sub-zero temperatures during winter and spring in all parts of the species’ range, cold tolerance is not selected against in populations experiencing overall warmer climatic conditions. These contrasting results from two species inhabiting similar ecological niches lead to the question if in amphibians physiological adaptations are more often genetically fixed than behavioural ones. Clearly, our data do not allow to distinguish between phenotypic plasticity and genetic adaptation as origin of the observed behavioural thermoregulation in high-elevation common frogs. Genetic adaptation to altitude has been found for several physiological traits such as sexual maturity and UV resistance (Miaud and Merilä, 2001), larval growth rate and larval period (Muir et al., 2014b). In Scandinavia adaptations in a range of larval fitness traits across a latitudinal gradient have been found (Laugen et al., 2003a, 2003b; Palo et al., 2003). Further work examining the genetic or plastic nature of behavioural traits crucial to the survival in stressful environments is needed for the assessment of the future evolutionary potential of species.
4.3. Conclusion Our results show that subalpine and alpine R. temporaria do not use glucose as a cryoprotectant as observed in the closely related North American wood frog. Instead they exhibit a series of behavioural adaptations that enable them to survive and thrive in cold environments. Our results highlight the complex habitat requirements of R. temporaria in alpine environments.
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Acknowledgment We thank Dr. M. Seewald and his team from the Alpenzoo Innsbruck for performing the implantation of transmitters. Thanks to N. Schintgen, D. Anker, D. Hittler and E. Ludwig for assistance with fieldwork, R. Ludwig for laboratory support, and M. Sztatecsny for providing telemetry equipment. Fieldwork was financially supported by the (Grant number 134263) and the Institute of Zoology, University of Innsbruck. G. Ludwig was supported by a PhD scholarship from the University of Innsbruck. All protocols used in this study were in accordance with the Austrian Legislation for animal experiments and approved by the ethical committee of the former Austrian Federal Ministry for Science and Research (BMWF-66.008/0010-II/3b/2011, BMWF-66.008/0012-II/ 3b/2012, BMWF-66.008/0017-II/3b/2012) as well as the Department of Environmental Protection of Tyrol.
References Aebli, H., 1966. Rassenunterschiede in Bezug auf Entwicklungsgeschwindigkeit und Geschlechtsdifferenzierung bei Rana temporaria in den Tälern des Kantons Glarus (Schweiz). Rev. Suisse Zool. 73, l-37. Angelier, E., Angelier, M.L., 1968. Observations sur le développement embryonnaire et larvaire de Rana temporaria L. (Batracien, Anoure). Ann. Limnol. 4, 113–131. Augert, D., Joly, P., 1993. Plasticity of age at maturity between two neighbouring populations of the common frog (Rana temporaria L.). Can.J. Zool. 71 (1), 26–33. Buckley, L.B., Jetz, W., 2007. Environmental and historical constraints on global patterns of amphibian richness. Proc. R. Soc. B 274 (1614), 1167–1173. Bea, A., Rodriguez-Teijeiro, J.D., Jover, L.L., 1986. Relations between meteorological variables and the initiation of the spawning period in populations of Rana temporaria L. in the Atlantic Region of the Basque Country (Northern Spain). Amphib.-Reptil. 7, 23–31. Beebee, T., 1994. Amphibian breeding and climate. Nature 374, 219–220. Bellis, E.D., 1962. The influence of humidity on wood frog activity. Am. Midl. Nat. 68 (1), 139–148. Boutilier, R.G., Tattersall, G.J., Donohoe, P.H., 2000. Metabolic consequences of behavioural hypothermia and oxygen detection in submerged overwintering frogs. Zool.: Anal. Complex Syst. 102, 111–119. Brand, M., Grossenbacher, K., 1979. Untersuchungen zur Entwicklungsgeschwindigkeit der Larven von Triturus a. alpestris (Laurenti, 1768), Bufo b. bufo (Linnaeus, 1758) und Rana t. temporaria (Linnaeus, 1758) aus Populationen verschiedener Höhenstufen in den Schweizer Alpen. Universität Bern (PhD Thesis). Carey, C., Alexander, M.A., 2003. Climate change and amphibian declines: is there a link? Divers. Distrib. 9, 111–121. Chubbs, T.E., Phillips, F.R., 1998. Distribution of the wood frog, Rana sylvatica, in Labrador: an update. Can. Field Nat. 112 (2), 329–331. Costanzo, J.P., Lee, R.E., Wright, M.F., 1992. Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs. J. Exp. Zool. 261 (4), 373–378. Carroll, E.A., Sparks, T.H., Collinson, N., Beebee, T.J.C., 2009. Influence of temperature on the spatial distribution of first spawning dates of the common frog (Rana temporaria) in the UK. Glob. Chang. Biol. 15, 467–473. Dallinger, R., Höckner, M., 2013. Evolutionary concepts in ecotoxicology: tracing the genetic background of differential cadmium sensitivities in invertebrate lineages. Ecotoxicology 22 (5), 767–778. Elmberg, J., 1990. Long-term survival, length of breeding season, and operational sex ratio in a boreal population of common frogs, Rana temporaria L. Can. J. Zool. 68, 121–127. Elmberg, J., Lundberg, P., 1991. Intraspecific variation in calling, time allocation and energy reserves in breeding male common frogs Rana temporaria. Ann. Zool. Fenn. 28, 23–29. Gasc, J.P., Cabela, A., Crnobrnja-Isailovic, J., Dolmen, D., Grossenbacher, K., Haffner, P., Lescure, J., Martens, H., Martinez-Rica, J.P., Maurin, H., Oliveira, M.E., Sofianidou, T.S., Veith, M., Zuiderwijk, A. (Eds.), 1997. Atlas Atlas of Amphibians and Reptiles in Europe. Societas Europea Herpetologica & Museum National d’Histoire Naturelle (IEGB/SPN), Paris. Hagström, T., 1982. Winter habitat selection by some northern European amphibians. Br. J. Herpetol. 6, 276–277. Hangartner, S.B., Laurila, A., Räsänen, K., 2012. Adaptive divergence in moor frog (Rana arvalis) populations along an acidification gradient: inferences from QSTFST correlations. Evolution 66, 867–881. Ishikawa, M., 2003. Thermal regimes at the snow–ground interface and their implications for permafrost investigation. Geomorphology 52 (1–2), 105–120. Koskela, P., Pasanen, S., 1974. The wintering of the common frog, Rana temporaria L. in northern Finland. Aquilo Series Zoologica 15, 1–17. Laugen, A.T., Laurila, A., Merilä, J., 2003a. Latitudinal and temperature-dependent variation in embryonic development and growth in Rana temporaria. Oecologia
