Population divergence of life history traits in theendangered green toad: implications for a supportrelease programme

B. Rogell, A. Berglund, A. Laurila & J. Hoglund

Population Biology and Conservation Biology/Department of Ecology and Evolution, Evolutionary Biology Centre, Uppsala University,

Uppsala, Sweden

Keywords

local adaptation; peripheral populations;

genetic diversity; amphibians.

Correspondence

Bjorn Rogell, Population and Conservation

Biology/Department of Ecology and

Evolution, Evolutionary Biology Centre,

Uppsala University, Norbyvagen 18D,

SE-75236 Uppsala, Sweden.

Tel: +46 18 471 2637;

Fax:+46 18 471 6424

Email: bjorn.rogell@ebc.uu.se

Editor: Mark-Oliver Rodel

Received 13 February 2011; revised 10 March

2011; accepted 10 March 2011

doi:10.1111/j.1469-7998.2011.00843.x

Abstract

There is an increasing awareness that adaptive differences among local popula-tions may affect the success of translocation programmes. A mismatch in habitat

quality of the target localities and in the local adaptations of the translocatedindividuals may reduce the success rate of the translocation programme. The greentoad Bufo viridis is the most threatened amphibian in Sweden and has been the

focus of an extensive translocation programme of eggs, tadpoles and juvenile toadsto several localities with apparently favourable conditions for green toads.However, the success of these measures has been poor. In this study, we

investigated the extent of local adaptation in the green toad by examiningpopulation divergence and the effect of thermal and saline conditions on larvalperformance in four Scandinavian populations. In a common garden experiment,

we measured larval survival and development as well as the occurrence of spinaldeformations. In addition, we quantified pond temperature and water salinity, twoimportant environmental variables for larval performance in anurans in thebreeding ponds as well as in seven additional localities included in the conservation

programme. We found significant variation among the localities in water tempera-ture and salinity, and significant among-population divergence in larval life historytraits and spinal deformations, including both trait means and plastic responses to

salinity and temperature. The available evidence suggests that at least part of thisdivergence is adaptive. We did not find direct support for local adaptationaffecting the success of the translocations, however, we argue that the population

origin and the impact of rearing conditions on the fitness-related larval traitsshould be taken into account in the introduction measures of the Swedish greentoad conservation programme as well as in translocation programmes in general.

Introduction

When natural populations become threatened or extinct,translocations (here defined as releases of non-native ani-

mals in order to replace locally extinct populations orenhance population sizes of threatened populations) are awidely used practise in conservation biology (Griffith, 1989;Armstrong & Seddon, 2008; Germano & Bishop, 2009). The

positive effects of translocation actions on population sizeare often due to pure demographic effects (Smith, Peterson& Houston, 2003; Creel, 2006); however, several studies

have found associations between translocations and in-creased individual fitness, such as increased fecundity (Mad-sen, Stille & Shine, 1996; Westemeier et al., 1998) and loss of

defects (Pimm, Dollar & Bass, 2006) after translocations.Although an efficient management tool, translocations arecontroversial because artificially induced gene flow might

not only reduce the genetic load through the addition of

genetic variation, but also disrupt local adaptations (e.g.Boulding & Hay, 2001; Tufto, 2001; Tallmon, Luikart &Waples, 2004). As local adaptation is common in natural

populations (Leinonen et al., 2008), there is a risk thattranslocated individuals are maladapted to their new envir-onment, or that the existing local adaptations are swampedby gene flow caused by the released individuals (Tufto, 2001;

Ashley et al., 2004; Tallmon et al., 2004; Kinnison, Hendry& Stockwell, 2007).

