Are high-latitude individuals superior competitors? A test with Rana temporaria tadpoles
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- Lindgren, B. & Laurila, A. Evol Ecol (2010) 24: 115. doi:10.1007/s10682-009-9294-4
Species with a wide distribution over latitudinal gradients often exhibit increasing growth and development rates towards higher latitudes. Ecological theory predicts that these fast-growing genotypes are, in the absence of trade-offs with fast growth, better competitors than low-latitude conspecifics. While knowledge on key ecological traits along latitudinal clines is important for understanding how these clines are maintained, the relative competitive ability of high latitude individuals against low latitude conspecifics has not been tested. Growth and development rates of the common frog Rana temporaria increase along the latitudinal gradient across Scandinavia. Here we investigated larval competition over food resources within and between two R. temporaria populations originating from southern and northern Sweden in an outdoor common garden experiment. We used a factorial design, where southern and northern tadpoles were reared either as single populations or as mixes of the two populations at two densities and predator treatments (absence and non-lethal presence of Aeshna dragonfly larvae). Tadpoles from the high latitude population grew and developed faster and in the beginning of the experiment they hid less and were more active than tadpoles from the low latitude population. When raised together with high latitude tadpoles the southern tadpoles had a longer larval period, however, the response of high latitude tadpoles to the competition by low latitude tadpoles did not differ from their response to intra-population competition. This result was not significantly affected by density or predator treatments. Our results support the hypothesis that high latitude populations are better competitors than their low latitude conspecifics, and suggest that in R. temporaria fast growth and development trade off with other fitness components along the latitudinal gradient across Scandinavia.
KeywordsActivityGrowthIntraspecfic competitionLatitudinal clinesRana temporaria
Large-scale climatic variation is an important factor causing adaptive intraspecific variation in a wide range of organisms (e.g. Conover and Schultz 1995; Angilletta et al. 2003; Ashton 2004; Blanckenhorn and Dermot 2004). For example, the shorter growth season at higher latitudes often selects for latitudinal clines in growth and development rates in ectotherms, as successful completion of the juvenile stages is crucial for many organisms and increased body size often enhances over winter survival (Roff 1980; Arendt 1997; Altwegg and Reyer 2003; Munch et al. 2003; Sears 2005). Accordingly, increased growth and development rates towards higher latitudes have been found in a wide range of ectotherms, including insects (Roff 1980; Arnett and Gotelli 1999; Gilchrist et al. 2001), fish (Conover and Present 1990; Conover et al. 1997; Imsland et al. 2000) and amphibians (Berven and Gill 1983; Merilä et al. 2000).
While many morphological, behavioural and physiological traits can affect the competitive ability of individuals in exploitative competition, in mobile organisms body size and activity level are often positively correlated with competitive ability. Large animals are competitively superior because of their greater capacity to forage (Connell 1983; Schoener 1983; but see Persson 1985). High activity levels also increase competitive ability by improving harvesting rate (Werner 1992, 1994; Grill and Juliano 1996). Therefore, the higher growth and activity rates of high latitude populations may make them competitively superior over slower growing low latitude populations. Such a competitive superiority of fast-growing genotypes is supported by studies making use of among-strain variation of hatchery and wild salmonid fishes (e.g. Devlin et al. 1999; Biro et al. 2004, 2006).
The relationship between latitudinal variation in growth and development and other important ecological traits, such as competitive ability or predator avoidance, are seldom studied. However, such information would be valuable if we are to understand how phenotypic variation along climatic gradients is maintained or how intra- and inter-specific interactions are modified if the climate changes. In the only study we are aware of that investigated competitive ability along a latitudinal gradient, James and Partridge (1998) found that high latitude Drosophila melanogaster populations were poorer competitors at high temperatures than low latitude populations when tested against a common laboratory strain. To our knowledge, no studies examining the competitive ability of conspecific animal populations along a latitudinal gradient have been conducted.