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135, 548–554. Laugen, A.T., Laurila, A., Räsänen, K., Merilä, J., 2003b. Latitudinal countergradient variation in the common frog (Rana temporaria) development rates – evidence for local adaptation. J. Evol. Biol. 16, 996–1005. Layne, J.R., Lee, R.E., 1987. Freeze tolerance and the dynamics of ice formation in wood frogs (Rana sylvatica) from southern Ohio. Can. J. Zool. 65 (8), 2062–2065. Lillywhite, H.B., 1970. Behavioral thermoregulation in the bullfrog, Rana catesbeiana. Copeia, 158–168. Lillywhite, H.B., 1975. Physiological correlates of basking in amphibians. Comp. Biochem. Physiol. 52 (A), 323–330. Ludwig, G., Sinsch, U., Pelster, B., 2013. Migratory behaviour during autumn and hibernation site selection in common frogs (Rana temporaria) at high altitude. Herpetol. J. 23, 121–124. Manis, M.L., Claussen, D.L., 1986. Environmental and genetic influences on the thermal physiology of Rana sylvatica. J. Therm. Biol. 11 (1), 31–36. MacArthur, D.L., Dandy, J.W.T., 1982. Physiological aspects of overwintering in the boreal chorus frog (Pseudacris triseriata maculata). Comp. Biochem. Physiol. 72A, 137–141. Miaud, C., Merilä, J., 2001. Local adaptation or environment induction? Causes of population differentiation in alpine amphibians. Biota 2 (1), 31–50. Muir, A.P., Biek, R., Mable, B.K., 2014a. Behavioural and physiological adaptations to low-temperature environments in the common frog, Rana temporaria. BMC Evol. Biol. 14, 110. Muir, A.P., Biek, R., Mable, B.K., 2014b. Local adaptation with gene flow: temperature parameters drive adaptation to altitude in the common frog (Rana temporaria). Mol. Ecol. 23, 561–574. Nöller, H.G., 1958. Eine einfache Technik der Blutentnahme beim Frosch. Pflügers Archiv 269 (1), 98–100. Palo, J.U., O'Hara, R.B., Laugen, A.T., Laurila, A., Primmer, C.R., Merilä, J., 2003. Latitudinal divergence of common frog (Rana temporaria) life history traits by natural selection: evidence from a comparison of molecular and quantitative genetic data. Mol. Ecol. 12 (7), 1963–1978. Pasanen, S., Karhapää, M., 1997. Can boreal common frog (Rana temporaria L.) survive in frost? Ann. Zool. Fenn. 34, 247–250. Pasanen, S., Sorjonen, J., 1994. Partial terrestrial wintering in a northern common frog population (Rana temporaria L.). Ann. Zool. Fenn. 31, 275–278. Patrelle, C., Hjernquist, M.B., Laurila, A., Söderman, F., Merilä, J., 2012. Sex differences in age structure, growth rate and body size of common frogs Rana temporaria in the subarctic. Polar Biol. 35 (10), 1505–1513. Pinder, A.W., Storey, K.B., Ultsch, G.R., 1992. Estivation and hibernation In: Feder, M. E., Burggren, W.W. (Eds.), Environmental Physiology of the Amphibians. The University of Chicago Press, Chicago, pp. 250–274. Reading, C.J., 1998. The effect of winter temperatures on the timing of breeding activity in the common toad Bufo bufo. Oecologia 117, 469–475. Reading, C.J., 2003. The effect of variation in climatic temperature (1980–2001) on breeding activity and tadpole stage duration in the common toad, Bufo bufo. Sci. Total Environ. 310, 231–236. Rödder, T., Kneissel, C., 2012. Influence of snow cover and grain size on the ground thermal regime in the discontinuous permafrost zone, Swiss Alps. Geomorphology 175–176, 176–189. Ryser, J., 1989. Weight loss, reproductive output, and the cost of reproduction in the common frog, Rana temporaria. Oecologia 7, 264–268. Ryser, J., 1996. Comparative life histories of a low- and a high-elevation population of the common frog Rana temporaria. Amphib.-Reptil. 17, 183–195. Schmid, W.D., 1982. Survival of frogs in low temperature. Science 215, 697–698. Sinsch, U., 1983. Wasserhaushalt und tagesperiodisches Verhalten von drei europäischen Rana-Arten (Amphibia: Anura).. Mat-Nat-Fak., Universität zu Köln (Ph. D. thesis). Sinsch, U., 1984. Thermal influences on the habitat preference and the diurnal activity in three European Rana species. Oecologia 64, 125–131. Sinsch, U., Leskovar, C., 2011. Does thermoregulatory behaviour of green toads (Bufo viridis) constrain geographical range in the west? A comparison with the performance of syntopic natterjacks (Bufo calamita). J. Therm. Biol. 36, 346–354. Storey, K.B., Storey, M., 1984. Biochemical adaption for freezing tolerance in the wood frog, Rana sylvatica. J. Comp. Physiol. B 155, 29–36. Storey, K.B., Storey, M., 1988. Freeze tolerance in animals. Physiol. Rev. 68, 27–84. Terhivuo, J., 1988. Phenology of spawning for the Common Frog (Rana temporaria) in Finland from 1846–1986. Ann. Zool. Fenn. 25, 165–175. Tryjanowski, P., Rybacki, M., Sparks, T., 2003. Changes in the first spawning dates of
common frogs and common toads in western Poland in 1978–2002. Ann. Zool. Fenn. 40, 459–464. Vences, M., Galán, P., Palanca, A., Vieites, D.R., Nieto, S., Reye, J., 2000. Summer habitat use and diel activity cycles in a high altitude Pyrenean population of Rana temporaria. Herpetol. J. 10, 49–56. Vences, M., Galán, P., Vieites, D.R., Puente, M., Oetter, K., Wanke, S., 2002. Field body temperatures and heating rates in a montane frog population: the importance of black dorsal pattern for thermoregulation. Ann. Zool. Fenn. 39, 209–220. Viitanen, P., 1965. Hibernation and seasonal movements of the viper, Vipera berus berus (L.) in southern Finland. Ann. Zool. Fenn. 4, 472–546. Voituron, Y., Barré, H., Ramløv, H., Douady, C.J., 2009. Freeze tolerance evolution among anurans: frequency and timing of appearance. Cryobiology 58, 241–247. ZAMG. Annual climate reports. 〈http://www.zamg.ac.at/cms/en/climate/climateoverview/current_climate/annual_climate.〉 retrieved Feburary 3, 2015. Zhang, T., 2005. Influence of the seasonal snow cover on the ground thermal regime: an overview. Rev. Geophys. 43, RG4002. http://dx.doi.org/10.1029/ 2004RG000157.