The global decline of amphibians is a major conservation

concern, and translocations are often used in conservationprogrammes to support or restore amphibian populations(Germano & Bishop, 2009). Amphibians differ from many

other vertebrate groups in their limited dispersal ability anduse of ponds and other freshwater habitats for breeding. Thebreeding habitats often differ widely in characteristics

Journal of Zoology

Journal of Zoology 285 (2011) 46–55 c� 2011 The Authors. Journal of Zoology c� 2011 The Zoological Society of London46

Journal of Zoology. Print ISSN 0952-8369

(Beebee, 2005), which is predicted to favour local adaptation(Sultan & Spencer, 2002; Beebee, 2005). Accordingly, many

studies have found local evolutionary responses suggestingthat local adaptation in early life history traits in amphi-bians is common (Berven & Gill, 1983; Gomez-Mestre &

Tejedo, 2003; Palo et al., 2003; Ficetola & De Bernardi,2005; Rogell et al., 2009; Orizaola, Quintela & Laurila,2010), even at very small geographic scales (Skelly, 2004;

Lind & Johansson, 2007; Richter-Boix et al., 2010).In Sweden, the green toad Bufo viridis has declined

rapidly during the last 50 years, and the species is today themost threatened amphibian species in the country, with only

a handful of populations remaining (Gardenfors, 2005;Wiren, 2006). In order to save the Swedish green toadpopulations, several current and past green toad localities

have been restored or improved, and wild-collected, labora-tory-reared eggs, tadpoles and toadlets (young-of-the-yeartoads) have been released at these sites. However, only very

few of the hundreds of thousands of tadpoles and toadletsreleased have returned as breeding adults (Wiren, 2006).Since the localities differ widely in habitat characters, we

hypothesized that local adaptation to specific pond condi-tions could, in part, explain the poor success of the translo-cation programme. We examined this by quantifyingpopulation divergences across four populations and put

them in relation to environmental parameters at 12 localitiesincluded in the Swedish green toad project.

We quantified two environmental parameters known as

important agents of selection for amphibian larvae; watertemperature and salinity. Salinity and temperature werechosen due to the explicit a priori assumption that these

differ among our study populations. Temperature affectsmetabolism and thus the rate of development, with coldertemperatures leading to lower metabolism and slower devel-opment. Ectotherms frequently adapt to cool temperature

habitats by growing faster than conspecifics from warmerhabitats when reared in a common environment, thuscompensating for the time loss associated with low tempera-

tures (Conover, Duffy & Hice, 2009). Anurans generallyperform poorly in saline conditions (Boutilier, 1992); how-ever, the green toad is one of the few anurans known to

inhabit saline estuarine habitats (Katz, 1973). Additionally,both temperature and salinity are selection pressures towhich amphibians can locally adapt, as shown in the closely

related natterjack toad Bufo calamita (Gomez-Mestre &Tejedo, 2003; Rogell et al., 2009, but see Rogell et al., 2010).

Green toad tadpoles from four Scandinavian populationswere raised until metamorphosis in a common garden

experiment using two temperature and three salinity treat-ments, size at and timing of metamorphosis was recorded aswell as survival, which are important fitness components in

anurans (Smith, 1987; Berven, 1990; Altwegg & Reyer,2003). Previous studies have demonstrated local adaptationin these traits (e.g. Palo et al., 2003; Richter-Boix et al.,

2010). If our study populations are locally adapted to theirnative environment, we predicted that tadpoles from coldlocalities have faster development and growth rates than

tadpoles from warmer localities. Similarly, we expected

tadpoles from saline environments to have a higher salinitytolerance in terms of survival and degree of malformations,

and experience less change in reaction norms in response tosaline conditions. We especially focused on differencesbetween one specific population (Limhamn quarry, see

below), which has acted as the main source of the Swedishtranslocation programme, and the other populations. Largedifferences in larval life history between Limhamn and the

other populations would suggest that Limhamn can be apoor source population for translocations.

Materials and methods

Study species

The green toad occurs from western Central Europe (i.e.Germany, eastern France and Italy) to Central Asia in the

east and North Africa in the south (Arnold & Ovenden,2004). The northernmost populations occur in Sweden,Denmark, the Baltic States and Russia. The cause for the

decline of the green toad in Sweden is habitat degradation,as grazed oligotrophic coastal meadows with shallow pondshave disappeared due to eutrophication and changes in land

management (Gardenfors, 2005; Wiren, 2006).The vast majority of released green toads in the Swedish

support release programme originate from one population,the Limhamn limestone quarry (Wiren, 2006). The environ-

ment at the Limhamn quarry is very different from the othergreen toad habitats within its northern range, raising ques-tions of this population’s suitability as a source population.

