Can age and growth patterns explain the geographical variation in the body size of two toad species?

Abstract

Determining both the age structure and growth pattern allows to establish the causal factors, environmental and/or genetic, that eventually may be responsible for the observed pattern of divergence. We examined the variation in age structure and growth pattern across populations of two toad species, Pelobates cultripes and Epidalea calamita that exhibit a geographic variation in body size in southern Spain. For both species, populations differed in mean age but age structure did not correlate with body size variation across populations. Although the population with the youngest females found for E. calamita was the smallest in body size, the oldest males for both species were found in a small body size population. The growth pattern fit well to a von Bertalanffy growth model and interdemic divergence were found for both the asymptotic body size (S_m ) and the growth coefficients (k). As expected, Large-Bodied populations of both species attained higher S_m but, Small-Bodied population had higher, although non significantly different, k growth coefficients. Also, the Small-Bodied population attained sexual maturity sooner but had also high longevity. The observed pattern may reflect both environmental variations in resources availability affecting body size observed across populations, but also different growth and maturity pathways that may respond to contrasting selective pressures.

Key words
Age; Anura; body size; Epidalea calamita; growth; Pelobates cultripes

INTRODUCTION

Body size is a complex attribute and many factors are responsible for its variation (Angilletta & Dunham 2003). Environmental conditions are determinant of ectotherms body size variation and current knowledge considers temperature as a main driver of large-scale size clines. There is a widespread plastic response of body size to rearing temperatures, with larger individuals occurring at lower temperatures and smaller individuals at warmer temperatures (temperature-size rule hypothesis, Atkinson 1994). For endotherms, Bergmann’s rule (Bergmann 1847), states that body size increases with latitude or altitude. Ray (1960) and Lindsey (1966) suggested that ectothermic species also follow this rule and display intra- and inter-specific body size variation as a response to environmental gradients. While the validity of Bergmann’s rule in endothermic species is well-documented (e.g. Ashton 2002a, Freckleton et al. 2003), in ectothermic vertebrates, such as amphibians, conflicting evidences were found for both Bergmann and converse Bergmann clines, reflecting inconsistent large scale patterns across taxa (e.g. Adams & Church 2008, Ashton 2002b, Cvetkovic et al. 2009, Sinsch et al. 2010). In addition, amphibians also may exhibit striking body size variation at smaller spatial scale, for nearby locations with similar climate characteristics, but with variation in microhabitat conditions, such as substrate nature, that may impose nutritional and/or hydric constraints (Marangoni et al. 2008, Leclair et al. 2005). Body size also varies according to sex: sexual size dimorphism is a population property that describes the intraspecific differences in overall size and shape traits between males and females (Fairbairn 2007). The evolution of sexual size dimorphism is an indirect consequence of the different reproductive roles of males and females (Andersson 1994), emerging from sexual differences in life history (Halliday & Verrell 1988, Halliday & Tejedo 1995, Monnet & Cherry 2002).

Amphibians appear to adapt to temporally uncertain environments with life-history trade-offs, such as variable age at maturity (Wilbur & Rudolf 2006), a strategy called “bet hedging” by Slatkin (1974). Differences in age-related parameters (e.g. age at maturity and longevity) could explain population divergence if individuals from small-bodied populations mature earlier or have a lower survival rate (i.e. reduced longevity), compared to large-bodied ones (e.g. Bruce & Hairston 1990, Cogălniceanu & Miaud 2003). For example, reduced life-span was suggested as the main factor of body size reduction of Pelobates cultripes inhabiting sandy substrates of Sierra de Ariça (Portugal) (Leclair et al. 2005). A significant reduction in body size was reported in both P. cultripes and Epidalea calamita populations in southwestern Spain, over a mere 60 km transect associated to a geological transition from Hercinic to sandy substrates. Such reduction in body size was accompanied by a drastic change in clutch mass, clutch size, and egg size in both species (Marangoni et al. 2008). One potential explanation is that body size reduction in these populations is the result of adaptive covariation among several life-history traits aimed at optimizing fecundity (Sinsch et al. 2010) or simply the result of phenotypic plasticity induced by changes in any environmental factors (e.g. substrate) affecting growth rates (van Kleunen & Fischer 2005). For example, in ectotherms, sexual maturity is delayed and occurs at smaller size when growth rate is lowered by low food availability, while it occurs also later but at larger sizes when growth rate is lowered by decreased temperature (Berrigan & Charnov 1994).