In anuran tadpoles, intraspecific competition can be intense and negatively affect growth and survival, especially when mortality due to predation is low (see Wilbur 1980; Skelly and Kiesecker 2001 for reviews). Density-dependence during the aquatic larval stage can have a major effect on population regulation in amphibians (Wilbur 1980; Smith 1983; Berven 1990; Loman 2004), although more recent studies have also emphasized the importance of the terrestrial stages for population regulation (Hellriegel 2000; Biek et al. 2002; Vonesh and De la Cruz 2002).
Growth and development rates of larval common frogs (Rana temporaria) increase along the latitudinal gradient across Scandinavia (e.g. Merilä et al. 2000; Laugen et al. 2003; Lindgren and Laurila 2005). Furthermore, high latitude tadpoles exhibit higher activity levels (Laurila et al. 2008), which, together with their larger body size, is likely to increase the competitive ability of high latitude tadpoles as compared to low latitude conspecifics. Importantly, high latitude tadpoles maintain their higher growth and development rates as well as higher activity over a range of temperatures (e.g. Laugen et al. 2003; Lindgren and Laurila 2005; Laurila et al. 2008) strongly suggesting adaptation to season length rather than to prevailing temperature (Conover and Schultz 1995; Laugen et al. 2003; Palo et al. 2003). High latitude tadpoles also have higher growth efficiency turning ingested food more effectively into biomass than low latitude conspecifics (Lindgren and Laurila 2005). In R. temporaria, intraspecific competition among tadpoles can be intense, and Loman (2004) found evidence for density-dependent regulation at the larval stage in several ponds in southern Sweden, suggesting that larval competition is an important ecological factor affecting population size in this species.
Here we investigated intraspecific competition between and within two populations of R. temporaria tadpoles situated 1,000 km apart along the latitudinal gradient across Sweden. We conducted an outdoor experiment where we addressed two main questions. First, do the fast-growing and active northern tadpoles have a competitive advantage over their southern conspecifics? We predicted that competition has a stronger negative effect on the slow-growing, less active southern tadpoles when raised together with the faster growing, more active northern tadpoles. In mixed-population treatments, we assigned the population origin of the tadpoles by using a diagnostic microsatellite marker with no alleles shared between the populations. As size-dependent competitive ability may depend on the amount of resources available for each individual (Persson 1985), we conducted the experiment at two densities. Second, since predator presence has a strong effect on activity of R. temporaria tadpoles (e.g. Laurila et al. 2004), we conducted the experiment both in the presence and absence of a non-lethal (caged) insect predator and asked: does predation risk affect the competitive outcome between northern and southern tadpoles? We predicted that the northern tadpoles would be less inclined to reduce activity and sacrifice growth due to predator presence, leading to an increase of competitive superiority of the northern over the southern tadpoles in the presence of predators.
Materials and methods
R. temporaria has a large distribution area in Europe from northern Spain to northernmost Norway and breeds in a wide range of aquatic habitats (Gasc et al. 1997). Based on our previous studies along the latitudinal gradient (Merilä et al. 2004, A. Richter-Boix et al. in preparation), we chose two representative populations from the southern and northern parts of the gradient. The low-latitude population was obtained by collecting 12 pairs of adult R. temporaria in a pond outside Uppsala (Stora Almby, 59°51′ N, 17°28′ E; hereafter U population) on April 15 2004. This population has growth and development rates typical to an open-canopy pond in this area (A. Richter-Boix et al., manuscript in preparation) The high latitude population was obtained by collecting ca. 500 eggs from each of 12 freshly laid egg clutches near Kiruna (Jukkasjärvi, 67°51′ N, 21°02′ E; hereafter K population) on May 20. Also this population has growth and development rates typical to this geographic area (population N5 in Merilä et al. 2004). The K population is located ca. 1,000 km N of the U population. The growth season length (daily mean temperature over 5°C) is ca. 194 and 113 days in U and K populations, respectively (Odin et al. 1983). Both populations breed in permanent, open-canopy ponds. The populations are fairly large (ca. 100 breeding females in U, >50 females in K), and larval competition is likely to occur within both populations. In addition, large populations of R. arvalis and Bufo bufo tadpoles may compete with R. temporaria tadpoles in U, but R. temporaria is the only amphibian species in K. Both ponds have large dragonfly and diving beetle predators, however, predator density is higher in U (Laurila et al. 2008).