The sheltered bottom of the quarry, 60m below sea level, iswarm, oligotrophic and alkaline. It is inhabited by severalplant and animal species that are rare or absent elsewhere in

Sweden (Wiren, 2006, see supporting information AppendixS1 for closer description of the site).

We included one Danish and three Swedish populations(Fig. 1, Table 1, supporting information Appendix S1) in the

common garden experiment. The three Swedish localitiesare the only ones currently harbouring large and viablegreen toad populations in the country. We collected envir-

onmental data on these and seven additional localities (Fig.1, Table 1, supporting information Appendix S1) includedin the Swedish conservation action plan for green toad

(Andren, Nilson & Larsson, 2000).

Pond characters

Salinity and temperature were measured in 12 ponds [onepond per locality, except at Limhamn where three pondswere measured, Limhamn A, B and C (only salinity)] inApril–August 2008. At each locality we selected the most

important breeding pond for green toads. In localities with-out reproduction the pond with the highest potential(judged on the basis of suitable habitats according to Wiren,

2006) was selected. We measured salinity with a Cond 340iSalinometer (WTW, Weilheim, Germany). One data-log-ging thermometer (HOBOs U22 water temp pro V2 logger,

Onset Computer Corp., Bourne, MA, USA) was placed in

Journal of Zoology 285 (2011) 46–55 c� 2011 The Authors. Journal of Zoology c� 2011 The Zoological Society of London 47

Population divergence of green toadB. Rogell et al.

each pond at the start of the breeding season in April. Thethermometers were set to record every 15thminute andplaced at a depth of c. 20 cm. The thermometers were

retrieved after the completion of larval development inAugust. Data from the period 18April to 10August 2008,when all the retrieved loggers were functional, were used inthe analyses.

Common garden experiment

In spring 2008 we collected 20 freshly laid eggs from each of 10egg-strings (full-sib families) at each of the four localities (seesupporting information Appendix S1) and transported them

to the laboratory facilities in Uppsala. The eggs and newlyhatched larvae were reared at 20 1C in 0.9L plastic containers

Figure 1 Map over the localities included in the study. 1, Limhamn (original population); 2, Eskilstorp (original population); 3, Utklippan (original

population); 4, Monnet (original population); 5, Falsterbo (original population); 6, Hogaborg; 7, Lernacken; 8, Ravlunda; 9, Brantevik; 10,

Landskrona; 11, Vik (see supporting information Appendix S1 for details).

Table 1 Salinity and temperature in ponds where green toad Bufo viridis breed, have previously bred, or have been introduced

Locality Salinity % Mean T Max T Min T SD T Collection date

Eskiltorpa,b 0.2 – – – – 21/4

Limhamn Aa,b 0 19.8 29.4 3.7 4.4 21/4

Limhamn Ca,b 0.2 – – – – 21/4

Limhamn Ba,b 1.8 15.8 26.3 2.7 3.9 21/4

Monneta,b 1.1 – – – – 2/5

Utklippana,b 1.5 18.2 29.9 7.9 4.2 25/4, 10/5

Falsterboc,b 0 17.9 27.9 8.4 3.6 –

Hogaborgc,b 0 17.2 26.8 9 3.8 –

Lernackenc,b 0 18.3 34.6 9 4.2 –

Ravlundac 0 19.4 33.9 9.7 4.5 –

Brantevikc 1.7 – – – – –

Landskronac 0 – – – – –

Vikd 0.3 13.7 21.1 5.2 3.1 –

aLocalities included in the common garden experiment.bLocalities with existing green toad population.cLocalities used in the introduction programme.dLocalities with extinct green toad population.

Mean T, Max T and Min T are the mean, maximum and minimum recorded water temperatures as 1C. The SD T is the standard deviation of the

mean temperature. In Limhamn, salinity was measured in three and temperature in two ponds.