Extreme body size variation in four amphibian species was reported from the sandy substrates from Doñana area, southwestern Spain. Populations of the Southern Marbled Newt (Triturus pygmaeus) and Bosca’s Newt (Lissotriton boscai) have a decrease in snout-vent length (SVL) of 29.0 % in males and 32.2 % in females for T. pygmaeus (García-París et al. 1993, Díaz-Paniagua et al. 1996, Díaz-Paniagua & Mateo 1999). Populations of two toad species, the Iberian Spadefoot Toad (Pelobates cultripes) and the Natterjack Toad (Epidalea calamita) (Marangoni & Tejedo 2008, Marangoni et al. 2008), exhibit a decrease in body mass of 71.6% and 76.1% and in SVL of 36.8% and 35.6% for P. cultripes and E. calamita, respectively, in nearby populations (less than 30 km) inhabiting Hercinic substrates (Marangoni & Tejedo 2008, Marangoni et al. 2008). This variation in body size over a small spatial scale appears to be coupled with changes in age-related parameters, with earlier age of maturity and shorter longevities in dwarf populations of the newt T. pygmaeus (Díaz-Paniagua et al. 1996). This suggests an important aspect when establishing geographical variation in amphibian body size; because their indeterminate pattern of growth, implying that body-size continues to increase throughout life, even after reaching sexual maturity, determine the attainment of a larger asymptotic body size (Stamps 1993, Halliday & Tejedo 1995). In addition, as in most ectotherms, clutch and, sometimes, egg size are positively correlated with amphibian female size (Duellman & Trueb 1986, Wells 2010). Analyzing clinal variation in size and the environmental determinants of intraspecific body size variation requires a parallel knowledge of the relationships between populational variation in age and their correspondence with size and related traits, such as reproductive outputs. In this study, we tested whether interpopulational divergence in age structure and growth are responsible for the variation in body size and reproductive output in two toad species, the Iberian Spadefoot Toad (Pelobates cultripes) and the Natterjack Toad (Epidalea calamita) along a 120 km transect in southern Spain.

MATERIALS AND METHODS

Area and species studied

Based on the previous studies done in the region (Marangoni & Tejedo 2008, Marangoni et al. 2008), we selected six populations for each species at locations distributed along 120 km stretch encompassing two areas with contrasting geological substrates: old Hercinic granite schist substrates of the Sierra Morena (Sevilla province) and Sandy substrates from Quaternary aeolian deposits in the Doñana region (Huelva province) (Fig. 1). The three populations from Hercinic substrate were categorized as Large-Bodied populations (hereafter): Pedroso, Navas and Aznalcóllar, with a range of altitudes between 100 and 420 m. Similarly, the three populations from the Sandy substrates were categorized as Small-Bodied population (hereafter): Bodegones, Abalario and Reserva Biológica de Doñana (RBD) with a range of altitudes between 20 and 63 m (Fig. 1). Two out of six E. calamita populations (Navas and RBD) were included in a previous study by Sinsch et al. (2010). All studied populations of both species breed in small and shallow temporary ponds that flooded by rainfalls and dry up in the summer and their breeding is synchronic (Marangoni & Tejedo 2008, Marangoni et al. 2008). The main climatic variables do not vary significantly along the transect: annual average rainfall was 640 mm and 585 mm, while average monthly temperature was 25.7⁰C and 24.5⁰C in July, and 9.6⁰C and 10.6⁰C in January, for Navas and RBD populations, respectively (Díaz-Paniagua 1986, F. Marangoni & M. Tejedo, unpublished data).

Figure 1
Location and geological substrate of the studied Epidalea calamita and Pelobates cultripes populations. Large-Bodied population (open circle) and Small-Bodied population (solid circle). Abbreviated names of sampling populations, geographic coordinates (Coordinates UTM x/y in meters, Datum European 1950, Spain and Portugal, Zone: 30) and elevation, are as follows: PED (255170/4190574, 395 m); NAV (229255/4187617, 420 m); AZN (210199/4158023, 130 m); BOD (175577/4120711, 32 m); ABA (174267/4115417, 63 m); RBD (188450/4102197, 24 m).