To be able to conduct a competition study between two phenologically separated populations, the tadpoles from the two populations need to hatch at approximately the same time. By collecting adults in U and storing them in the laboratory we delayed their reproduction until the breeding started in K. Our original plan was to collect adults also in K and make artificial crossings in the same manner as in the southern population. Unfortunately, we were unable to capture a sufficient number of adult individuals in K and used field-collected fresh eggs instead.
To evaluate the possibly harmful effects of time delay in breeding of the U population, we conducted the crosses in two batches. Five pairs were crossed the following day after being captured (April 16, with methods described in Räsänen et al. 2003) to produce five full-sib families which acted as treatment controls. The remaining seven pairs were kept in the laboratory at 4°C in plastic boxes filled with moist peat moss (Sphagnum) until they were crossed on May 20 to produce seven full-sib families for the experimental U population.
For both populations ca. 500 eggs per family were raised in the laboratory (18°C, 18 h light:6 h dark photoperiod) in two 3 l vials (250 eggs per vial) filled with reconstituted soft water (RSW; APHA 1985) until they reached Gosner stage 25 (Gosner 1960, gills fully absorbed). The water in the vials was changed completely every 3 days. To investigate whether the extended storing of the U adults affected the larval life history traits of the offspring, we reared U tadpoles from both the early and late crosses until metamorphosis in temperature-controlled laboratory (18°C, 18 h light:6 h dark photoperiod), corresponding to the conditions in Uppsala area during the late larval period (Orizaola and Laurila 2009; A. Richter-Boix and A. Laurila, unpublished). About 10–11 (early) or 5–8 (late cross) U tadpoles from each family were raised individually in 1 l vials filled with 0.8 l of RSW. The vials were arranged on four shelves (blocks) to account for a known vertical temperature gradient within the room. The tadpoles were fed ad libitum chopped spinach and the water was changed every 4 days.
The experiment was conducted outdoors in a fenced field close to Uppsala. Due to logistic limitations we could not replicate the experiment under high latitude conditions, however, our laboratory studies indicate that K individuals maintain higher growth and development rates over a range of relevant temperatures (14–22°C; e.g. Laugen et al. 2003; Lindgren and Laurila 2005; Laurila et al. 2008) suggesting that our results are robust in respect to the location of the experiment. We filled 60 opaque plastic tanks (36 × 40 × 90 cm) with 80 l of tap water 2 weeks before the start of the experiment and added 1 l of pond water as an algal oculum. We added 10 g of dried aspen (Populus tremula) leaves and 6 g of rabbit pellets to act as a nutrient base and to provide food and shelter for the tadpoles. The tanks were covered with mosquito net to prevent colonisation by insects. The photoperiod during the experiment corresponded to late larval period in both populations (day length in June in Uppsala corresponds roughly to August day length in Kiruna), suggesting that the populations were likely to experience the time of season in a similar manner. Moreover, in laboratory the effects of photoperiod on the growth and development in R. temporaria tadpoles are considerably smaller than the genetic differences found between southern and northern populations (Laurila et al. 2001; A. Laurila and S. Pakkasmaa, unpublished data) suggesting that photoperiod cues played only a minor role in the responses.
The two populations reached Gosner stage 25 simultaneously, and the experiment was started 2 days later (June 3, day 0 of the experiment). A 2 × 2 × 2 × 2 randomized block design consisting of two populations (U, K), two densities (low and high), two between-population competition treatments (mixed and alone), two predator treatments (present or absent) and five spatial blocks was used. The tanks were placed in five rows (the blocks) 1 m apart, with one randomly placed replicate of each treatment combination within each block giving us five replicates per treatment and a total of 60 tanks (note that the mixed-populations treatment tanks with U and K tadpoles together were the same for the two populations). For each population we pooled tadpoles in equal amounts from each family to one container before adding them to the experimental tanks. Each tank received either 10 (low density) or 30 (high density) tadpoles and the populations were allocated to these treatments either alone (only U or K tadpoles) or as mixes of the two in equal proportions (i.e. 5 or 15 tadpoles from both populations).