Journal of Zoology 285 (2011) 46–55 c� 2011 The Authors. Journal of Zoology c� 2011 The Zoological Society of London48

Population divergence of green toad B. Rogell et al.

until reaching stage 25 (Gosner, 1960, i.e. start of feeding).The tadpoles were then transferred to 0.9L individual vials in

a common garden experiment with a factorial design consist-ing of two temperature and three salinity treatments. Thetemperature treatments were (1) a warm temperature treat-

ment in six heated-water baths (water temperature 27� 1 1C,henceforth warm treatment); (2) a cold treatment in a climateroom (20� 0.5 1C, henceforth cold treatment). The levels of

the temperature treatment were chosen based on a prioriassumptions regarding the mean and high temperatures inthe generally warm ponds where green toads breed and theseassumptions were supported by the field data (Table 1). The

salinity treatments were 0, 1.8 and 4.8% (henceforth fresh-water, medium and high salinity treatment, respectively). InScandinavia, the natterjack toad B. calamita often coexists in

the same ponds with the green toad, and the salinity levelswere chosen on the basis of salinity levels the natterjack toadtolerate and may locally adapt to (Gomez-Mestre & Tejedo,

2003; Rogell et al., 2009). There were 30 tadpoles from eachpopulation in each treatment combination (three per full-sibfamily). A randomized block design, with six spatial blocks in

the cold treatment and five in the warm treatment, was used inorder to account for temperature deviations within eachtemperature treatment.

Reconstituted soft water (RSW; APHA, 1985) was used

throughout the experiment to assure homogenous waterquality. The salinity treatments were prepared by adding2 g (low salinity) or 5 g (high salinity) of Sea Salt (Tropic

Marines, Wartenberg, Germany) per litre RSW, yieldingrealized salinity levels of 0, 1.8 and 4.8%. The tadpoleswere fed a mix of spinach and commercial crayfish food

(Aller Aquas, Christiansfeld, Denmark) ad libitum at aweight ratio of 20:1, mixed to a homogenous puree. Thewater was changed every 4 days, and in the warm treatmentthe vials were topped up daily with deionized water to

replenish water lost due to evaporation and to keep salinityconstant.

When reaching metamorphosis (defined as the emergence

of the first forelimb, Gosner stage 42) the tadpoles wereremoved from the experiment and weighed with an electro-nic balance with a precision of 0.1mg. Four response

variables were analysed: survival, mass at metamorphosis(g), larval period (days) and ordinal level of spinal deforma-tion. During the course of the experiment, we found that

several larvae suffered from spinal deformations (henceforthscoliosis, a known problem in both nature and in theSwedish translocation programme; Wiren, 2009), and itwas evident that the most severely affected tadpoles were

unable to function normally. In order to analyse the pre-sence of these deformities at metamorphosis, we devised ascale, the scoliosis index (henceforth SI) from 0 to 5 where 0

was no spinal deformities and 5 was severe spinal deformi-ties in the tail and back. As the tadpoles with a SI of 5 wereunable to function normally, they were removed from the

experiment and statistical analyses, except for calculationsconcerning the deformation index and survival. The deter-mination of SI is presented in the supporting information

Appendix S1.

Statistical analyses

Pond water temperature data on daily maximum, minimum

and mean temperatures were analysed using a linear mixedeffect (LME) model with water temperature as responsevariable, pond identity as fixed factor and measurement timeas random effect. Significance of the model was tested using a

mixed effect ANOVA. To test whether natural toad occur-rence (defined as a binary variable, presence/absence ofbreeding) was explained by pond temperature, we modelled

the toad occurrence with the explanatory variables tempera-ture (fixed), pond identity (random) and day (random). Beinga binary variable, occurrence was analysed using a binomial

error distribution and a logit link. The model was fitted usingLaplace estimation and statistical significance was verifiedusing the z-value of the regression coefficient for temperature.

The analyses were performed on daily minimum, maximumand mean temperatures. To test whether salinity affected toadoccurrence we analysed toad occurrence as explained bysalinity using a generalized linear model with a binomial error

distribution. Statistical significance was verified using the t-value of the regression coefficient.