Assessing age-related parameters

We collected 248 Epidalea calamita and 149 Pelobates cultripes adults from their breeding ponds from fall 1999 to winter 2004. Toad collection methods and body size measurements are detailed in Marangoni et al. (2008). Briefly, calling males or pairs in amplexus were collected during chorusing nights, to ensure that all individuals were mature. Upon capture animals were measured and weighted, and a toe was cut and stored in 70% alcohol for age assessment. All individuals were released back into their original ponds within 24-48 h after their capture. The protocol used for skeletochronology followed the method proposed by Castanet & Smirina (1990) with small modifications (Stănescu et al. 2016). Two independent observers recorded the presence/absence of the line of metamorphosis (LM) and counted the lines of arrested growth (LAG). In those individuals with no remnant LM, we estimated the degree of resorption by osteometrical analysis following Sagor et al. (1998), so that endosteal resorption did not prevent age estimation. We distinguished annual growth marks (i.e., LAGs sensu stricto) from non-annual ones (i.e., irregular interruptions during periods of inactivity), using the method described in Sinsch et al. (2007). Annuli (sensu Peabody 1958) were easily distinguishable from actual LAGs since they always stained weaker than true LAGs and were often broader, as previously described by Leclair et al. (2005) and Sinsch et al. (2007) in temperate species. Occasionally, in some individuals, one or two annuli were visible within the summer growth period. We assumed that the number of LAGs is equivalent to the number of aestivations experienced by each individual, thus giving a direct estimation of individual age.

Demographic life history traits

For each reproductive individual analyzed we obtained the following five, sex-specific and age-related, variables (sensu Leskovar et al. 2006): (1) age at sexual maturity: the minimum number of LAGs; (2) longevity: the maximum number of LAGs; (3) potential reproductive lifespan: the difference between longevity and age at sexual maturity; (4) mean lifespan: mean of age distribution; and, (5) size at sexual maturity: the average snout-vent length of all first breeders with the minimum number of LAGs.

Growth Estimation

We estimated the average body growth of toads (SVL) using von Bertalanffy growth equation (von Bertalanffy 1938):

$$S_t=S_m–(S_m–S_0)e^{–k(t–t0)}$$

where t = number of growing seasons experienced (age); t ₀ = age at metamorphosis (proportion of the growing season already elapsed at metamorphosis); S_t = average body size after having experienced t growing seasons; S_m = average maximal body size; S₀ = average body size at metamorphosis; k = growth coefficient, defining the slope of the growth curve, measuring the exponential rate of approach to S_m . Fair estimates of S₀ for each population, and thus allowing a better model adjustment, were obtained as average body size at metamorphosis from two independent larval growth experiments (P. cultripes: Marangoni & Tejedo 2008, E. calamita: Marangoni 2006, see Table IV). Additionally, for fitting P. cultripes growth models, we used mean body size of 5-month-old juveniles, obtained from another study (Marangoni 2006).

Table I
Age and size-related parameters of Small (Sandy substrate) and Large-Bodied populations (Hercinic substrate) of Epidalea calamita and Pelobates cultripes. AM = age at sexual maturity of the youngest first breeders; PRLS = potential reproductive lifespan, computed as the difference between longevity and AM; SVL = snout-vent length (mm), BM: body mass (g). SDI: sexual dimorphism index. All values for Age, SVL, BM and Minimum SVL at AM are expressed as mean ± 1 SE, [n]: sample size.