As predators we used late-instar dragonfly larvae (Aeshna sp.) collected in ponds near Uppsala. In the experiment, the predators were kept in transparent plastic cages (Ø 80 mm, height 210 mm) with a double net bottom (mesh size 1.5 mm) allowing the tadpoles to receive both visual and chemical cues from the predator. One cage was placed in the middle of each tank, hanging ca. 7 cm from the bottom. Each cage in the predator treatment received one dragonfly larva, whilst in the no-predator treatment the cage was left empty. The predators were fed every second day with ca. 300 mg of R. temporaria tadpoles.
Tadpole behaviour was observed around noon for three consecutive days during two periods: days 4–6 (period 1) and days 14–16 (period 2) of the experiment. On each observation day the number of visible (i.e. not hiding) and the number of active (moving tail) tadpoles was recorded in each tank at five occasions separated by 30 min.
After the first individuals metamorphosed (stage 42, Gosner 1960; occurrence of at least one forelimb), the containers were checked daily for metamorphs. Metamorphosed tadpoles were sacrificed using MS-222 and preserved in 70% ethanol. From the preserved individuals we later measured body length (BL; from the tip of the snout to the cloaca) using a digital calliper to the nearest 0.01 mm, and body mass (BM) to the nearest 0.1 mg. Larval period (LP) was measured as days elapsed from the start of the experiment to metamorphosis. Growth rate (GR) was estimated by dividing BM with LP. Tissue samples were taken from the metamorphs in the mixed treatments in order to establish population origin by using molecular methods (see below).
Allele frequencies and sample size (individuals) analyzed for RtμH microsatellite locus in the U parental generation, K tadpoles of known family origin and experimental individuals in the mixed populations-treatment
Sample size (N)
We used mixed model ANOVAs with REML estimation (proc Mixed in SAS 9.1) to test if storing the parental U individuals in the laboratory affected metamorphic body weight, body length, growth rate or larval period of the tadpoles. Cross type and block were treated as fixed effects and family (nested under cross type) as a random effect.
The behavioural and life history data from the outdoor competition experiment were analysed using MANOVAs followed by univariate ANOVAs. Our experimental design was 2 × 2 × 2 × 2 × 5 randomized block design. However, since the two populations in the mixed treatment could not be differentiated in the behavioural analyses, separate analyses were run for behavioural and life history data. In the behavioural analyses we had four factors: mix, with three levels [U alone, both populations (mix), and K alone], density, predator and block. For each tank, we calculated the mean proportion of active and visible individuals in relation to initial tadpole density during each 3-day period. The behavioural data were arcsin-squareroot transformed before the analyses. Six tanks were excluded from the behavioural analyses because algal blooming interfered with the observations. Removal of these tanks did not affect the results qualitatively. Tank means were used as response variables in the life history trait analyses. Survival was determined as the number of metamorphosing tadpoles divided by the original number of tadpoles in the experimental unit and analysed with type III general linear models with a logit link function and binomial error structure using GENMOD procedure in SAS.
Effects of crossing date on U life history
MANOVA table for the effects of block, density, mix (competition with own or foreign population) and predator presence on the behaviour of R. temporaria tadpoles during the two time periods (1 and 2)
Density × Mix
Mix × Predator
Density × Predator
Density × Mix × Predator
Density had a strong effect on tadpole behaviour during period 2. In contrast to period 1, tadpoles were now more active and hid less at low density (Table 2; Fig. 2). The main effects of predator and mix were not significant, but there was a significant density × mix × predator interaction, with tadpoles in the low density-mixed treatment combination being less active and hiding more than tadpoles in the single population treatments in the presence of a predator (Table 2; Fig. 2). Moreover, the opposite, although not as strong, pattern emerged in the absence of a predator, i.e. the tadpoles hid less and were more active in the mixed than in the single population treatment (Fig. 2). Block had no significant effect on any of the behavioural traits (Table 2).