We analysed the effects locality, temperature and salinity on

larval survival, life-history traits and scoliosis by fitting linearmodels to the common garden data. For survival, larval periodand mass, we used LMEs with block (nested under tempera-

ture) fitted as a random factor to the data. For the continuousresponse variables (larval period and mass) the LMEs werefitted using restrictedmaximum likelihood. In order to validatethe LME assumptions regarding homoscedasticity among

treatment combinations and normality of residual distribu-tions, larval period was log transformed andmass at metamor-phosis was square root transformed. Survival was analysed

using a binomial error distribution and a logit link. Thesurvival model was fitted using Laplace estimation. SI, a factorranked on an ordinal scale, was analysed using a proportional

odds logistic regression, assuming no influence of the nestedvariable block. All LME and proportional odds logisticregression models were simplified based on the sequential

removal of non-significant terms using likelihood ratio tests(Crawley, 2007). All statistical analyses were done with R (RDevelopment Core Team, 2006). Throughout the analyses, allexplanatory variables were treated as categorical variables. The

final models contained the parameters presented in Tables 2–4and are presented in the supporting information Appendix S1.

Results

Environmental variation

Only eight of the 12 thermometers were retrieved, as those

placed in Eskilstorp, Monnet, Landskrona and Brantevikdisappeared during the summer. There were significantdifferences in temperature among the ponds both in daily

mean (F7,696=524.2, Po0.0001, Table 1), daily minimum(F7,695=229.6, Po0.0001, Table 1) and daily maximum(F7,695=222.0, Po0.0001, Table 1). Neither minimum

(b=0.012, z=0.003, P=0.997), maximum (b=0.039,

Journal of Zoology 285 (2011) 46–55 c� 2011 The Authors. Journal of Zoology c� 2011 The Zoological Society of London 49

Population divergence of green toadB. Rogell et al.

z=0.013, P=0.990) nor mean (b=0.040, z=0.012,P=0.990) temperature explained natural toad occurrence.

Salinity varied among the localities, and within the Lim-hamn quarry (0–1.8%, Table 1). However, salinity did notexplain toad occurrence (b=0.44, t=0.46, P=0.654).

Common garden experiment

Larval period was significantly affected by temperature andsalinity with longer larval periods in the cold treatment and

the two saline treatments (Table 2, Fig. 2). Monnet andUtklippan had significantly longer larval periods than the

tadpoles originating from Limhamn and Eskilstorp (Table 2,Fig. 2). This was especially the case in low temperature andhigh salinity as indicated by significant locality� tem-

perature and locality� salinity� temperature interactions(Table 2, Fig. 2).

Mass at metamorphosis did not differ between localities

(Table 2, Fig. 2), but was significantly lower in the warmtreatment and saline treatments (Table 2, Fig. 2). There wasa significant interaction between locality and salinity indi-cating that the Monnet population had a steeper decline in

mass at metamorphosis than the other populations (Table 2,Fig. 2). The significant temperature� salinity interactionindicated that the decline in mass with increasing salinity

was stronger in the cold treatment (Table 2, Fig. 2).Survival was significantly higher in the warm treatment

and significantly lower with increasing salinity (Table 3,

Fig. 3). Locality had no significant effects on survival andwas not included in the model.

The degree of scoliosis increased significantly at higher

salinity and temperature (Table 4, Fig. 4). There was nosignificant locality main effect. The significant locality� sali-nity interaction indicated that, in contrast to the other popula-tions, the degree of spinal deformation in Utklippan tadpoles

did not increase with increasing salinity (Table 4, Fig. 4).

