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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Reproductive Investment of Female Green Toads (Bufo viridis) Author(s): Sergio Castellano, Marco Cucco, Cristina Giacoma Source: Copeia, 2004(3):659-664. 2004. Published By: The American Society of Ichthyologists and Herpetologists DOI: http://dx.doi.org/10.1643/CE-03-182R2 URL: http://www.bioone.org/doi/full/10.1643/CE-03-182R2 BioOne (www.bioone.org ) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use . Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.

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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, researchlibraries, and research funders in the common goal of maximizing access to critical research.

Reproductive Investment of Female Green Toads (Bufo viridis)Author(s): Sergio Castellano, Marco Cucco, Cristina GiacomaSource: Copeia, 2004(3):659-664. 2004.Published By: The American Society of Ichthyologists and HerpetologistsDOI: http://dx.doi.org/10.1643/CE-03-182R2URL: http://www.bioone.org/doi/full/10.1643/CE-03-182R2

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, andenvironmental sciences. BioOne provides a sustainable online platform for over 170 journals and books publishedby nonprofit societies, associations, museums, institutions, and presses.

Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance ofBioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use.

Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiriesor rights and permissions requests should be directed to the individual publisher as copyright holder.

q 2004 by the American Society of Ichthyologists and Herpetologists

Copeia, 2004(3), pp. 659–664

Reproductive Investment of Female Green Toads (Bufo viridis)

SERGIO CASTELLANO, MARCO CUCCO, AND CRISTINA GIACOMA

Females of iteroparous species may compromise between contrasting reproduc-tive strategies. They should balance the amount of energy devoted to reproductionagainst the energy saved for growth and survival, and they should compromise be-tween the quantity versus the quality of offspring. In the present paper, we analyzethese trade-offs in European Green Toads (Bufo viridis). We define reproductiveinvestment in terms of clutch dry mass, number of eggs, and mean ovum dry mass,whereas we describe the amount of resources saved for growth and survival (somaticinvestment) with a fat index derived from the total body electrical conductivity (TO-BEC) of postspawning females. Our results indicate that larger individuals producelarger clutches, both in the number and size of eggs. Females with higher repro-ductive effort, however, also show a larger somatic investment regardless of size.We show that females of similar size invest similar proportions of resources in re-production, so that those in good condition not only show larger reproductive effortthan lower-quality females but also store larger amounts of resources for growth,survival, and future reproduction.

AN understanding of life-history evolution re-quires analysis of the balance of energy de-

voted to growth, development, and reproduc-tion and how this balance can be adjusted tomaximize lifetime reproductive success(Stearns, 1992; Roff, 2002). From a life-historyperspective, reproduction requires animals tocompromise between contrasting alternatives.For instance, there should be a trade-off withrespect to the time and energy allotted to re-production versus the time and energy directedtoward growth, survival, and future reproduc-tion. Furthermore, for females there is often atrade-off between the quantity versus the qualityof their offspring.

For female anurans without parental care, re-productive effort depends largely on the totalamount of energy allocated to oocytes (Grafe etal., 1992). These resources are known to be di-rectly affected by female condition, in particularby the amount of energy stored in fat bodies( Jørgensen, 1992). However, several allocationstrategies might result in a positive relationshipbetween body-condition and clutch-size. Figure1 models three alternative hypotheses in whichfemales can adjust reproductive effort to theiramount of available energy: (A) Females showa high, condition-dependent reproductive in-vestment and a low, condition-independent so-matic investment; (B) Females short of allocableenergy invest less in reproduction (both in ab-solute and relative terms) than females withmore resources, so that, after spawning, femalesin poor condition will have more amounts ofenergy for growth and future reproduction; or(C) Females with abundant resources show

greater reproductive effort than females withscarce resources, but, at the same time, theysave more energy for somatic growth and, afterreproduction, they still maintain higheramounts of available energy.

Female reproductive success depends notonly on the total amount of resources allocatedto reproduction but also on the way these re-sources are distributed among offspring (i.e.,on the number and quality of zygotes). Femalecontrol over offspring fitness, therefore, can besignificantly influenced by allocation of energyreserves (Kaplan, 1998) and potentially by ge-netic quality through mate choice (Andersson,1994; Welch et al., 1999). For example, largereggs tend to produce larger tadpoles, whichgrow and develop faster, resulting in an in-creased probability of reaching metamorphosisthan tadpoles hatching from smaller eggs (Lau-gen et al., 2002; Loman, 2002; but for environ-mental effects on the egg-size versus larval sur-vival relationships, see Kaplan, 1992).