Species / Populations	Sex	Age (LAGs)	AM (LAGs)	Longevity (LAGs)	RCM	PRLS (yr)	SVL (mm)	BM (g)	Minimum SVL at AM	Maximum SVL (age)
E. calamita
Large-Bodied	♂	2.46 ± 0.11 [39]	1	4		3	72.64 ± 0.53 [177]	32.61 ± 0.92 [115]	61.5 ± 4.5 [2]	88(3)
	♀	3.6 ± 0.17 [43]	2	8	0.41 ± 0.02 (77)	6	71.43 ± 0.71 [98]	28.5 ± 0.92 [77]	67.4 ± 3.75 [5]	85(5)
	♂+♀	2.94 ± 0.12 [82]	1	8		7	72.24 ± 0.43 [275]	31.04 ± 0.68 [192]	---	---
	SDI	+1.46					-1.01	-1.14	+1.09	-1.04
Small-Bodied	♂	3.79 ± 0.18 [89]	1	7		6	55.04 ± 0.60 [147]	12.79 ± 0.42 [147]	51.89 ± 1.34 [9]	77(4)
	♀	3.27 ± 0.19 [77]	1	8	0.47 ± 0.02 (102)	7	53.53 ± 0.65 [105]	11.09 ± 0.44 [105]	50.55 ± 2.92 [11]	86(6)
	♂+♀	3.52 ± 0.13 [166]	1	8		7	54.41 ± 0.44 [252]	12.08 ± 0.31 [252]	---	---
	SDI	-1.16					-1.03	-1.15	-1.03	+1.12
P. cultripes
Large-Bodied	♂	4.16 ± 0.34 [19]	2	8		6	85.92 ± 0.76 [101]	45.46 ± 1.28 [101]	75[1]	99(5)
	♀	4.64 ± 0.41 [11]	2	6	0.35 ± 0.13 (4)	4	96.02 ± 2.03 [37]	60.14 ± 5.67 [27]	85[1]	125(6)
	♂+♀	4.11 ± 0.28 [30]	2	8		6	88.63 ± 0.86 [138]	48.56 ± 1.64 [128]	---	---
	SDI	+1.12					+1.12	+1.32	+1.13	+1.26
Small-Bodied	♂	4.67 ± 0.18 [72]	1	8		7	67.30 ± 0.58 [191]	22.0 ± 0.52 [188]	57[1]	86(6)
	♀	4.83 ± 0.2 [47]	2	8	0.29 ± 0.02(64)	6	71.52 ± 0.98 [134]	23.16 ± 0.89 [112]	79 ± 1 [2]	97(6)
	♂+♀	4.72 ± 0.14 [119]	1	8		7	68.85 ± 0.55 [325]	22.33 ± 0.47 [300]	---	---
	SDI	+1.03					+1.06	+1.05	+1.39	+1.13

Reproductive effort

We used relative clutch mass (RCM) as an estimate of reproductive effort, calculated as the ratio of clutch mass to spent body mass (Shine 1992, Bonnet et al. 2003). Reproductive traits of the amplectant pairs such as clutch and egg size were obtained following Marangoni et al. (2008). Briefly, the amplectant pairs were assigned to plastic containers where they quickly went back into amplexus and oviposition took place within 12 h. The resulting egg clutches were photographed and the eggs counted and measured. Clutch mass was obtained by subtracting the female body mass before and after deposition. All individuals were released back into their original ponds within 24-48 h after their capture.

Statistical analyses

We computed a sexual dimorphism index (SDI) for SVL, body mass and average age, with the results arbitrarily defined as positive when females are larger than males and negative in the converse situation (Lovich & Gibbons 1992), since it allows to compare all three parameters (but see Ranta et al. 1994):

$$\mathrm{SDI}=\frac{{\mathit{\text{Mean size}}}_{\mathit{\text{larger sex}}}}{{\mathit{\text{Mean size}}}_{\mathit{\text{smaller sex}}}}$$

Where “Mean size” refers to either SVL, body mass or average age. The value of SDI is equal (if there is not sexual dimorphism) or greater than unit (when one of the sexes has a larger mean size). The results are defined arbitrarily as positive when females are larger than males and negative when males are larger.

All variables were log-transformed to achieve normality. Two-tailed t-test compared mean age between sexes and species and the Kolmogorov–Smirnov tested populational variation in age distribution frequencies. Univariate analyses of variance (ANOVA) tests for differences in mean age and RCM between the sexes, environments (Hercinic versus Sandy substrates) and populations, within environments. Population was considered a random effect throughout the analyses. We adjusted P-values by the sequential Bonferroni procedure, when multiple comparisons or tests were performed (Rice 1989). The von Bertalanffy growth model was fitted employing the least square procedure. We used type III general linear models using the STATISTICA 6.0 statistical package (StatSoft Inc. 2001). Mean values were compared using post-hoc Scheffé multiple comparison test, at α = 0.05. We used linear regression to test the association between age and body size. All results are expressed as mean ± 1 standard error (SE).