Discussion

We found that the Scandinavian green toad populationsdiffered in larval life history traits known to be important

Table 2 ANOVA tables of the effects of population, salinity and

temperature on larval life history traits

Numd.f. Dend.f. F P

(a) Larval period

Locality 3 592 6.2 0.0004

Temperature 1 9 800.3 o.0001

Salinity 2 592 6.5 0.0016

Locality� temperature 3 592 28.8 o.0001

Locality� salinity 6 592 0.4 0.8558

Temperature� salinity 2 592 0.7 0.4883

Locality� temperature� salinity 6 592 3.6 0.0017

(b) Mass

Locality 3 599 1.4 0.2283

Temperature 1 9 412.6 o.0001

Salinity 2 599 72.6 o.0001

Locality� salinity 6 599 3.2 0.0041

Temperature� salinity 2 599 13.4 o.0001

Numd.f., numerator degrees of freedom; dend.f., denominator degrees

of freedom.

Figure 2 Reaction norms for larval period (a),

and mass at metamorphosis (b) for the four

populations in the three different salinity treat-

ments. Solid lines indicate the cold treatment

and dashed lines the warm treatment. Error

bars (very small in some cases) represent �1

standard error.

Journal of Zoology 285 (2011) 46–55 c� 2011 The Authors. Journal of Zoology c� 2011 The Zoological Society of London50

Population divergence of green toad B. Rogell et al.

fitness traits in amphibians. We also found differences intemperature and salinity among the breeding ponds suggest-

ing that the selection regimes among the ponds may differ.Consequently, because ectotherms frequently exhibit localadaptations to temperature (Conover et al., 2009), and

anurans can adapt to saline conditions (Gomez-Mestre &Tejedo, 2003), some of the divergence found among thepopulations could be attributed to local selection and may

thus have important implications for the success of thecurrent translocation project.

The ponds without historical records of occurrence, butwhere green toads have been introduced, did not deviate in

either temperature or salinity from the ponds with currentgreen toad populations. The coolest pond by far was Vik, alocality where the green toad is now extinct. However, one

of the Limhamn ponds (55MS, see supporting informationAppendix S1), where breeding occurs yearly, also had low

temperatures. Both ponds are shaded by trees, which islikely to decrease water temperature (Skelly, 2004; Richter-Boix et al., 2010). While there is no historical data on the

temporal development of vegetation around the ponds inVik, flight photos from the 1930s indicate a strong increaseof vegetation coverage (from 30 to 50% total coverage by

trees and large bushes) in the coastal areas in the immediatevicinity of Vik (http://www.giscentrum.lu.se). Thus, de-creasing water temperature in the breeding pond due toincreased shading by growing vegetation can be a contribut-

ing factor and, considering the low temperature in the Vikpond, even a potential explanation for the extinction of thispopulation. Keeping in mind the strong effect that low

temperature had on green toad development rates in thecommon garden experiment, it is important to avoid shad-ing vegetation around the breeding ponds to create bene-

ficial developmental conditions for the larvae.Both temperature and salinity significantly affected larval

performance. High temperature led to higher survival,

shorter larval period, lower mass at metamorphosis andhigher amount of spinal deformation. Higher survival in thewarm temperature suggests that this temperature was lessstressful for the tadpoles. However, the increased amount of

spinal deformities and the smaller size at metamorphosis inthis temperature are likely to affect fitness negatively at theterrestrial stage (e.g. Berven, 1990; Altwegg & Reyer, 2003).

Indeed, the fact that metamorphic size was large and SI waslow at 20 1Cmay suggest that these conditions are preferable

Table 3 GLMM parameter estimates for survival

Coefficient SE z P SD

Intercept 1.92 0.26 7.38 0 –

Temperature warm 1.21 0.28 4.32 o0.0001 –

Salinity low �0.8 0.28 �2.83 0.0047 –

Salinity high �1.45 0.27 �5.41 0 –

Random effect

variance: block

0.086 – – – 0.293

The default contrast matrix in R (‘contrast treatments’, Crawley, 2007)

was used to generate the estimates. The cold freshwater treatment

was set as intercept and the parameter estimates (coefficient)

represent the difference to the intercept. SE is the standard error for

the coefficient, z is its associated z-value.

