Animal Conservation (2005) 8, 33–40 C© 2005 The Zoological Society of London. Printed in the United Kingdom DOI:10.1017/S1367943004001805

Supplementation or in situ conservation? Evidence of localadaptation in the Italian agile frog Rana latastei andconsequences for the management of populations

Gentile Francesco Ficetola† and Fiorenza De Bernardi

Dipartimento di Biologia, Universita degli Studi di Milano. V. Celoria 26, 20133 Milano, Italy

(Received 25 February 2004; accepted 8 June 2004)

AbstractRelocation of endangered species can be an effective conservation tool if it does not mix populationsthat represent significant intraspecific variation. The threatened Italian agile frog, Rana latastei, has smallpopulations with low genetic diversity: translocation has been proposed to improve the likelihood of survivalof populations. Using a common environment experiment and field surveys, we investigated whether therewere differences in larval growth and developmental rate between foothill and lowland R. latastei populations,to evaluate if they are evolutionarily significant units. In nature, the colder climate of the foothills causesdelayed metamorphosis. Conversely, in a common environment, larvae from foothill populations show fastergrowth and development. We did not find a significant egg-size related maternal effect or any differences insize at metamorphosis. We hypothesise that counter-gradient selection promoted fast growing phenotypes in acold environment, where low temperatures slow down larval development. Foothill populations, despite beingonly a small geographical distance away from lowland populations, seem to be adapted to a colder climate andrepresent an evolutionarily significant unit. Different populations should, therefore, be managed independently,avoiding translocation. We suggest that evolutionary divergence between populations should be verified priorto planning relocation programmes, to prevent the risk of genetic homogenisation.

INTRODUCTION

Relocation of animals is a popular management toolfor the conservation of threatened species. For example,in past decades there have been many attempts to re-introduce species to areas from which they had becomeextinct, with successful results, at least in some cases (forreviews, see Griffith et al., 1989; Fischer & Lyndenmayer,2000). Supplementation is another relocation actionfrequently used in conservation biology (Storfer, 1999).In supplementation, individuals are added to an existingpopulation of conspecifics. If the source population iswild ranging, the movement of the animals is termedtranslocation (for definitions, see IUCN, 1995).

In supplementation actions, individuals can be movedin areas where habitat deterioration or hunting hascaused a decrease in population size. Supplementationcould reduce the vulnerability of recipient populationsto environmental and demographic stochasticity, allowingfaster population growth and the recolonisation of newlysuitable habitat (Lubow, 1996). Moreover, supplement-ation has been proposed as a tool for managing popu-lations that are facing the problems of inbreeding

†All correspondence to: G. F. Ficetola. Tel. +39 (0)250314729;Fax +39 (0)250314802; E-mail: francesco.ficetola@unimi.it

depression (Hedrick, 2001). Such small, isolated popula-tions can be threatened by the loss of genetic diversitycaused by genetic drift, by the fixation of deleteriousmutations caused by the low efficiency of natural selectionand by increased inbreeding. Since these phenomena,together, can cause loss of fitness (see Keller &Waller, 2002), introducing individuals from populationswith higher genetic diversity potentially could restore thegenetic diversity and fitness of small, isolated populations.

However, supplementation could also have detrimentaleffects on endangered species, because it leads to areduction in the genetic differences between populations(Moritz, 2002). Intraspecific genetic diversity is recog-nised as a central conservation problem (Huges, Daily &Ehrlich, 1997; Sinclair, 2001) and the potential fora species’ evolutionary success is maximised throughthe maintenance of adaptive diversity (Frankel, 1974;Hedrick, 2001). Populations representing significantadaptive variation are not ecologically exchangeable(Crandall et al., 2000): several authors define them as‘evolutionarily significant units’, suggesting that theyshould be independently managed to avoid genetichomogenisation (Ryder, 1986; McKay & Latta, 2002;but see also Moritz, 2002). Evaluating whether differentpopulations are evolutionary independent is, therefore, animportant question to resolve prior to supplementation.

34 G. F. FICETOLA AND F. DE BERNARDI

Nevertheless, experimental tests of adaptive divergencebetween populations are scarce in endangered species(Hedrick, 2001).