Body size and body condition are known toplay an important role in influencing energeticallocation to offspring in female anurans. Forexample, there is typically a positive associationbetween fecundity and snout–vent length (SVL;review in Duellman and Trueb, 1986), whichmay be an important determinant of sexual sizedimorphism (Howard, 1988). The relationshipbetween ovum-size to body size is not as clearas the relationship between clutch-size andbody-size, because of the negative correlationbetween ovum size and clutch size (Duellmannand Trueb, 1986; Jørgensen, 1992) and the highwithin-individual variation of ovum size (reviewin Jørgensen 1992; Kaplan and King, 1997).

660 COPEIA, 2004, NO. 3

Fig. 1. Models of condition-dependent reproduc-tive allocation strategies. Females with differentamounts of available resources move along differentdiagonal lines. The horizontal dash line representsthe minimum somatic investment compatible withsurvival, whereas the vertical dash line represents theminimum amount of resources necessary for repro-duction. The stippled area represent potential repro-ductive production. Three strategies are presented,all resulting in a positive correlation between repro-ductive effort and female condition. According to theHypothesis A, females invest most of their availableresources in reproduction, and similar amounts of re-sources in growth and survival. According to Hypoth-esis B, females in poor condition save larger amountsof resources for growth and survival than those in bet-ter condition. According to Hypothesis C, females in-vest similar proportions of available energy to repro-duction, those having invested more in reproductionsaved also larger amounts of energy for growth.

We studied female reproductive investment ina population of the European Green Toad (Bufoviridis). The aim of this research was twofold.First, we analyzed the relationship between re-productive and somatic investment to deter-mine which of the patterns presented in Figure1 best explained the allocation strategy of fe-male green toads. Second, we investigated theeffects, if any, of body size and postspawningbody condition on the trade-off between num-ber and size of eggs. We described the repro-ductive effort in terms of clutch dry mass anddistinguished between the number of eggs (fe-male fecundity) and mean ovum size (offspringquality). We quantified the amount of resourcessaved for growth and survival (somatic invest-ment) by means of a fat index, derived from thetotal body electrical conductivity (TOBEC) ofpostspawning females.

MATERIALS AND METHODS

In April and May 2002, amplectant male andfemale Green Toads were caught in a pond nearZucchea (NW Italy, 448499N, 78269E), kept in amoist sack at 5 C to prevent egg deposition, andcarried to the laboratory. There, pairs were set-tled into separate tanks (60 3 40 cm, 30 cm tall)with shallow water (whose temperature variedbetween 12 and 16 C), and left there overnight.Spawning usually started a few hours later andegg deposition was completed by the followingmorning. Pairs were released in the same placewhere they were captured the day before.

Clutch weight and number of eggs.—Egg stringswere divided into 4–6 portions of similar length.Each segment was coiled in a glass dish, driedwith blotting paper, weighed to the nearest 0.1mg, and photographed with a digital camera.Half the clutch portions were dehydrated in afan convection oven at 60 C for about 24 h toobtain dry-mass. To minimize our impact on theGreen Toad population, the remaining clutchportions were placed into 80-liter aquaria andallowed to hatch and tadpoles develop to stage24 of Gosner (1960), before their release intothe parental pond. To infer the dry mass ofthese clutch portions, we multiplied their wetweight by the dry mass to wet mass ratio of thedehydrated portions.

To quantify individual female reproductive in-vestment, we measured three parameters: (1)clutch dry-mass; (2) number of eggs per clutch;and (3) mean dry-mass of a single egg. Wecounted the total number of eggs per clutch onthe digital pictures of coiled egg string portionsusing the software Image Tool 3.0 (University ofTexas Health Science Center in San Antonio).This program automatically counted the eggs itidentified and showed their outlines on theoriginal picture. When necessary, we adjustedthe automatic result by manually counting thoseeggs that the program failed to identify. Themean dry mass of a single egg was obtained byaveraging the mean individual egg mass valuesfrom each dehydrated portion.

Female size and condition.—To characterize sizeand condition of females, we employed threeparameters: (1) snout–vent length (SVL), mea-sured with a digital calliper to the nearest 0.01mm on anaesthetized individuals (see below);(2) body mass, measured with a digital balanceto the nearest 0.1 g, both before and after eggdeposition; and (3) an after-spawning Fat Index,calculated as the residuals of a regression be-tween total body mass and an indirect estimate

661CASTELLANO ET AL.—TOAD REPRODUCTIVE INVESTMENT

TABLE 1. DESCRIPTIVE STATISTICS OF THE VARIABLES EMPLOYED TO DESCRIBE THE REPRODUCTIVE INVESTMENT

AND THE PHENOTYPE OF FEMALES.

n Minimum Maximum Mean SD CV (%)