Gerda Ludwig: My research interests are behavioural and physiological adaptations of amphibians to extreme environments. I am currently finishing my PhD on the hibernation ecology and physiology of common frogs in alpine regions at the Institute of Zoology at the University of Innsbruck, Austria.
Ulrich Sinsch: My research interests are behaviour and ecology of amphibians since Diploma and PhD thesis at the University of Cologne, Germany, which dealt with the behavioural regulation of body temperature and osmotic homeostasis. As a postdoc at the Max-Planck Institute of Behavioural Physiology at Seewiesen and at the University of Bonn focus changed to the orientation behaviour and population ecology of amphibians. As a Full Professor of Zoology at the University of KoblenzLandau, I have served as head of the Department of Biology and as Dean of the Faculty of Sciences. My current research focuses on the population ecology and biodiversity of palearctic and neotropical anurans, and on the limnology of temperate lakes.
Bernd Pelster: 1985: PhD; Institute for Zoology IV of Heinrich-Heine-Universität, Düsseldorf, Germany Supervisor: Prof. Dr. M.K. Grieshaber. 1985–1995: Research assistant; Institute for Physiology; Ruhr-University Bochum, Germany. 1989–1990: PostDoc; Department of Zoology, University of Massachusetts, Amherst, USA. 1996-current: Full professor and Head of the Division Animal Physiology at the Institute of Zoology and Limnology; Leopold-Franzens-University Innsbruck, Austria. 2001–2005: Chair of the Institute of Zoology and Limnolog. 2004–2008: Dean of the Faculty of Biology. 2013-current: Chair of the Institute of Zoology.
Contents lists available at ScienceDirect
Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio
Behavioural adaptations of Rana temporaria to cold climates Gerda Ludwig a,n, Ulrich Sinsch b, Bernd Pelster a a b
Institute of Zoology, University of Innsbruck, Technikerstr. 25, A-6020 Innsbruck, Austria Institute of Sciences, Department of Biology, University of Koblenz-Landau, Universitätsstraße 1, D-56070 Koblenz, Germany
art ic l e i nf o
a b s t r a c t
Article history: Received 3 November 2014 Received in revised form 12 February 2015 Accepted 13 February 2015 Available online 14 February 2015
Environmental conditions at the edge of a species’ ecological optimum can exert great ecological or evolutionary pressure at local populations. For ectotherms like amphibians temperature is one of the most important abiotic factors of their environment as it influences directly their metabolism and sets limits to their distribution. Amphibians have evolved three ways to cope with sub-zero temperatures: freeze tolerance, freeze protection, freeze avoidance. The aim of this study was to assess which strategy common frogs at mid and high elevation use to survive and thrive in cold climates. In particular we (1) tested for the presence of physiological freeze protection, (2) evaluated autumnal activity and overwintering behaviour with respect to freeze avoidance and (3) assessed the importance of different high-elevation microhabitats for behavioural thermoregulation. Common frogs did not exhibit any signs of freeze protection when experiencing temperatures around 0 °C. Instead they retreated to open water for protection and overwintering. High elevation common frogs remained active for around the same period of time than their conspecifics at lower elevation. Our results suggest that at mid and high elevation common frogs use freeze avoidance alone to survive temperatures below 0 °C. The availability of warm microhabitats, such as rock or pasture, provides high elevation frogs with the opportunity of behavioural thermoregulation and thus allows them to remain active at temperatures at which common frogs at lower elevation cease activity. & 2015 Elsevier Ltd. All rights reserved.
Keywords: Amphibians Rana temporaria Freeze protection Freeze avoidance Behavioural thermoregulation Microhabitat selection
1. Introduction Environmental conditions at the edge of a species’ ecological optimum can exert great ecological or evolutionary pressure at local population level. In this case adaptive or plastic responses to stressor at individual level facilitate the survival of populations (Hangartner et al., 2012; Dallinger and Höckner, 2013). Exploring the mechanisms that allow a species to persist in challenging environments is crucial for understanding how species deal with ecological and evolutionary pressure, especially in the light of global climate change. For ectotherms ambient temperature is one of the most important physical aspects of their environment. It affects directly physiological processes such as metabolism, growth, development and reproduction, muscle contraction, oxygen transport, and digestion (Carey and Alexander, 2003; Laugen et al., 2003a) and thus influences timing of hibernation and reproduction (e.g. Terhivuo, 1988; Beebee, 1994; Reading, 1998, 2003; Tryjanowski et al., 2003; Carroll et al., 2009) and global species richness patterns (Buckley and Jetz, 2007). Shorter n
Corresponding author. E-mail addresses: [email protected] (G. Ludwig), [email protected] (U. Sinsch), [email protected] (B. Pelster). http://dx.doi.org/10.1016/j.jtherbio.2015.02.006 0306-4565/& 2015 Elsevier Ltd. All rights reserved.