Among vertebrates, amphibians are considered bysome authors to be potential candidates for relocationprogrammes (Marsh & Trenham, 2001; Semlitsch, 2002;but see Seigel & Dodd, 2002). Since each femaleproduces many eggs, it is relatively easy to rear them andto have large stocks for reintroduction or supplementation:therefore, several projects on the relocation of threatenedamphibians have been carried out and/or are ongoingworldwide (see Burke, 1991; Semlitsch, 2002). The Italianagile frog, Rana latastei, is a brown frog that is endemicto the lowlands of northern Italy (Padano-Venetian plain)and adjacent countries. It lives in riparian woodlands,breeding in river washes and ponds (Pozzi, 1980).These lowlands are currently dominated by agricultureand urbanisation and now R. latastei survives onlyin a limited number of frequently small and isolatedwoodlands. Moreover, the isolation of suitable patcheshas increased the probability of extinction of thesepopulations (Ficetola & De Bernardi, 2004). Fewer than100 breeding sites have been recorded for this species inthe European herpetological atlas; therefore R. latastei isconsidered to be one of the most threatened amphibians inEurope (Grossembacher, 1997) and is rigorously protectedin the European Union (EC 43/1992). Plans are ongoingfor its conservation (Gentilli et al., 2003).

Recent studies have revealed a loss of genetic diversityin peripheral populations in the north-western edge ofR. latastei’s range, when compared to populations witha more central or eastern range (Garner, Angelone &Pearman, 2003; Garner, Pearman & Angelone, 2004).Moreover, preliminary data suggest that some peripheralpopulations that are isolated and on the northern edge ofthe range, could have a lower survival than populationswith a more central location (Ficetola et al., 2003).Translocation of individuals from southern to northernpopulations has been, therefore, proposed to increasegenetic diversity and to avoid the risk of inbreedingdepression (Garner et al., 2003).

We investigated differences in larval performancebetween foothill and lowland populations of R. latasteiin Lombardy (Northern Italy), to evaluate whetherthey belonged to different evolutionary units. Foothillpopulations are small in size and are on the northernedge of the species distribution. Conversely, the southernlowland populations that we studied are larger andlive along rivers. The geographical distance betweenthe populations is small (< 60 km); hence the lowlandpopulations could be good sources of individuals fortranslocation to increase the genetic diversity of foothillpopulations (Garner et al., 2003). However, it is alsopossible that these populations evolved in slightly differentclimates or habitats. Prior to making translocation plans,it is important to evaluate whether the populations haveheritable differences caused by evolutionary processes. Ifdifferences exist, the populations should be consideredto be evolutionarily independent units and they shouldbe protected per se, avoiding translocation; in situ

conservation would be more appropriate. We measuredlarval life-history traits (growth and development rates,size at metamorphosis) since these can evolve in relationto climatic differences (Conover & Shultz, 1995) andare important traits affecting the fitness of anurans(Semlitsch, 2002 and references therein). We comparedlarval performance of hill and lowland populations innature and under common laboratory conditions. Wealso measured maternal effects (see Methods, below) toevaluate the differences between populations excludingpotential environmental effects.

METHODS

Study area and population sampling

Five localities in Northern Italy where R. latasteipopulations live were sampled (Fig. 1). These populationshave different geographical settings: three are in thehills of Brianza (AL, CU and MZ) at altitudes of 320,300 and 175 m above sea level (a.s.l.), respectively;while the remaining two populations (TC and ZB) arefound in the River Po lowlands at altitudes of 69 and76 m a.s.l., respectively. The hill populations are on thenorthernmost range edge and near to the altitudinal limitof R. latastei distributions (Grossenbacher, 1997). Thenumber of breeding females, as estimated by the number ofegg masses, was 43, 10 and 150, respectively, in the popu-lations AL, CU and MZ, and 300 and 1000, respectively,in the populations TC and ZB. All of these populationsbreed in wetlands in or very near to hornbeam andpedunculate oak woods and all are protected in NaturalParks. During early March 2003, we gently removedfrom each population a small fraction (58 ± 5 eggs) fromeach of five spawns laid during the night before thesampling day. The eggs were transferred into 200 mlplastic containers and returned the day after to thelaboratory.

Larval performance measures in the field

To evaluate differences in age at metamorphosis betweenpopulations in the field we dip-netted one hill population(AL) and one lowland population (ZB). During each dip-netting session we captured and briefly examined at least50 tadpoles from the different microhabitats of these twosites, in order to evaluate their development stage. All ofthe tadpoles were liberated immediately after examination.This dip-netting was carried out every 2nd–4th day fromMay 2003, until the first metamorph was found (Gosner(1960) developmental stage 42: emergence of forelimbs).Only two sites were examined, since at the sites CUand TC, R. latastei is syntopic with R. dalmatina: thetadpoles of these two species are similar and it is notpossible to unambiguously distinguish between them (e.g.Vercesi, Bernini & Barbieri, 2000). At the MZ site ananthropogenic pond drying killed all tadpoles prior to themetamorphosis.