Body size and conditionSVL (mm)After-spawning Fat IndexBefore-spawning body mass (g)After-spawning body mass (g)

28282827

59.5923.4721.615.3

77.142.65

52.637.8

70.0420.0238.726.5

4.351.697.95.4

6.2

20.320.6

Reproductive investmentEgg dry mass (mg)Number of eggsClutch dry mass (mg)

272727

0.4456872718.3

0.72176028203.3

0.5789135089.0

0.0825501316.0

13.528.625.8

Fig. 2. Relationship between female SVL andclutch size (r 5 0.748; F 5 30.464; df 5 1, 24; P ,0.001).

of the lean body mass. The composition of thebody was evaluated nondestructively by electro-conductivity with the TOBEC (Total Body Elec-trical Conductivity) system. This device gives anindication of the lean body mass, since the con-tribution of lipid tissue to conductivity is negli-gible (Walsberg, 1988; Castro et al., 1990; An-gilletta, 1999).

To measure the electrical conductivity index,each individual was placed in the detectionchamber (44 mm diameter) of a SA-3000 SmallAnimal Body Composition Analyzer (EM-SCAN,Inc., Springfield, Illinois) for 5–6 sec. TOBECvalues were derived from the formula: TOBECindex 5 (S—E) / R, where S 5 measurementwith the specimen, E 5 empty measurement,and R 5 reference number (detailed instruc-tions in the EM-SCAN operator’s manual). Foreach individual the measurement was repeatedthree times and the average value was employedin successive analyses. As body electrical con-ductivity is sensitive to temperature, hydrationand position of the subject in the measurementchamber, toads were measured at a constant

room temperature of about 20 C. In addition,TOBEC measures of each toad were taken with-in 3 to 4 min subsequent to being held in waterfor 5–10 min to control for hydration. Followingthe protocol developed by Fisher et al. (1996)for aquatic species, we sedated toads in a 0.2%solution of MS222 (Sandoz) to avoid move-ment, and each individual was centered headfirst on the carrier device of the apparatus. Wechecked machine calibration following readingstaken on every third toad by using a calibrationtube supplied with the device.

RESULTS

Table 1 shows descriptive statistics of the var-iables employed to describe the phenotype andthe reproductive investment of females. Clutchmass shows high individual variation (CV 525.9%), which is mostly caused by variation ofeggs per clutch (CV 5 28.6%) rather than bythe mass variation of individual eggs (CV 513.6%) within the clutch. Because of egg de-position, females lose on average 12.2 g, whichis about a quarter (23%) of their mass. Howev-er, a large portion of this loss is water, becausethe mean dry mass of clutches (5.1 g) repre-sents only 41.8% of the total loss.

Factors affecting variation of clutch mass.—A mul-tiple regression analysis between clutch mass(dependent variable) and SVL and Fat Index(independent variables) was highly significant(R2 5 0.864; F2,23 5 73.071; P , 0.001): largerclutches tended to be laid by larger females(Fig. 2; partial regression coefficient [bSVL] 5283.05; P , 0.001) with higher values of thepostspawning Fat Index (partial regression co-efficient [bFat Index] 5 170.785; P 5 0.013). Thus,independent of size, females with higher repro-ductive effort retained more resources for

662 COPEIA, 2004, NO. 3

Fig. 3. Pattern of association among the variablesemployed to describe female state (SVL), somatic ef-fort (postspawning fat index), and reproductive effort(number and mean size of eggs).

growth and future reproduction, as predictedby Hypothesis C (Fig.1).

Clutches may differ in dry mass because theycontain a different number of eggs and/or be-cause their eggs differ in size. Simple regressionanalyses show the former of the two hypothesesas the most important to explain variation infemale reproductive investment. As expected,the number of eggs correlated significantly tothe clutch dry mass (R2 5 0.745; b 5 0.445; F1,25

5 72.9; P , 0.001), whereas the mean mass ofsingle eggs had no significant effect (R2 50.056; b 5 4002.5; F1,25 5 1.5; P . 0.1). Al-though ovum-mass had minimal effect onclutch-size variation, it was significantly differentamong females (Kruskal-Wallis: x2 5 66.3; df 527; P , 0.001), suggesting that females differednot only in the number of eggs they producedbut also in the amount of resources they invest-ed per single eggs.

Factors affecting egg mass and number.—The mul-tiple regression analysis between the number ofeggs per clutch (dependent variable) and SVLand Fat Index (independent variables) washighly significant (R2 5 0.604; F2,23 5 17.565; P, 0.001). However, only SVL appeared to affectthe dependent variable (partial regression co-efficient bSVL 5 451.4; P , 0.001); the Fat Indexdid not show any significant effect (partial re-gression coefficient bFat Index 5 337.54; P . 0.1).