growing seasons impose time constraints on growth and development (Laugen et al., 2003b; Muir et al., 2014a), sub-zero temperatures during winter pose the risk of freezing and set geographical limits to the distribution of species. Only two amphibian species, the North American wood frog (Lithobates [Rana] sylvaticus) and the European common frog (Rana temporaria) are able to inhabit regions north of the Arctic Circle. Both species exhibit a wide geographic distribution: L. sylvaticus occurs from Alaska to Labrador, south to New Jersey, northern Georgia and northern Idaho (Chubbs and Phillips, 1998). R. temporaria is found from north of the Arctic Circle in Scandinavia to northern Spain, and in mountainous regions up to an elevation of 2600 m (Gasc et al., 1997). In both species populations are subject to a variety of climatic conditions, which substantially modify their annual activity cycle. For example, at the southern edge of their distribution, common frogs do not hibernate and breeding is initiated at the beginning of winter (Bea et al., 1986). In lowland populations in Switzerland and France common frogs can be found breeding as early as February (e.g. Ryser, 1989; Augert and Joly, 1993). With increasing latitude or altitude breeding starts later (e.g. Elmberg, 1990; Elmberg and Lundberg, 1991) and can occur as late as May in northern or high alpine populations. In general, there are three ways amphibians can cope with temperatures below zero (Pinder et al., 1992): (1) tolerate freezing
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of extra-cellular body fluids (freeze tolerance) (Storey and Storey, 1984); (2) production of cryoprotectants to prevent cells from freezing (freeze protection) (Storey and Storey, 1988) and (3) choosing hibernation sites where temperatures do not drop below zero (freeze avoidance) (Boutilier et al., 2000; Sinsch and Leskovar, 2011). The behavioural and physiological adaptations of L. sylvaticus to sub-zero temperatures are well studied. Wood frogs cease activity when temperatures drop below 4.4 °C (Bellis, 1962). Their ability to tolerate extra-cellular freezing and to prevent intra-cellular freezing by using glucose as cryoptrotectant (e.g. Storey and Storey, 1984, 1988; Layne and Lee, 1987; Costanzo et al., 1992), allows for terrestrial hibernation underneath leaf litter and snow, where temperatures drop as low as 7 °C (Schmid, 1982; MacArthur and Dandy, 1982). In contrast, our understanding of how R. temporaria deals with low temperatures is less clear. So far studies in high elevation or northern populations have focused on summer microhabitat use and diel activity (Vences et al., 2000), age structure (Ryser, 1996; Patrelle et al., 2012), breeding behavior (Elmberg, 1990; Elmberg and Lundberg, 1991) and larval development (e.g. Aebli, 1966; Angelier and Angelier, 1968; Brand and Grossenbacher, 1979; Laugen et al., 2003a; Muir et al., 2014a). With respect to the hibernation ecology of R. temporaria, the occurrence of both, aquatic and terrestrial hibernation has been reported (Viitanen, 1965; Koskela and Pasanen, 1974; Hagström, 1982; Elmberg, 1990; Pasanen and Sorjonen, 1994). Voituron et al. (2009) found 100% mortality in adult common frogs after eight hours of complete freezing of body fluids. Pasanen and Karhapää (1997) observed that adult R. temporaria survived 24 h exposure to sub-zero temperatures, but died within three days after exposure. Muir et al. (2014a) found that 82% of R. temporaria larvae from low-elevation sites survived short periods of freezing. From high elevation sites, only 32% survived. However, data on physiological and behavioural responses to low temperatures of wild adult common frogs are lacking. This gap of knowledge in the general biology of R. temporaria limits our ability to link local responses to phenotypes and thus assess the impact of environmental change on this species. Therefore the objectives of the present study were to compare mid- and high-elevation adult common frogs in terms of their physiological and behavioural adaptations to cold climates. In particular we (1) tested for the presence of physiological freeze protection, (2) evaluated autumnal activity and overwintering behaviour with respect to freeze avoidance and (3) assessed the importance of different high-elevation microhabitats for behavioural thermoregulation.
2. Material and methods 2.1. Study areas The study was conducted in two common frog populations at different elevations. The high elevation population is located in an alpine basin in the Fotsch Valley in the northern Stubai Alps, Austria (11°12′53″E, 47°08′59″N) at an elevation of 2300 m. A detailed description of this site is found it Ludwig et al. (2013). Based on annual egg mass counts during the breeding periods between 2011 and 2014 population size is estimated to be around 700 adults. Reproductive activity usually starts in mid to late May. The subalpine population is located in a small wetland area in the northeast of the city of Innsbruck, Austria (11°24′58″E, 47°17′11″N) at an elevation of 650 m. Several small ponds are surrounded by marsh area and mixed forest. Based on egg mass counts in spring 2013 and 2014 the population size is estimated to be around 200 adults. Breeding usually starts in mid-March.