RESULTS

Lines of Arrested Growth (LAGs) were well defined in the periosteal bone and allowed a proper age estimation in all individuals of both species. For those specimens in which resorption occurred during the formation of endosteal bone, at least 76% and 85% of the first LAG (E. calamita and P. cultripes, respectively) the LM was never completely eroded. The age structure and age-size related parameters are presented in Table I and Fig. 2. The differences between frequency distributions of age between substrates (Hercinic vs. Sandy) by sex and, between sexes within substrate are presented, in Table II. Body size differed between populations occurring at different soil substrates, populations with smaller individuals of both species were found in the Sandy substrates populations (E. calamita, SVL: F ₁,₂₁₃ = 128.99, P < 0.0001, BM: F ₁,₂₁₃ = 211.59, P < 0.0001; P. cultripes: SVL: F ₁,₁₁₂ = 124.73, P < 0.0001, BM: F ₁,₁₁₂ = 175.72, P < 0.0001, Table I). The sexual dimorphism index (SDI) showed differences in body size between the sexes in P. cultripes, with females larger than males, whereas in E. calamita there are no differences in SVL between sexes, but males have higher body mass compared to females (Table I; see also Table I in Marangoni et al. 2008). Age class distributions between Large and Small-Bodied populations varied only in E. calamita males, but not in E. calamita females and both sexes in P. cultripes (Table II). As a consequence, mean age showed a significant “sex x substrate” interactions in E. calamita (Table III, Fig. 4). E. calamita males from Small-Bodied populations were on average older than Large-Bodied ones, but females from different sized populations were identical in mean age (Figure 4, Table I and Table III). Additionally, this “sex x substrate” interaction indicates that females were significantly older than males in Large-Bodied populations, but not mean age differences between sexes were found in Small-Bodied ones (Table I, Fig. 4).

Figure 2
Age distribution of Small-Bodied (Upper) and Large-Bodied (Lower) populations of males and females of Epidalea calamita and Pelobates cultripes.

Figure 3
Mean age variations among populations of Epidalea calamita and Pelobates cultripes. Values for both sexes are pooled. Open = Large-Bodied populations, solid = Small-Bodied populations. All values are means ± 1 SE. Different letters mean significant differences using a Scheffe´ post hoc multiple comparison test at α = 0.05.

Figure 4
Effects of sex x substrate interaction on age in Epidalea calamita.

Table II
Comparison of frequency distributions of age between substrates (Hercinic; large-bodied populations; Sandy: Small-bodied populations) by sex (a), and between sexes within substrate. n_L and n_S, frequency of Large and Small-bodied individuals, respectively (b). Kolmogorov-Smirnov test frequency. Significant values are highlighted in boldface.

Epidalea calamita	nL	nS	Dmax	P
Sex / Substrate	Large-Bodied vs Small-Bodied
♂	36	80	-0.469	<0.001
♀	26	73	0.216	>0.10
♂+♀	62	153	-0.246	< 0.01
Substrate /Sex	Males vs Females
Large-Bodied	36	26	-0.483	<0.005
Small-Bodied	80	73	0.132	>0.10
Large + Small Bodied	124	99	-0.055	>0.10
Pelobates cultripes
Sex / Substrate	Large-Bodied vs Small-Bodied
♂	13	67	-0.259	>0.10
♀	5	49	-0.159	>0.10
♂+♀	18	116	-0.218	>0.10
Substrate /Sex	Males vs Females
Large-Bodied	13	5	-0.323	>0.10
Small-Bodied	67	49	-0.060	>0.10
Large + Small Bodied	80	54	-0.083	>0.10

Table III
Results of two-way mixed model nested ANOVAs for responses of Epidalea calamita and Pelobates cultripes populations to substrate (Hercinic, Large-Bodied population vs Sandy, Small-Bodied populations), accounting for variation among sexes. Response variable are Mean Age and Relative Clutch Mass (RCM). Significant values are highlighted in boldface.

Source	Mean Age								Mean RCM
	E. calamita				P. cultripes				E. calamita				P. cultripes
	d.f.	F	P		d.f.	F	P		d.f.	F			d.f.	F	P
Substrate	1	0.16	0.719		1	0.04	0.845		1	3.11	0.180		1	0.13	0.727
Population (Substrate)	3	31.34	0.009		4	3.24	0.140		3	1.24	0.297		2	4.19	0.020
Sex	1	2.25	0.217		1	0.01	0.937
Sex x Population (Substrate)	3	1.49	0.217		4	1.66	0.162
Sex x Substrate	1	14.54	0.023		1	0.61	0.448
Error	238				135				174				64

Table IV
Estimated parameters from von Bertalanffy’s equation, for body growth (SVL) of Epidalea calamita and Pelobates cultripes. S _m = average maximal body size, S ₀ = body size at metamorphosis (see text for details). k = growth coefficient, defining the shape of the growth curve, r ² = model fit. SDI: sexual dimorphism index. All values are means ± 1 SE (CI 95%).