0.5

0.6

0.7

0.8

0.9

1.0

20°C

Pro

port

ion.

sur

viva

l

0‰ 1.8‰ 4.8‰

MonnetEskiltorpLimhamnUtklippan

0.5

0.6

0.7

0.8

0.9

1.0

27°C

Pro

port

ion.

sur

viva

l

0‰ 1.8‰ 4.8‰

Figure 3 Reaction norms for proportion survival

of the four populations in the three different

salinity treatments. The results are presented

separately for each temperature. Error bars

(very small in some cases) represent �1 stan-

dard error.

Journal of Zoology 285 (2011) 46–55 c� 2011 The Authors. Journal of Zoology c� 2011 The Zoological Society of London 51

Population divergence of green toadB. Rogell et al.

for raising tadpoles in the laboratory for translocationpurposes. The natural fluctuations in temperature of the

breeding ponds were very large, and this may have an effectof the performance of larval amphibians. For example,Niehaus, Wilson & Franklin (2006) showed that the larval

period of Limodynastes peroniiwere shorter and that mass atmetamorphosis was smaller in a fluctuating thermal condi-tion as when compared with a stable temperature treatment.

Local adaptation to fluctuating thermal conditions mayoccur as an alternative to adaptation to mean temperatures.

Salinity was a considerable stressor to the larval green

toads as evident by lower survival under more saline condi-tions, and the non-adaptive plastic responses away from theoptimum (Ghalambor et al., 2007). The larvae in the salinetreatments had longer larval period, smaller mass at meta-

morphosis and increased degree of scoliosis as comparedwith larvae in the freshwater treatment. While green toadhas a higher salinity tolerance than most other amphibians

(Katz, 1973), even moderate salinity appears stressful for thetadpoles. Reduced tadpole performance in saline pondsshould also be taken into account also in translocation

programmes, and introducing tadpoles in saline pondsshould be avoided.

In all traits except survival, there were significant effectsof locality and/or interactions between locality and other

factors, indicating that the populations differed in the traitmeans and in the plastic responses to the treatments. As the

local breeding habitats differed, the differences found inlarval performance can indicate local adaptation. However,because only four populations were examined, caution is

needed when interpreting the adaptive value of the popula-tion differences found in the present study. We were alsonot able to perform crosses between populations for

logistical and ethical reasons and could hence not controlfor the putative presence of maternal effects (Rasanen &Kruuk, 2007). Keeping these caveats in mind, we will discussthe differences in larval performance found among the

populations.The Eskilstorp population had shorter larval period than

the other populations. Because the Eskilstorp population

inhabits shallow ponds with high pond desiccation risk(Wiren, 2006), conditions known to select for higher growthrates and shorter development times (Lind & Johansson,

2007; Rogell et al., 2009), it seems likely that these traitvalues are the result of adaptation to the high desiccationrisk found at this locality.

Utklippan was the only population in which scoliosis didnot increase in the saline treatments. Interestingly, one ofthe highest salinities was recorded in Utklippan, which is anisland population breeding in a rock pool close to the sea.

We suggest that low incidence of scoliosis in the salinetreatments in Utklippan indicates local adaptation to themore saline conditions faced by this population.

Also the divergence between Utklippan and Limhamnpopulations in larval period is interesting given the supportrelease of Limhamn males and juveniles into the Utklippan

population (see supporting information Appendix S1). Thepresent Utklippan population is likely to be a hybrid popula-tion between the historical Utklippan population and Lim-hamn. This suggests that the putative local adaptations in

scoliosis tolerance and larval life history have persisted despitethe gene flow brought about by translocated individuals thatwere not locally adapted to the island habitat.

Although our data suggest local adaptation in some ofthe green toad populations, we did not find any support forLimhamn being a poor source for the support release stock.

Temperature and salinity varied among the Limhamnponds: while one of the ponds had the highest salinity andthe second lowest temperature, the other pond had the

warmest mean temperature and zero salinity. If the toads inLimhamn quarry exchange individuals among breedingponds, the expectation is that plastic responses should befavoured over adaptations to specific conditions (Sultan &

Spencer, 2002). Contrary to our initial hypothesis, ourresults suggest that Limhamn is a suitable source forintroduction into non-specific habitats.