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2.2. Experiments to test for freeze protection In 2013 17 adult common (16 males, 1 female) frogs were caught in breeding ponds at the beginning of spawning at the subalpine site and their blood glucose levels measured. At the alpine site 23 adults (21 males, 2 females) were caught on snow or ice-slush right after emergence from overwintering and tested for elevated blood glucose levels. Blood was drawn into a heparinized capillary tube from the Vena angularis as described by Nöller (1958) and transferred to a blood glucose meter (Accu-Chek Performa Nano, Roche Diagnostics GmbH, Germany, measuring range: 10–600 mg/dl; measuring precision according to EN ISO 15197:2003 standard: 95% of the individual glucose results shall fall within 7 15 mg/dL of the results of the manufacturer's measurement procedure at glucose concentrations of o75 mg/dL). Sampling was done in situ and did not last longer than 1 min. In addition we recorded air and water temperature using a digital thermometer (RS Digital Thermometer AH-50A Typ K, RS Components, Austria; measuring range: 50 °C to 200 °C; measurement precision: 0.1 °C; measuring error: þ/ 1.5 °C). To test for seasonal changes, we repeated blood glucose measurements in the alpine population in June (N ¼29, 19 males, 29 females) and late August (N ¼ 19, all males). These individuals were all caught in ponds. Blood glucose levels were compared using One-Way ANOVA. Significance level was set to alpha ¼0.05. 2.3. Experiments to test for freeze avoidance Over a period of three years (2011–2013) a total of 46 adult R. temporaria (mass 440 g; 31 males, 15 females) in both populations were marked with radio transmitters (BD-2H, 3.4 g and BD-2TH, 2.9 g, Holohil Systems Ltd., Canada) to monitor their movements. In 2011 transmitters were attached externally. The method of external attachment is described in Ludwig et al. (2013). In 2012 and 2013 transmitters were implanted into the body cavity. For surgery, frogs caught in the field were brought back to the lab and anaesthetized by submersion in a buffered solution of MS-222 (4 mg/L), through a small incision transmitters were implanted into the body cavity. The incision was closed using absorbable sutures (Novosyn HS 15, Braun, Spain) and frogs were rinsed under fresh water until fully recovered. After surgery frogs were kept in a tempered climate chamber (18 °C, L:D¼13:11) for three days to facilitate wound healing and then released at the site of capture. Marked frogs were located at least once a week. At each relocation we recorded the frog's position using GPS (Garmin, eTrex). In addition, we measured the line-of-sight distance each individual had moved between two observations. An animal moving more than 5 m between relocations was classified as active. When an individual remained sedentary for two or more consecutive relocations we assumed it had reached its hibernation site. At each site one data logger (TG-4100 Tinytag Aquatic 2, Gemini Data Loggers Ltd., UK) was placed on the ground and one in the stream to record air and water temperature during fall, winter and spring. Every year data loggers were placed in the same spots on the ground and in streams. Data loggers recording air temperature were placed directly on the ground shaded by a rock (alpine site) or shrubs (subalpine site). Temperatures were recorded in 2 h intervals from the beginning of September until the end of March in the subalpine population and the end of June in the alpine population. We used the telemetry data to calculate the autumnal activity period for each marked frog, defined as the number of days from September 16 (by this date all frogs had been released back into the field) until it reached its hibernation site. For each year in each population we calculated daily average, minimum and maximum temperature between September 16 and December 10. As activity patterns and hibernation behaviour in the alpine population were
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not sex-biased (Ludwig et al., 2013), male and female data were pooled for further analysis. We tested for differences in length of the activity period, and daily mean and daily maximum temperature between years within populations and between populations using One-Way ANOVA and Kruskal–Wallis Test. Significance level was set to alpha ¼0.05. Every spring we recorded the date of emergence from hibernation (high elevation population only) and appearance of first egg masses (both populations). 2.4. Microhabitat potential for behavioural thermoregulation To assess the importance of behavioural thermoregulation based on microhabitat selection, we conducted a series of solar exposure experiments during the 2014 breeding period in the alpine population. For this purpose, 20 adult common frogs were caught in and around breeding ponds and each frog's core, dorsal and ventral temperature was measured in situ using a thermistor (RS Digital Thermometer AH-50A Typ K, RS Components, Austria; measuring range: 50 °C to 200 °C; measurement precision: 0.1 °C; measuring error: þ/ 1.5 °C). Core temperature was measured by inserting the thermistor into the cloaca. After measurements were completed each animal was anaesthetized by submersion in a buffered solution of MS-222 (4 mg/L). When the amphibian failed to respond to a light pinch on a toe-tip, it was placed on snow, rock, pasture or underneath dwarf shrubs. After a 10 min acclimatisation period (Lillywhite, 1975; Sinsch, 1983) we again recorded air and substrate surface temperature as well as the animal's core, ventral and dorsal temperature. Then the animal was moved to the next substrate type and again left to acclimatize for 10 min before temperature measurements were repeated. This procedure was repeated until each individual had passed all four substrate types. The sequence of substrates tested, snow, rock, pasture and shrubs, was the same for all individuals. We tested for differences in substrate, air, core, dorsal and ventral temperature using One-Way ANOVA. To achieve normal distribution, all data sets were ln transformed. In addition we also tested for differences in frogs’ core, dorsal and ventral temperature between substrates using One-Way ANOVA on Ranks. Significance level was set to alpha ¼0.05. All statistical analyses were performed using the software package SigmaPlot 12.5 (Systat Software Inc., USA).
3. Results 3.1. Freeze protection In the high elevation population blood glucose concentration did not differ significantly immediately after emergence from hibernation in May, during spawning (June) and in fall (August) (One-way ANOVA F2,67 ¼1.879, p ¼0.161). All frogs caught right after emergence were found on snow or in ice-slush and were the only ones experiencing substrate surface temperature around 0 °C (mean¼ 2 °C, range: 0.3 to 5.2 °C). Their mean blood glucose level was 37.1 mg/dl (SD ¼16.1). In June and late August mean blood glucose levels were 42.9 mg/dl (SD ¼18.5) and 32.4 mg/dl (SD ¼6.9), respectively. Corresponding mean water temperatures were 9.6 °C and 12.3 °C. In the subalpine population mean blood glucose level during spawning was 46.3 mg/dl (SD ¼ 13.6). It differed marginally (p ¼0.043) from the fall blood glucose level in the high elevation population (Fig. 1). 3.2. Freeze avoidance Out of the 46 frogs originally marked, we were able to follow 25
Fig. 1. Box plots comparing blood glucose concentration of adult common frogs at different times of the year in the alpine and subalpine population. Solid lines inside the box show medians. Lower and upper boundary of the box indicates the 25th and 75th percentile. Whiskers indicate the 90th and 10th percentiles.