Species / Populations	Sex	SVL (mm)
		S m	S 0	k	r 2
E. calamita
Large-Bodied	♂	69.94 ± 1.18 (67.59 – 72.29)	13.28 ± 0.54 (12.2 – 14.35)	1.51 ± 0.23 (1.05 – 1.97)	0.988
	♀	74.77 ± 1.94 (70.92 – 78.62)	13.29 ± 0.69 (11.90 – 14.67)	0.81 ± 0.13 (0.56 – 1.06)	0.982
	♂+♀	71.78 ± 1.10 (69.61 – 73.95)	13.31 ± 0.74 (11.85 – 14.76)	1.20 ± 0.15 (0.91 - 1.50)	0.979
	SDI	+1.07		-1.86
Small-Bodied	♂	59.99 ± 0.66 (58.72 – 61.27)	12.94 ± 0.82 (11.31 – 14.57)	1.43 ± 0.15 (1.13 - 1.72)	0.972
	♀	55.33 ± 0.82 (53.70 – 56.95)	12.86 ± 0.99 (10.92 – 14.82)	1.90 ± 0.31 (1.28 - 2.52)	0.956
	♂+♀	58.05 ± 0.59 (56.89 – 59.22)	13.01 ± 1.01 (11.02 – 15.01)	1.53 ± 0.15 (1.24 - 1.82)	0.940
	SDI	-1.08		+1.33
P. cultripes
Large-Bodied	♂	100.08 ± 3.64 (92.88 – 107.28)	29.95 ± 0.57 (28.83 – 31.07)	0.40 ± 0.05 (0.31 – 0.49)	0.959
	♀	118.00 ± 8.17 (102.65 – 134.98)	30.49 ± 0.59 (29.31 – 31.66)	0.26 ± 0.04 (0.17 – 0.35)	0.950
	♂+♀	101.99 ± 3.42 (95.23 – 108.75)	29.81 ± 0.68 (28.47 – 31.16)	0.41 ± 0.05 (0.32 – 0.50)	0.961
	SDI	+1.17		-1.54
Small-Bodied	♂	73.05 ± 0.82 (71.45 – 74.66)	29.70 ± 0.38 (28.95 – 30.46)	0.63 ± 0.04 (0.55 – 0.71)	0.967
	♀	79.22 ± 1.24 (76.78 – 81.67)	29.65 ± 0.42 (28.81 – 30.48)	0.55 ± 0.04 (0.46 – 0.63)	0.961
	♂+♀	74.96 ± 0.81 (73.37 – 76.55)	29.52 ± 0.47 (28.59 – 30.46)	0.64 ± 0.05 (0.56 – 0.73)	0.961
	SDI	+1.08		-1.15

There was mean age populational variation within particular substrate in both E. calamita (Population (substrate): P <0.01, Table III, Fig. 3) and P. cultripes, (only examined within the Sandy substrates), F ₂,₁₁₀ = 7.521, P < 0.001). Both species showed that ABA population has the older mean age differing from all analyzed populations (post hoc Scheffe´ multiple comparison test, the lowest P value found, E. calamita: P < 0.0001, P. cultripes: P = 0.002), (Fig. 3, Appendix A). Finally, no other sources of mean age variation, such as sex or substrate main effects were significant (Table III).

RCM variation was not explained by population origin, substrate / size for both species and only a population effect was found in P cultripes (Table III). Within the Small-Bodied sandy substrate populations, RBD (the Smallest-Bodied population) showed the highest reproductive effort (0.40 ± 0.02 (7)), even higher than Large-Bodied population, although it only was significantly different from BOD population (post-hoc comparisons, Scheffé range test, RCM: RBD vs BOD, P = 0.0398). Also, despite “population within environment” effects were not observed in E. calamita, the RBD (the Smallest-Bodied population) also showed the highest reproductive effort among all populations (0.49 ± 0.03 (35)).

The patterns of growth for body size fits well with a von Bertalanffy’s growth model (Table IV, Fig. 5) and interpopulation divergences were found for both the estimated asymptotic body size (S_m ) and the growth coefficient (k). As expected, Large-Bodied populations of both species attained higher asymptotic size (S_m ), but Small-Bodied population had higher growth coefficients, although the differences are not significant (Table IV and Table V).