A number of recommendations directly applicable in thegreen toad conservation programme rise from the presentstudy. First, we suggest that the divergence in fitness-related

traits and its possible adaptive nature is acknowledged infuture conservation efforts of Swedish green toads. Ifmaladapted genotypes are released they will pay a demo-

graphic cost of selection and, possibly, spread maladapted

Table 4 Proportional odds logistic regression coefficients for degree

of scoliosis

Coefficient 2.5% 97.5%

Temperature warm 1.90 1.55 2.27

Salinity low 1.28 0.52 2.05

Salinity high 1.20 0.44 1.97

Pop Es �0.12 �0.89 0.66

Pop Li �0.55 �1.38 0.26

Pop Ut �0.44 �1.20 0.32

Salinity low�Pop Es �0.82 �1.90 0.26

Salinity high�Pop Es 0.09 �1.00 1.17

Salinity low�Pop Li �0.09 �1.20 1.03

Salinity high�Pop Li 0.51 �0.59 1.61

Salinity low�Pop Ut �1.61 �2.71 �0.52

Salinity high�Pop Ut �1.20 �2.30 �0.11

Intercept a SE t

0|1 1.64 0.32 5.19

1|2 2.30 0.33 7.17

2|3 2.84 0.34 8.69

3|4 3.70 0.39 10.69

4|5 5.15 0.39 13.17

The general model is logitðPðY � jÞÞ ¼ aj þ bx, and a coefficient (b) is

thus interpreted as the e(�1� b) decrease in odds to place a larvae in

category j or lower. 2.5 and 97.5% represent the lower and upper

95% confidence interval levels for the coefficient. The default con-

trast matrix in R (‘contrast treatments’, Crawley, 2007) was used to

generate the estimates, that is comparing categorical levels to an

intercept set to population Monnet in the cold freshwater treatment. ais the intercept value, SE is the standard error for the intercept.

Pop Es, population Eskilstorp; Pop Li, population Limhamn; Pop Ut,

population Utklippan.

Journal of Zoology 285 (2011) 46–55 c� 2011 The Authors. Journal of Zoology c� 2011 The Zoological Society of London52

Population divergence of green toad B. Rogell et al.

genes within the population (Tallmon et al., 2004). Forexample, if future translocations are planned to areas withsaline breeding ponds, Utklippan may be a suitable source

population. Secondly, we found that the Limhamn popula-tion is a suitable source of introduction into most green toadhabitats. The poor success of the green toad translocation

programme may thus be explained by other factors thanpopulation origin. For example, the success at the terrestrialstages can be of great importance for amphibian popula-

tions (Harper & Semlitsch, 2007; Harper, Rittenhouse &Semlitsch, 2008), and needs to be investigated in more detailalso in Swedish green toads. Thirdly, our data suggest thatclearing the surroundings of ponds from shading vegetation

can be beneficial in order to create a more beneficialmicroclimate for larval development. Finally, toadletsreared as tadpoles in 20 1C low salinity conditions had less

scoliosis and a higher mass at metamorphosis, suggestingthat these conditions can be preferable when rearing greentoads for release into the wild.

Acknowledgements

We thank Petter and Sara Albinsson, Lars Briggs, BrittaJohansson, Johan Wallen and Mats Wiren for logisticalhelp. Trevor Beebee, Mattias Hagman, Marten Hjernquist,

Ulrich Sinch, Amber Rice and Mats Wiren commented onearlier versions of this paper. Simon Karvemo analysedflight photos. The project was funded by Formas (to

J. Hoglund) and Zoologiska stiftelsen (B. Rogell). The study

was conducted under permissions from the Ethical Commit-tee for Animal Experiments in Uppsala County and thecounty board in Skane.

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Supporting information

Additional Supporting Information may be found in theonline version of this article:

Appendix S1. Localities included in the study.

As a service to our authors and readers, this journalprovides supporting information supplied by the authors.

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Journal of Zoology 285 (2011) 46–55 c� 2011 The Authors. Journal of Zoology c� 2011 The Zoological Society of London 55

Population divergence of green toadB. Rogell et al.