individuals to their hibernation sites. The transmitter signal of 14 animals was lost during the course of the study. 2 frogs were found dead. 1 animal was trapped and killed in ice when ponds started to freeze over. 3 individuals were preyed upon by an unknown predator and their transmitters located in a deserted marmot cave. From one individual the transmitter had to be removed after the antenna got tangled up in grass. Hibernation site selection did not vary between populations. In the alpine population 11 individuals hibernated in streams and a slow flowing spring, three individuals hibernated in ponds. In the subalpine population 10 animals hibernated in the stream, one individual retreated to a pond. Terrestrial hibernation did not occur in our study. The length of autumnal activity periods did not differ significantly between years and between populations (One-Way ANOVA F3,22 ¼ 2.8, p ¼0.064, Table 1). In the alpine population the first individual began hibernation on October 4, the last one on November 28. In 2013 early build-up of snow led to a sudden end of the active period on November 7 (Julian day 307; Fig. 2). Subalpine frogs retreated to the stream between October 18 and November 27 (Fig. 3). Mean daily temperature and daily maximum temperature did not differ significantly between years within populations, but did differ between low and high elevation population (Kruskal–Wallis Test, H ¼76.87, p o0.05, Table 1). In 2012 high elevation frogs emerged from streams on May 25 and started spawning on May 27. In 2014 emergence started on May 20, spawning on May 25. Due to the permanent ice and snow cover during winter, stream temperature was not influenced by fluctuations of ambient temperature until ice break in spring. Stream temperature fluctuated between 0.3 °C and 1.0 °C in 2012, and 1.0–3.2 °C in 2014. Corresponding spring air temperature on the ground varied between 1 °C and 1 °C as long as data loggers were covered by snow (Julian day 1–151 in 2012, Julian day 1–144 in 2014) (Fig. 4). The dense vegetation and the lack of snow on the ground did not permit us to observe emergence from hibernation at the subalpine site. Therefore only the onset of spawning was recorded each year. In 2013 spawning started on March 13, in 2014 on March 11. Although the low elevation stream did not freeze over during winter, water temperature remained fairly constant and fluctuated around 5 °C in both years. Due to the lack of a permanent snow cover air temperature near the ground fluctuated during each study period and varied between study periods. In
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Table 1 Mean, minimum and maximum values for length of autumnal activity period (d), mean daily temperature (°C) and mean daily maximum temperature (°C) in the alpine population (2300 m asl) and subalpine population (650 m asl). High elevation population 2011
Length of activity period (d) Mean temperature (°C) Mean maximum temperature (°C)
Low elevation population 2013
2012
2013
Mean (SD)
Range
Mean (SD)
Range
Mean (SD)
Range
Mean (SD)
Range
56.9 (9.8) 1.9 (4.7) 5.3 (5.1)
41–76 6.4–11.6 2.7–15.7
40.7 (13.2) 2.6 (2.8) 7.1 (8.9)
18–53 0.2–10.0 0.2–30.0
54.0 (14.4) 7.3 (5.3) 10.5 (6.6)
40–74 3.4–21.1 3.1–24.2
56.4 (9.8) 7.0 (6.6) 9.4 (5.7)
38–62 3.6–16.3 1.3–19.2
2013 sub-zero temperatures occurred frequently. In contrast, in 2014 temperatures did not drop below 0 °C except for the beginning of January (Fig. 5). 3.3. Microhabitat potential for behavioural thermoregulation The surface temperature of pasture was the highest of all four substrates (mean ¼26.4 °C, SD ¼8.9) reaching maxima of 39.4 °C and being significantly higher than corresponding mean air temperature (One-Way ANOVA F2,56 ¼9.99, p o0.001). Also rock surface temperature was significantly above air temperature (OneWay ANOVA F2,56 ¼6.36, p ¼0.003). Shrubs were the thermally most balanced microhabitat. Mean substrate and air temperature did not differ significantly (One-Way ANOVA F2,56 ¼1.42,
p¼ 0.250). The snow's surface was the coldest and significantly lower than air temperature (One-Way ANOVA F2,56 ¼106.06, po 0.001; Fig. 6). Exclusively on snow the frogs’ core, dorsal and ventral temperature differed significantly (One-Way ANOVA F2,56 ¼ 40.8, po0.001), with dorsal temperature being the highest and ventral temperature being the lowest. On rock, pasture and underneath shrubs there was no significant differences between core, dorsal and ventral temperature (One-Way ANOVA, rock: F2,56 ¼ 0.95, p ¼0.40; grass: F2,56 ¼ 0.10, p ¼0.91; shrubs: F2,56 ¼0.02, p¼ 0.98; Fig. 6). When comparing substrate types, core, ventral and dorsal temperature differed significantly between pasture and snow and pasture and shrubs as well as between rock and snow. Between rock and shrubs only core and dorsal temperature differed
Fig. 2. Daily mean (solid line), maximum (upper dashed line) and minimum (lower dashed line) fall air temperature at ground level in the alpine population in 2011 (a) and 2013 (b). Date is shown in Julian days (1 ¼January 1; 259 ¼ September 16; 344¼ December 10). Symbols and numbers indicate when how many frogs retreated into hibernation.
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Fig. 3. Daily mean (solid line), maximum (upper dashed line) and minimum (lower dashed line) fall air temperature at ground level in the subalpine population in 2012 (a) and 2013 (b). Date is shown in Julian days (1 ¼ January 1; 259 ¼September 16; 344¼December 10). Symbols and numbers indicate when how many frogs retreated into hibernation.
Fig. 4. Mean daily stream temperature (dashed lines) and air temperature at ground level (solid lines) at the alpine site between January 1 and May 31 in 2012 (thin lines) and 2014 (bold lines). Date is shown in Julian days (1 ¼ January 1). Triangles indicate the emergence from hibernation, circles indicate the onset of spawning (open symbols ¼ 2012, closed symbols ¼ 2014).