Local adaptations in Rana latastei 35

SwitzerlandAustria

SloveniaCroatia

Switzerland

AL

MZ

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CU

12°

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Fig. 1. Location of sampled populations (Lombardy Region, Northern Italy) and average daily temperature (◦C) in July. Temperature mapwas redrawn from Belloni & Annovazzi (1999). TC and ZB are lowland sites, AL, CU and MZ are foothill sites.

Larval performance measures in the laboratory

Spawn samples were reared under standard laboratoryconditions (constant 20 ◦C with 12-h light–dark cycles).Immediately after hatching, 10 tadpoles were randomlyselected from each clutch and put in the same plasticcontainer filled with 1.5 l of aged tap water. Thus, theexperimental design was 10 tadpoles per clutch, fiveclutches per population, five populations, giving a totalof 250 tadpoles. The containers were randomly sortedover the same bench in the laboratory. After reachingGosner’s stage 25, the tadpoles were fed ad libitumwith rabbit chow and lettuce and the water was changedweekly.

The tadpoles were weighed to the nearest 0.01 g onreaching stage 25 in order to evaluate the tadpole startingweight. They were weighed again at 24, 31, 37 and 45 daysafter hatching. During weighing, each tadpole was indi-vidually removed from the water, carefully blotted driedand weighed; the tadpole was then immediately returnedin its container. Age in days from hatching until meta-morphosis, (defined as the almost complete tail resorption(Gosner’s stage 45)), was recorded and metamorphswere weighed to the nearest 0.001 g. After the meta-morphosis all of the froglets were liberated in their wetlandof origin.

Tadpole weight 37 days after hatching was taken asa measure of tadpole growth in terms of weight gainrate, since soon after this the fastest developing tadpolesbegan metamorphic climax and lost weight (Fig. 2). Weconsidered that the age at metamorphosis was a measureof developmental rate.

00 20 40 60

250

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Days

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pole

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Fig. 2. Growth curves and weight at metamorphosis of tadpolesreared in a common environment. Open diamonds, tadpolesfrom foothill populations; filled diamonds, tadpoles from lowlandpopulations. Error bars = 2 standard errors; horizontal errorbars = 2 standard errors of age at metamorphosis. Weight atmetamorphosis was taken from animals already living in a subaerialenvironment (i.e. metamorphosis was complete and the frogletswere now air-breathers).

Statistical analysis

In all our analysis, we used the average value for thetadpoles from each container for each larval performancemeasure (starting weight, weight during growth, ageat metamorphosis, weight at metamorphosis), since thetadpoles in the same container were siblings. Towards

36 G. F. FICETOLA AND F. DE BERNARDI

the end of the experiment, a few tadpoles died in somecontainers resulting in reduced densities in some of thereplicates. Therefore, we added the final density of eachtadpole family (DENSITY) as a covariate in our analysis.

We used mixed model analysis of variance to evaluatethe effects of geographical position (GEOG: hill/lowland),population of origin (POP), tadpole starting weight(W START) and final density (DENSITY) on larval per-formance features of R. latastei tadpoles. We used GEOGas a factor and considered POP nested within GEOG as arandom factor; we used W START as a covariate to checkfor maternal effects (see Parichy & Kaplan, 1992;Zeisset & Beebee, 2003 for a similar approach), sincetadpole hatching size is strongly correlated with eggsize (Kaplan, 1998; Laugen, Laurila & Merila, 2003a).As dependent variables, we used the weight 37days after hatching (W 37D), age at metamorph-osis (AGE METAM) and weight at metamorphosis(W METAM). The residuals were normally distributed.All means are ± 1 standard error.

RESULTS

Field study

All of the populations laid eggs approximately in the sameperiod (March 1–15). We caught the first metamorph in thelowland site ZB on 17 May. During the same day, the mostdeveloped tadpoles we caught in the hill site AL were atdevelopmental stage 33 (toe development of hind limbs).We caught the first metamorph in the hill site AL on18 June. During the same day, we did not find anyR. latastei tadpoles in the lowland site ZB, probablybecause they had all metamorphosed prior to this date.Therefore, in the field, AL (hill) tadpoles appeared totake approximately 1 month longer to complete the meta-morphosis than did ZB (lowland) tadpoles.