To analyse the causal factors responsible foregg-mass variation, we used as predictors of themultiple regression model SVL, Fat Index, andthe total number of eggs per clutch. The anal-ysis showed highly significant results (R2 50.715; F3,22 5 18.4; P , 0.001), with significanteffects of all three independent variables: SVL(bSVL 5 0.023; P , 0.001) and the Fat Index(bFat Index 5 0.015; P 5 0.016) weighed positivelyon the mean egg mass, whereas the number ofeggs per clutch weighed negatively (begg-number

520.001; P , 0.001).

DISCUSSION

Our study showed that (1) large females in-vested more in reproduction than small fe-males; (2) independent of body size; females ingood condition laid larger clutches and storedlarger amounts of energy for growth and surviv-al than females in poorer condition; (3) inde-pendent of body size and condition, femalesthat produced a larger number of eggs investedless energy per single egg. Figure 3 summarizesthe pattern of associations between body sizeand reproductive and somatic effort of females.

In Green Toads, as in many other anuran spe-

cies (review by Duellman and Trueb, 1986),body size is the most important phenotypic traitaffecting variation in female reproductive ef-fort. In other anurans (review in Halliday andVerrel, 1988) and in Green Toads as well (Cas-tellano et al., 1999), body size is often found tocorrelate positively (though weakly) to age;thus, the body-size to clutch-size positive asso-ciation may reveal an increase of reproductiveeffort with age. Large, old females, due basedon their reduced expected future life, shouldinvest proportionally more in reproduction andless in growth and survival than their youngercounterparts. Our results, however, by failing tofind any negative association between reproduc-tive and somatic storage, provide no evidenceto support this hypothesis and suggest that thestrong positive correlation between body sizeand clutch size may result from a developmentalconstraint caused by the allometric relation-ships between gonad and overall body size. Bet-ter conditioned females, as more efficient con-sumers of environmental resources, can growfaster than females of poor quality, and attain alarger size, therefore affording them greater re-productive investment.

The large among-female variation in repro-ductive effort was more likely caused by varia-tion in the number rather than by the size(mass) of eggs. In our study, the largest clutchhad three times more eggs than the smallestclutch, whereas the highest ovum dry mass was

663CASTELLANO ET AL.—TOAD REPRODUCTIVE INVESTMENT

less than twice as heavy as the lightest egg.Large clutch size variation has also been ob-served within other conspecific populations.Jørgensen (1984), in comparing the reproduc-tive investment of Danish and Israeli femalegreen toads, found the mean number of full-grown vitellogenic oocytes per clutch amount-ing to about 11,000 in the Danish and 9000 inthe Israeli populations. Furthermore, in the Is-raeli toad population a negative correlation wasfound between the number and size of eggs ina clutch. A similar negative relationship was ob-served also in Rana temporaria (Gibbons and Mc-Carthy, 1986) and Rana sylvatica (Berven, 1988),where variation was correlated with age: clutchegg number decreasing and egg mass increas-ing with age.

Differing from the observed Israeli popula-tion data, we did not find a negative associationbetween the number and size of eggs, unless wetook into account the effect of both body sizeand postspawning body condition. Indeed,large females with large amounts of allocableenergy not only recruited more oocytes to vitel-logenic growth but also produced larger eggsthan smaller females. Moreover, we found thatresources were equally partitioned to both re-productive and somatic investments, which sup-ports the condition-dependent reproductive hy-pothesis (Fig. 1, Hypothesis C). This strategy al-lows females to retain a proportion of theiravailable resources for future reproductive con-tributions while contributing to the current re-productive cohort (van Noordwijk and de Jong,1986).

ACKNOWLEDGMENTS

We thank A. Rosso for technical support anddiscussion and two anonymous reviewers forhelpful comments and the River Po RegionalPark for the permission of catching and han-dling Green Toads. This research was financedby MIUR 2001 to CG.

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(SC, CG) DIPARTIMENTO DI BIOLOGIA ANIMALE E

DELL’UOMO, UNIVERSITA DI TORINO, VIA AC-CADEMIA ALBERTINA, 17, 10123 TORINO, ITALY;AND (MC) DIPARTIMENTO DI SCIENZE E TEC-

NOLOGIE AVANZATE, UNIVERSITA DEL PIEMONTE

ORIENTALE, VIA CAVOUR 84, 15100 ALESSAN-DRIA, ITALY. E-mail: (SC) [email protected]; (MC) [email protected]; and (CG)[email protected]. Send reprint re-quests to SC. Submitted: 18 July 2003. Ac-cepted: 24 April 2004. Section editor: S. F.Fox.