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Fig. 5. Mean daily stream temperature (dashed lines) and air temperature at ground level (solid lines) at the subalpine site between January 1 and March 31 in 2013 (thin lines) and 2014 (bold lines). Date is shown in Julian days (1 ¼ January 1). Circles indicate the onset of spawning (open symbols ¼ 2013, closed symbols ¼2014).
significantly. Between shrubs and snow only ventral temperature differed significantly (One-way ANOVA on Ranks H3 ¼41.45; p o0.05). No significant difference in core, dorsal and ventral temperature was found between rock and pasture.
4. Discussion At first glance the presence of a large and stable common frog population at a high elevation study site is surprising considering the harsh local climate. Under such extreme environmental conditions amphibians are required to develop a series of adaptations to grow, accumulate energy reserves and reproduce despite overall low temperatures and short growing seasons. 4.1. Freeze protection and freeze avoidance The absence of seasonal blood glucose variation indicates that even high altitude common frogs do not mobilize glycogen as a cryoprotectant. Blood glucose concentrations of emerging high altitude common frogs experiencing temperatures around or below zero were not elevated compared to those of conspecifics tested in summer and fall (Fig. 1) and corresponded to blood glucose levels measured in wood frogs kept at 23 °C (52.02 mg/dl; Storey and Storey, 1984). In comparison, blood glucose concentration in frozen wood frogs reached up to 333.3 mg/dl (Storey and Storey, 1984). Although we were not able to measure blood glucose levels directly during hibernation and therefore cannot exclude the possibility of a change in blood glucose levels between hibernation and emergence, the results of our telemetry study show that common frogs retreat to frost-free hibernation sites during winter months where there is no need for freeze protection. In contrast, emerging frogs can be found on snow or in slush were the surrounding temperatures are around or below 0 °C. If these frogs do not exhibit elevated blood glucose levels it is unlikely that blood glucose levels of frogs exposed to constant water temperature above 0 °C are elevated. These findings suggest that common frogs in subalpine and alpine regions use behavioural
adaptations alone to avoid freezing and to thrive in cold climates. At the first glance air temperature at ground level during winter seem surprisingly mild, especially at the alpine study site, and may raise questions of how representative these data are. Based on the annual climate report of the Austrian Institute for Meteorology and Geodynamics, winter 2011/12 and 2012/2013 were typical winters. Winter of 2013/2014, however, was one of the warmest years in the past 246 years (ZAMG, 2015; online information) The mild subzero temperatures at ground level in the alpine population, can be explained by the fact that dataloggers were snowed in for most of the winter in 2011/12 (Julian day 344 in Fig. 2a to Julian day 150 in Fig. 4) in and again between Julian day 284 and 294 (Fig. 2b) and from Julian day 307 (Fig. 2b) to Julian day 141 (Fig. 4) in 2013/2014. Depending on the overall weather conditions and the presence or absence of frozen soil at the onset of snowfall, air temperature at the snow- soil interface usually fluctuates around 0 °C (Ishikawa, 2003; Zhang, 2005; Rödder and Kneissel, 2012). Due to the lack of a permanent snow cover in the subalpine study area air temperature shows great daily fluctuations and has overall lower temperature minima than alpine air temperature (Fig. 3(a) and (b), Fig. 5). The mild winter of 2013/2014 is reflected in the overall warmer air temperature at the subalpine site (Fig. 5). However, because of the insulation effect of the snow cover (Ishikawa, 2003; Zhang, 2005; Rödder and Kneissel, 2012), the overall milder weather conditions during the winter months in 2013/2014 are not as clearly visible in the data from the alpine site. The lack of freeze protection in subalpine and alpine adult common frogs agrees with the altitudinal variation in freeze protection found in common frog larvae (Muir et al., 2014a). Larvae from breeding sites at 703 m asl. exhibit a lower level of freeze protection than larvae from breeding sites between 77–222 m asl. Muir et al. (2014a) suggested that due to the lack of an insulating snow and ice cover during winter, complete freezing of ponds occurs more frequently at low altitudes. Therefore freeze protection may be selected for at low elevation. A similar evolutionary divergence seems possible for adult common frogs. Whereas
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Fig. 6. Box plots comparing surface temperature of snow (a), rock (b), pasture (c) and shrubs (d) with the core, dorsal and ventral temperatures of common frogs. Solid lines inside the box show medians, dashed lines means. Lower and upper boundary of the box indicates the 25th and 75th percentile. Whiskers indicate the 90th and 10th percentiles.