Figure 5
Growth curves fitted to the von Bertalanffy model, for body growth (SVL) in Epidalea calamita (left panels) and Pelobates cultripes (right panels). Large-Bodied population – top-line; Small-Bodied population – bottom line.

Table V
T-student test for differences between Large-Bodied (LBP) and Small-Bodied populations (LBP) in estimated parameters from von Bertalanffy equations, for body growth (SVL) of Epidalea calamita and Pelobates cultripes. S _m = average maximal body size, k = growth coefficient, defining the slope of the growth curve. Significant values are highlighted in boldface.

Species	Sex	S m (LBP vs SBP)				k (LBP vs SBP)
		t	df	P		t	df	P
E. calamita	♂	4.978	114	<0.001		0.086	114	0.466
	♀	7.945	97	<0.001		-0.805	97	0.211
	♂+♀	7.040	213	<0.001		-0.355	213	0.361
P. cultripes	♂	8.606	78	<0.001		-0.350	78	0.364
	♀	7.610	52	<0.001		-0.326	52	0.373
	♂+♀	7.970	132	<0.001		-0.283	132	0.389

DISCUSSION

Our study showed that interdemic differences in age and growth are only partially responsible for the extreme reduction in body size in two toad species over a short geographical range associated to soil substrate differences. Thus, the Small-Bodied populations compensate for smaller size by achieving earlier sexual maturity. This allows for a longer potential reproductive lifespan, thus compensating for the lower number of eggs deposited during a single annual reproductive event (fig. 6). Thus, females from both populations tend towards depositing a similar number of eggs during their life-span by either growing a larger body size (and producing a larger clutch size), or maturing earlier and increasing the reproductive life-span.

Figure 6
A schematic comparison of size and age-related parameters in Large and Small-Bodied populations.

Several factors can contribute to small adult sizes in amphibians: reduced egg size, reduced larval and/or juvenile growth and early sexual maturation. Small females often tend to lay smaller eggs (Semlitsch & Gibbons 1990, Michimae 2007, Davenport & Summers 2010, Vignoli et al. 2018) which in turn tend to yield smaller metamorphs (Bernardo 1996). Variation in larval growth may affect adult size, especially when compensatory post-metamorphic growth does not take place (Metcalfe & Monaghan 2001), but differences in growth rate between juveniles may fill the body size gap (Vignoli et al. 2018, Székely et al. 2020). Post-metamorphic individuals exhibit indeterminate growth that declines when sexual maturity is attained (Hemelaar 1988). Differences in larval growth partially explain the divergence in size at metamorphosis, accounting for 20% of total population divergence in adult size found between the toad populations from different substrates, as indicated by common garden experiments (Marangoni & Tejedo 2008). Our study highlighted that age-related parameters are partly responsible for this pattern of size variation.

There are differences between sexes in mean age, with males having a lower mean age, except for Small-Bodied E. calamita populations, where males live longer. Longevity (i.e. highest age class observed) is higher in females in E. calamita. The differences between sexes in mean age are highest in the Large-Bodied populations. The differences in mean age and longevity are smaller in P. cultripes, compared with

E. calamita. Both species present a steep reduction in body size and body mass, within just about 30 km, in association with the Sandy substrates (Marangoni et al. 2008). Such reduction in body size also causes a parallel decrease in reproductive output and particularly on egg size in both species (Marangoni et al. 2008), and a reduced body size at metamorphosis in both species (Marangoni 2006, Marangoni & Tejedo 2008). Since juveniles from the Small-Bodied populations metamorphose later and at a smaller size compared with those from Large-Bodied populations (Marangoni & Tejedo 2008), they compensate by reaching sexual maturity rapidly, after the first year, and afterwards almost stop growing, as indicated by the higher k values. Thus, most energy is directed towards reproduction and little to growth in Small-Bodied populations.

Age and body size at sexual maturity indicate how fast individuals can start reproducing and are also an indicator of their reproductive output, since fecundity is often associated with body size (Plaistow et al. 2004). A study of two syntopic species of the genus Pelobates that differ in their sexual size dimorphism showed that different growth rates before sexual maturity and differences in energy allocation between growth and reproduction after sexual maturity are responsible for the differences in adult size (Cogălniceanu et al. 2014).