Life history and survival
MANOVA table on the effects of block, population, density, mix (competing with own or foreign population) and predator presence on R. temporaria larval life history traits
Population × Mix
We found evidence for high latitude R. temporaria tadpoles being stronger competitors than low-latitude conspecifics. U tadpoles had significantly longer larval periods when raised together with K individuals, whereas the effect of the U population on the K population did not differ from the single-population K treatment. As in previous studies (Laugen et al. 2003; Lindgren and Laurila 2005; Laurila et al. 2008), high latitude tadpoles had higher growth and development rates, and early in the experiment they were more active and hid less than low latitude tadpoles, likely contributing to the higher competitive ability of the K population. A second factor increasing the competitive ability of the high latitude tadpoles is their higher growth efficiency (Lindgren and Laurila 2005), which allows more effective transformation of ingested food into biomass. Our ability to draw conclusions is somewhat weakened by the fact that only two populations were used in this study. However, individual growth and development rates in the present populations are similar to those found in other R. temporaria populations within these geographical areas (Merilä et al. 2004; A. Richter-Boix et al. in preparation), suggesting that our results are representative for the populations living in these areas. The results are also in accordance with previous studies from other systems showing that individuals or populations with higher somatic growth rates are competitively dominant (e.g. Devlin et al. 1999; Biro et al. 2004, 2006).
The competitive effect of K tadpoles on U tadpoles was longer larval period. While the effect was relatively mild (ca. 3 days later metamorphosis in U in the presence of K), this may have negative consequences on later life stages as studies in other amphibian species have found that timing of metamorphosis affects juvenile survival and age at maturation (Smith 1987; Altwegg and Reyer 2003; but see Relyea 2007). At any rate, the delay in metamorphosis induced by competition by K tadpoles is likely to increase the competitive disadvantage of metamorphosing U tadpoles during the terrestrial phase as, depending on the treatment combination, U tadpoles will metamorphose 4–7 days later than K tadpoles (Fig. 3). Interestingly, we found no evidence for the K population negatively affecting size or growth of U individuals (or vice versa) suggesting that in response to competition by K tadpoles, U individuals metamorphosed later rather than at smaller size. This is line with the notion that post-metamorphic fitness effects are more strongly correlated with size than larval period (reviewed by Relyea 2007). Nevertheless, size and growth—but not larval period—were strongly negatively affected by density in both populations, indicating that density effects were mediated in a different way than the effects of population-specific competitive ability. Hence, at high density U tadpoles paid an additive ‘double cost’ of competition in terms of both size and time, suggesting that the combined fitness consequences of inter-population competition were more severe at high density.
Events during larval life stages can have strong effects on amphibian population regulation because mortality during this stage is often high and because larval life history traits, often modified by density-dependent processes, influence fitness later in life (e.g. Smith 1983; Berven 1990; Altwegg and Reyer 2003; Loman 2004, see also Hellriegel 2000; Vonesh and De la Cruz 2002). While direct competition between the genotypes used in the present experiment is highly improbable due to the geographic distance, our results suggest that under a climate change scenario larval competition by low-latitude genotypes moving towards higher latitudes is unlikely to have negative effects on high-latitude R. temporaria unless other environmental conditions (e.g. predator density, see below) along the gradient are also changing. However, a more definitive test of this question could perhaps be done by using more closely located populations showing differentiation in behaviour and life history traits (see Relyea 2002; Van Buskirk and Arioli 2005 for amphibian examples) as these genotypes are more likely to compete with each other.
If we accept the negative effect by K tadpoles on the larval period of U tadpoles as an indicator of relative competitive ability between southern and northern genotypes, we may ask the question what prevents the fast-growing and -developing northern populations from spreading southwards? The explanation could be trade-offs arising via costs of high growth rates. These costs include e.g. higher vulnerability to starvation, decreased pathogen resistance, reduced strength of vital structures and increased risk of predation (reviewed by Arendt 1997). In the case of Scandinavian R. temporaria, the available data suggest that at least predation may play a role. In general, the high activity levels of fast-growing individuals make them more vulnerable to predation (Werner and Gilliam 1984; Mangel and Stamps 2001; Brodin and Johansson 2004) suggesting that the high growth rate of high latitude individuals trades off with higher vulnerability to predation. In R. temporaria, high latitude tadpoles are more vulnerable to predation, and predator densities are considerably lower at the northern end of the gradient (Laurila et al. 2008; see Lankford et al. 2001; Laurila et al. 2006 for other systems). Hence, larval growth and development rates along the gradient may be balanced against the predation risk in the breeding ponds. If predator densities will increase in the northern breeding ponds as a result of climate change, this will favour the southern genotypes, which have higher survival in the presence of predators (Laurila et al. 2008).