Laboratory study

None of the embryos from two spawn samples of thepopulation CU survived until stage 25, hypotheticallydue to inbreeding depression, therefore we dropped thesetwo spawns from all our analyses. Overall, we consideredtadpoles from 23 spawns: 13 from the foothill populationsand 10 from the lowland populations.

At 20 ◦C, the tadpoles from the foothill populationsgrew faster and metamorphosed earlier than those fromthe lowland populations (Fig. 2). Average weight 37 daysafter hatching was 449 ± 9 mg for foothill tadpoles and395 ± 13 mg for lowland tadpoles (Fig. 3(a)). The firsttadpoles metamorphosed 48 days after hatching: they werefrom the population AL (hill); the last one metamorphosed79 days after hatching: it was from the populationZB (lowland). On average, foothill tadpoles meta-morphosed 53.4 ± 0.5 days after hatching while lowlandtadpoles metamorphosed 57.2 ± 1.3 days after hatching:development rate was therefore 7.1% faster in the foothillpopulations (Figs 2, 3(b)). Weight 37 days after hatching

0

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ys)

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ght a

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is (

mg)

a a a bb

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(b)

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Fig. 3. Larval performance of R. latastei tadpoles from five popula-tions. Grey bars, foothill populations; black bars, lowland popula-tions. Error bars = 2 standard errors. (a) weight of tadpoles 37 daysafter hatching. (b) age at metamorphosis. (c) weight at meta-morphosis. a,bIndicate significant differences for the larval per-formance measure considered: all populations labelled ‘a’ aresignificantly different from all populations labelled ‘b’; populationssharing the same letter are not significantly different from oneanother.

and age at metamorphosis were strongly correlated (r =− 0.694, n = 23, P < 0.001): the tadpoles that grew fasterafter the hatching also metamorphosed earlier. Therelationship between growth rate and weight at meta-morphosis was not significant (r = 0.148, n = 23,

Local adaptations in Rana latastei 37

Table 1. Effects of various factors on different R. latastei tadpolelife history parameters

Factors F d.f. P

A. Dependent: W 37DGEOG 20.152 1,4.7 0.007POP 0.508 3,16 0.682W START 0.453 1,16 0.511DENSITY 4.730 1,16 0.045

B. Dependent: AGE METAMGEOG 9.838 1,6.1 0.020POP 0.294 3,16 0.829W START 0.702 1,16 0.415DENSITY 0.016 1,16 0.902

C. Dependent: W METAMGEOG 0.241 1,3.7 0.651POP 1.168 3,16 0.353W START 0.693 1,16 0.417DENSITY 55.521 1,16 < 0.001

GEOG, geographical position; POP, population; W START,starting weight; DENSITY, final density in experimentalcontainers; W 37D, weight 37 days after hatching; AGE METAM,age at metamorphosis; W METAM, weight at metamorphosis.

P = 0.50). We observed a tendency for the lastmetamorphosing tadpoles to be heavier than the first(r = 0.388, n = 23, P = 0.067).

We observed a significant effect of geographicalpositions on larval performance, but differences werenot present between populations within groups (Table 1).Tadpole starting weight did not have a significant effecton larval performances, suggesting a lack of egg-sizerelated maternal effect. Tadpoles from foothill populationsgrew faster and metamorphosed earlier than tadpoles fromlowland populations (Table 1, Figs 3(a), (b)). However,geographical position did not seem to influence the weightat metamorphosis (Table 1, Fig. 3(c)). Age at meta-morphosis was not related to final density in experimentalcontainers. However, the tadpoles from containers withlower density were the heaviest both 37 days afterhatching and at metamorphosis (Table 1). Since wedid not observe a significant variability within groupfor any measure of larval fitness, we repeated theanalysis pooling the data from populations with the samegeographical position. The results were the same as thoseobtained using the complete model. Weight 37 days afterhatching was significantly higher in foothill populations(F1,19 = 17.630, P < 0.001) but it did not seem to beaffected by starting weight (F1,19 = 0.023, P = 0.881).Foothill populations metamorphosed significantly earlierthan did lowland populations (F1,19 = 6.168, P = 0.023)but age at metamorphosis did not seem to be affectedby starting weight (F1,19 = 0.222, P = 0.643). Weight atmetamorphosis was not different between foothill andlowland populations (F1,19 = 0.0764, P = 0.786) nor didit seem to be affected by starting weight (F1,19 = 0.364,P = 0.553). Age at metamorphosis was not related to finaldensity (F1,19 = 0.129, P = 0.723); however the tadpoles

from lower density containers were the heaviest both37 days after hatching (F1,19 = 5.012, P = 0.037) and atmetamorphosis (F1,19 = 56.476, P < 0.001).