The reasons and implications of sexual size dimorphism in anurans have been extensively debated (Shine 1979, Woolbright 1983, Halliday & Verrell 1986, Monnet & Cherry 2002), but no clear pattern is apparent. It could be due to male–male interactions (Shine 1979), female choice for larger males (Halliday & Verrell 1988), or differences in age structure between sexes that lead to differences in body size (Miaud et al. 1999, Monnet & Cherry 2002, Liao & Chen 2012). Shine (1979) reviewed for the first time the literature on sexual size dimorphism and observed that in 90% of anurans, females grow larger than males. Sexual size dimorphism is present in P. cultripes with females larger and heavier than males, while in E. calamita sexual dimorphism is absent regarding body size, but, rather unusual, males are heavier than females. This suggests that age alone is not responsible for these differences.

Fecundity selection in females and sexual selection in males are considered the major evolutionary forces selecting for larger body size in most organisms (Blanckenhorn 2000). Halliday & Verrell (1988) suggested that female choice of larger males favors males with rapid juvenile growth, and not just older age. This might explain the rapid growth pattern of males in Large-Bodied populations of both species, as an advantageous trait for reproduction. The positive correlation between female body size and clutch size (Duellman & Trueb 1986) suggests a benefit to male–male competition for larger females. Selection for large body size is eventually counterbalanced by opposing selective forces, primarily viability selection (Andersson 1994, Blanckenhorn 2000). Increased body size will be favored only if it decreases mortality or enhances reproductive success sufficiently to compensate for the increased production required by a larger body mass (Brown & Sibly 2006).

Previous studies that estimated age related parameters through skeletochronology indicate important variation in both size and age among populations (Appendix A). The P. cultripes population from Serra da Arica (Leclair et al. 2005) is even smaller in size than our Small-Bodied populations. Overall, we report here for the first time age at sexual maturity of 1 year, smaller than the reported 2-4 years in other studies on P. cultripes or 2-7 in other species of the genus Pelobates. Longevity is also lower in the studied populations (8 years old) compared to a maximum of 12 years in a population from Madrid (Talavera 1990). A complete review of age-related studies in E. calamita was done by Sinsch (2015). Age at sexual maturity varies greatly and is usually higher than our reported 1 year, reaching up to 5 years old. Longevity varies largely: in three populations it was over 10 years, but in other populations is shorter, of even 4 years, i.e. half of our reported 8 years. Longevity is influenced by sample size, i.e. a larger sample means a higher probability to find older individuals, so comparisons should be made with caution.

The development and growth, and thus the size of most organisms, are affected by temperature and water availability (Sheridan & Bickford 2011). For ectotherms, metabolic rates directly scale with temperature, thus an increase in metabolism associated with climate warming will reduce the body size of ectotherms unless organisms can compensate with greater food intake or reallocating caloric resources. Increased temperatures can also lead to smaller ectotherms by increasing developmental rate (termed the temperature–size rule). Decreasing body size has been proposed as a universal response to increasing temperatures (Baudron et al. 2014). Body-size reduction is thus the third universal response to global warming, alongside changes in the phenology and distributions of species. Apart from the direct effect of temperature on body size, one other probable proximate cause of changing body size is a change in the availability or quality of food (Gardner et al. 2011). Since in our study air temperature does not differ between large and small-Bodied populations, we suggest that differences in food availability or quality caused by substrate are responsible for the observed variation of body size.

Overall, our study identified an interesting pattern in life-history strategies, with populations from two different species having similar trade-offs between size, growth and age, to compensate for environmental variation.

ACKNOWLEDGMENTS

We thank F. Campos and C. Díaz-Paniagua for their invaluable help at laboratory and skeletochronological method, respectively. F. Stănescu reviewed earlier drafts of this manuscript, providing useful comments that greatly improved the paper. Two anonymous reviewers provided constructive comments. We thank H. Duarte, a native speaker, for correcting the English draft of this manuscript. This work was supported by grant PB96–0861 from Dirección General de Investigación Científica y Técnica conceded to M. Tejedo. Thanks also to the Consejería de Medio Ambiente de la Junta de Andalucia and the Reserva Biológica de Doñana, for providing the corresponding permits and facilities. All animal experimentation was conducted in accordance with the legal standards of Spain.

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Publication Dates

• Publication in this collection
  04 June 2021
• Date of issue
  2021

History

• Received
  22 Apr 2019
• Accepted
  15 Aug 2019