subalpine and alpine individuals hibernating in streams never experience freezing, freeze protection may be present in common frogs exposed to milder climatic conditions during winter, that allow for terrestrial hibernation, but also bear the risk of occasionally occurring sub-zero temperatures. Further field studies are needed to test this hypothesis. 4.2. Microhabitat potential for behavioural thermoregulation Although mean ambient fall temperature at 2300 m asl. was on average 5 °C lower than at 650 m asl., autumnal activity periods in both populations were of similar length (Table 1). In fall timely migration to hibernation streams ensures that high-elevation common frogs can retreat into the water, whenever temperatures drop below zero, e.g. during night or episodic snow fall. Comparison of the 2013 maximum daily temperatures from the alpine site and the 2013 mean daily temperature from the subalpine site show that maximum values at high elevation are similar or above mean values at low elevation. This suggests that during the day warm microhabitats along the streams, e.g. rocks or pasture, provide high elevation animals with the opportunity for behavioural thermoregulation. The results from the solar exposure
experiments support this hypothesis and show that especially pasture and rock microhabitats allow frogs to raise their body temperature significantly (Fig. 6). Despite overall lower temperature these behavioural traits potentially allow high elevation common frogs to remain active for a similar period of time as their conspecifics at lower elevation and thus give them more time to build up energy stores for overwintering. Also in spring the availability of open water ensures the survival of common frogs at high altitude. At the high elevation site, the break-up of ice over hibernation streams coincides with the ice break on breeding ponds, so that frogs emerging from streams have access to open water during nocturnal temperature drops or cold spells. Since migration from hibernation sites to breeding ponds occurs before snowmelt on land is completed, animals must cross extended patches of snow. To minimise the risk of predation posed by a dark-coloured frog on white background, fast advancement is advantageous. However, in order to move quickly it is crucial for ectotherms to maintain a certain body temperature (Lillywhite, 1970; Vences et al., 2002). We observed two ways in which common frogs accomplish this goal while moving across snow: (1) during resting periods on snow common frogs show an
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atypical upright posture with their front legs completely stretched. This reduces the area of body surface in contact with snow and minimises heat loss through conduction. Our conclusion is supported by the results of the solar exposure experiments. Only on snow dorsal temperature was significantly higher than ventral temperature (Fig. 6). (2) Migrating high elevation common frogs use snow-free spots (e.g. pasture, wind or sun exposed places or small boulders) as heat islands to warm up. The thermal gain by this behaviour agrees with the results from our experiments. Surface temperatures of both pasture and rock were significantly higher than corresponding air temperature and in both cases frogs’ core temperature was significantly higher than air temperature (Fig. 6). In a laboratory-based study Sinsch (1984) found evidence of thermo-regulative behaviour in lowland frogs (60 m asl.). These frogs showed a shift in activity pattern from nocturnal at warmer temperatures to diurnal at colder temperatures. In addition they remained mostly in the terrestrial part of the experimental setup at lower temperatures, whereas the time spent in water increased with increasing temperature. These results suggest that thermoregulative behaviour is also present in lowland common frogs. However, we found that common frogs from the subalpine population did not use thermo-regulative behaviour to extend their autumnal activity period. Instead they ceased activity at temperatures at which their high elevation conspecifics remained active. Since emergence from overwintering occurs earlier and growing seasons are longer at lower elevations, low elevation animals do not need to remain active in fall for a longer time to accumulate sufficient energy reserves for overwintering. Our finding of intraspecific variation in cold tolerance based on behavioural thermoregulation disagrees with the pattern found in L. sylvaticus. In a laboratory based study, Manis and Claussen (1986) found no evidence of intraspecific variation in cold tolerance in individuals along a latitudinal gradient ranging from 37 °34′ N to 45°31′N. They concluded that since wood frogs encounter sub-zero temperatures during winter and spring in all parts of the species’ range, cold tolerance is not selected against in populations experiencing overall warmer climatic conditions. These contrasting results from two species inhabiting similar ecological niches lead to the question if in amphibians physiological adaptations are more often genetically fixed than behavioural ones. Clearly, our data do not allow to distinguish between phenotypic plasticity and genetic adaptation as origin of the observed behavioural thermoregulation in high-elevation common frogs. Genetic adaptation to altitude has been found for several physiological traits such as sexual maturity and UV resistance (Miaud and Merilä, 2001), larval growth rate and larval period (Muir et al., 2014b). In Scandinavia adaptations in a range of larval fitness traits across a latitudinal gradient have been found (Laugen et al., 2003a, 2003b; Palo et al., 2003). Further work examining the genetic or plastic nature of behavioural traits crucial to the survival in stressful environments is needed for the assessment of the future evolutionary potential of species.
4.3. Conclusion Our results show that subalpine and alpine R. temporaria do not use glucose as a cryoprotectant as observed in the closely related North American wood frog. Instead they exhibit a series of behavioural adaptations that enable them to survive and thrive in cold environments. Our results highlight the complex habitat requirements of R. temporaria in alpine environments.
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Acknowledgment We thank Dr. M. Seewald and his team from the Alpenzoo Innsbruck for performing the implantation of transmitters. Thanks to N. Schintgen, D. Anker, D. Hittler and E. Ludwig for assistance with fieldwork, R. Ludwig for laboratory support, and M. Sztatecsny for providing telemetry equipment. Fieldwork was financially supported by the (Grant number 134263) and the Institute of Zoology, University of Innsbruck. G. Ludwig was supported by a PhD scholarship from the University of Innsbruck. All protocols used in this study were in accordance with the Austrian Legislation for animal experiments and approved by the ethical committee of the former Austrian Federal Ministry for Science and Research (BMWF-66.008/0010-II/3b/2011, BMWF-66.008/0012-II/ 3b/2012, BMWF-66.008/0017-II/3b/2012) as well as the Department of Environmental Protection of Tyrol.
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Gerda Ludwig: My research interests are behavioural and physiological adaptations of amphibians to extreme environments. I am currently finishing my PhD on the hibernation ecology and physiology of common frogs in alpine regions at the Institute of Zoology at the University of Innsbruck, Austria.
Ulrich Sinsch: My research interests are behaviour and ecology of amphibians since Diploma and PhD thesis at the University of Cologne, Germany, which dealt with the behavioural regulation of body temperature and osmotic homeostasis. As a postdoc at the Max-Planck Institute of Behavioural Physiology at Seewiesen and at the University of Bonn focus changed to the orientation behaviour and population ecology of amphibians. As a Full Professor of Zoology at the University of KoblenzLandau, I have served as head of the Department of Biology and as Dean of the Faculty of Sciences. My current research focuses on the population ecology and biodiversity of palearctic and neotropical anurans, and on the limnology of temperate lakes.
Bernd Pelster: 1985: PhD; Institute for Zoology IV of Heinrich-Heine-Universität, Düsseldorf, Germany Supervisor: Prof. Dr. M.K. Grieshaber. 1985–1995: Research assistant; Institute for Physiology; Ruhr-University Bochum, Germany. 1989–1990: PostDoc; Department of Zoology, University of Massachusetts, Amherst, USA. 1996-current: Full professor and Head of the Division Animal Physiology at the Institute of Zoology and Limnology; Leopold-Franzens-University Innsbruck, Austria. 2001–2005: Chair of the Institute of Zoology and Limnolog. 2004–2008: Dean of the Faculty of Biology. 2013-current: Chair of the Institute of Zoology.