Our second prediction, the competitive effect of the K population on the U population is stronger in the presence of a predator, was not fulfilled. A possible explanation for this result is that as compared to previous studies (e.g. Laurila et al. 2004, 2008) predators had relatively little effect on tadpole behaviour. The weak antipredator response in U tadpoles may have been affected by the fact that the crosses were conducted very late in the season (see below). As we found increased growth rates in the late crosses in the laboratory experiment, it seems likely that the growth rates of the U tadpoles were similarly increased in the outdoor experiment. The high growth rate may have been coupled with increased activity levels and a weaker behavioural antipredator response (see Johansson et al. 2001; Altwegg 2002 for examples from other systems). In addition, stronger competition by K tadpoles may have induced higher activity in the U tadpoles in the mixed-populations treatments. Indeed, tadpoles in the low density-mixed populations-predator present treatment behaved more like high density tadpoles, suggesting that competition by K tadpoles affected the behaviour of U tadpoles in this treatment (Fig. 2). This notion is further supported by the observation that, although the three-way interaction was not statistically significant, U tadpoles tended to have longer larval periods in this treatment as compared to the predator absent treatment (Fig. 3).
U tadpoles from the late (experimental) crosses grew faster and were larger at metamorphosis, but developed at the same rate as U tadpoles from the early (control) crosses. Since the experimental crosses were conducted over a month later than the normal breeding time, it is possible that the increased tadpole growth is a consequence of maternal effects (Mousseau and Fox 1998). One possibility is that the delayed females made a higher hormonal or nutrient investment in their eggs to increase growth rate, and hence survival probability, of offspring produced late in the season (Uller et al. 2007). Notwithstanding a potentially interesting adaptive explanation and although the genetic basis of R. temporaria life history variation along the Scandinavian latitudinal gradient is well established (Laugen et al. 2002, 2005; Palo et al. 2003), this result calls for caution when interpreting intraspecific variation among and within amphibian populations.
As already stated above, the delay of the birth date of the experimental U tadpoles may have influenced our results. However, the increased growth rate of the experimental U as compared to control U tadpoles suggests that they performed as well, or perhaps better, in competition than if they were born at the natural time. Indeed, the low latitude tadpoles may have been induced to increase activity and/or physiological growth mechanisms and, consequently, growth by delaying the breeding by a month. This may have made our study a conservative assessment of the competitive effect along the gradient.
We found evidence that competition by fast-growing genotypes from a high-latitude population had a negative effect on development time of slow-growing genotypes from a low-latitude population in a widespread amphibian species. High-latitude tadpoles were larger and more active, which together with higher growth efficiency (Lindgren and Laurila 2005) gives them a competitive advantage in the absence of a lethal predator. However, the trade-off between predation risk and activity/growth may affect the outcome of competition in natural ponds. In this study, we chose to focus on competition over a range of relevant ecological conditions which vary along the present gradient (conspecific and predator density; Johansson et al. 2006; Laurila et al. 2008) and influence competitive interactions in tadpoles (e.g. Werner 1992, 1994). While there is generally no or very little overlap in growth and activity between the low and high latitude R. temporaria populations, two populations are clearly few to draw definitive conclusions about competitive ability along latitudinal gradients. More comprehensive studies including replicated populations and lethal predators will be necessary to fully understand how competition and predation influence life history and behaviour along climatic gradients in R. temporaria and other species with wide distribution along climatic gradients.
We thank Sofia Wennberg for help with the experiment, Gunilla Engström and Kerstin Santesson for help in the molecular laboratory, and Jon Loman, Gerard Malsher, German Orizaola and Katja Räsänen for valuable comments on earlier versions of the manuscript. This study was performed with the permission of the Ethical Committee for Animal Experiments in Uppsala County and funded by the Swedish Research Council (grant to AL) and Zoologiska Stiftelsen (BL).