DISCUSSION

Evolutionary meaning of between-population differences

Under the same laboratory conditions, foothill populationsof R. latastei showed significant differences from lowlandpopulations in larval performance traits. Ignoring poten-tial maternal effects, our results seem to have anunambiguous interpretation: foothill populations have acapacity for faster growth and development and thisdifferentiation is difficult to interpret other than in the lightof local adaptation to some selective factor (Merila et al.,2000; Laugen et al., 2003b). Egg size is a major sourceof maternal effects in amphibians (Kaplan, 1998). Startingweight did not significantly affect larval performance,thus, we suggest that differences in larval performancesbetween these populations are mainly caused by geneticdifferentiation and not because of egg-size relatedmaternal effects (c.f. Laugen et al., 2003b).

Many studies have demonstrated that growth and devel-opment rates strongly influence the fitness of amphibians.High growth and development rates enable tadpoles tometamorphose quickly to escape death in drying ephem-eral ponds, to escape aquatic predators, or to maximise sizeat metamorphosis, therefore these traits should be subjectto strong natural selection (Wilbur & Collins, 1973;Travis, Keen & Juilianna, 1985; Banks & Beebee, 1988;Newman, 1988a,b). Several factors could have caused theobserved differences between populations: among them,the effect of temperature on the selection of a fast-growingphenotype seems to be the most likely.

Water temperature strongly affects development andgrowth rate in amphibians: tadpoles living in a colderenvironment grow and develop slowly. A few studies haveoutlined that when compared in a common environment,larvae from populations living in colder areas showedthe genetic capacity to complete metamorphosis faster(Berven, Gill & Smith-Gill, 1979; Berven, 1982a; Loman,2002a). When geographical variation in genotypes is inopposition to environmental influence, a trait displays acountergradient variation (Conover & Shultz, 1995). Ourresults are consistent with this pattern and could be anew case of countergradient variation in amphibians. Inour study case, foothill populations of R. latastei livein a slightly colder climate, with average spring andsummer temperature being 1–2 ◦C lower than that forlowland populations (Belloni & Annovazzi, 1999; Fig. 1).Therefore, in nature, the tadpoles of foothill populationsmetamorphosed later than did the tadpoles from lowlandpopulations. However, in a common environment thefoothill populations showed the capacity to grow andmetamorphose earlier. Thus, it is likely that naturalselection favoured a fast-growing genotype in the foothillpopulations, living in a colder climate. Individuals notadapted to the hill environment are expected to have a

38 G. F. FICETOLA AND F. DE BERNARDI

slow growth and development rate if reared in the colderclimate of hills and, therefore, to have a too delayedmetamorphosis (Berven et al., 1979).

Our study dealt with only a relatively few sites,tadpoles were reared at only one temperature and thepopulations cannot be placed along a continuous climaticgradient. Therefore, it is possible that the differencesseen in larval performance are caused by forces otherthan countergradient selection. For example, it is possiblethat the populations are adapted to different thermaloptima: tadpoles develop relatively faster when they arereared at temperatures more similar to those of theirwetland of origin (Skelly & Freidenburg, 2000). Moreover,there is some evidence that frog populations living intemporary ponds can evolve a faster development rateor a higher developmental plasticity, to reduce the risk ofdesiccation (Wilbur & Collins, 1973; Laurila, Karttunen &Merila, 2002; Loman, 2002b). However, none of thewetlands of our study was temporary, therefore this latterhypothesis seems to be unlikely. The desiccation of siteMZ was an unusual event, caused by the inappropriatemaintenance of a pond, since this population resides ina urban park. Finally, other differences between sites cannot be ruled out, such as differences in predator abundance(Lardner, 1998), differences in food availability or otherunrecognised differences between habitats.

Local adaptation: implications for management

We observed variation that could be adaptive betweenpopulations of R. latastei living in sites quite close togetherand with rather small ecological differences. Thus, foothilland lowland populations are ecologically distinct and thereis not exchangeability between them (Crandall et al.,2000): the lowland populations should not be usedas a source of individuals in supplementation projectsaimed at increasing the size and/or genetic diversityof the foothill populations. Indeed, translocation couldhave negative effects for the conservation of R. latastei.First, the genetic homogenisation could undermine theevolutionary potential of the species. In a human-dominated environment, with fast and unpredictablemodifications, the evolutionary potential has pivotalimportance for the persistence of the species. Moreover,lowland individuals introduced to a hill site could havevery low fitness (home-site advantage), since they maybe poorly adapted to the new environment, sufferingunusually high mortality or lowered reproductive success(Montalvo & Ellstrand, 2000). For example, translocatedtadpoles could have delayed metamorphosis or highermortality (Berven et al., 1979). Similarly, foothill tadpolestransplanted to the lowland environment would not beadapted to the warmer climate, suffering a reduced abilityto cope with higher temperatures (Skelly & Freidenburg,2000). Furthermore, it is possible that populations differfor other life-history traits not investigated in this study,such as size and age at first reproduction (Berven,1982b) and, therefore, the home-site advantage could beeven stronger. Finally, mixing different populations could

result in the reduced fitness of hybrids, a phenomenoncalled outbreeding depression (Storfer, 1999; Montalvo &Ellstrand, 2000; Edmands & Timmerman, 2003).

The foothill populations are small and isolated andthey face a high risk of extinction due to environmentaland demographic stochasticity and due to potentialinbreeding depression. Since these populations seem torepresent significant adaptive variation for R. latastei, theirprotection should be prioritised for the conservation of thisthreatened frog. Translocation projects are not acceptable,thus different management proposals should be made,emphasising in situ conservation. First, we should con-sider habitat management, such as the creation of newponds, improvement in the suitability of existing wetlandsand management of terrestrial habitat (Semlitsch, 2002).The availability of larger patches and of a more suitablehabitat should allow an increase in the population size,however, the results of these actions will require a longtime. During the winter of 2001–2002, new wetlandswere excavated where the population AL lives. To date,this population seems to have reacted well to theseactions, since the number of breeding females grew from19 during 2001 to 43 during 2003 (A. Gentilli, pers.comm.). Obviously, an increase in genetic diversity willrequire a longer time than does demographic growth(Hedrick, 2001). Eventually, for a faster recovery of intra-population genetic diversity, translocation of individualswithin the foothill population group could be considered,since variability within group seems to be low in ourstudy (Table 1). These actions should be planned onlyafter more detailed studies on the differences betweenthese populations and, preferably, after molecular geneticstudies.

CONCLUSION

We showed that R. latastei populations less than 50–60 kmapart and living in similar environments have significantdifferences in possible adaptive traits. Therefore, manage-ment actions such as translocation could lead to a lossof intraspecific genetic diversity and evolutionary poten-tial and, eventually, to outbreeding depression. Localadaptation could be more frequent than previouslysuspected in animals with low mobility and fast generationtimes, resulting in significant adaptive differences beingpresent between populations that are close or livingin similar habitats. Many studies have used variationat neutral genetic markers to evaluate differences bet-ween populations prior to planning translocations as amanagement tool (for a review, see Crandall et al., 2000).However, the correlation between neutral and adaptivevariation can be low and the use of neutral markers onlywould not reveal the presence of variants representingimportant intraspecific diversity (for a discussion, seeCrandall et al., 2000; Montalvo & Ellstrand, 2000;Hedrick, 2001; Pearman, 2001; McKay & Latta, 2002 butsee also Moritz 1999, 2002). Therefore, the analysis ofevolutionary variation, as measured by life-history traits,could be a useful tool for the development of a correct

Local adaptations in Rana latastei 39

decisional pathway for the management and conservationof wild populations, for example in relocation plans.

Acknowledgments

We thank the managers of several Parks in Lombardy(Adda Sud, Valle del Ticino, Valle del Curone, Valle delLambro) who allowed us to perform this study. F. Bernini,A. Gentilli, E. Razzetti and A. Bonardi and the SocietasHerpetologica Italica-Lombardia helped us to locate thebreeding sites and provided useful information; S. Belloniprovided useful climatic data and advice. I. Mazzoleniand L. Nudo greatly helped during the laboratory work.The comments of P. B. Pearman, P. L. Starkweather,A. Gentilli, D. Fontaneto and two anonymous refereesgreatly improved earlier drafts of the manuscript.

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