Abstract. The survival of threatened species as the European tree frog (Hyla arborea) is strongly dependent on thegenetic variability within populations, as well as gene flow between them. In Switzerland, only two sectors in its westernpart still harbour metapopulations. The first is characterised by a very heterogeneous and urbanized landscape, while thesecond is characterised by a uninterrupted array of suitable habitats. In this study, six microsatellite loci were used toestablish levels of genetic differentiation among the populations from the two different locations. The results show thatthe metapopulations have: (i) weak levels of genetic differentiation (FST within metapopulation ≈ 0.04), (ii) no differencein levels of genetic structuring between them, (iii) significant (p = 0.019) differences in terms of genetic diversity (Hs) andobserved heterozygozity (Ho), the metapopulation located in a disturbed landscape showing lower values. Our results suggestthat even if the dispersal of H. arborea among contiguous ponds seems to be efficient in areas of heterogeneous landscape, aloss of genetic diversity can occur.
As a result of growing urbanization and sub-sequent habitat degradation and fragmentation,numerous animal species currently face extinc-tion or have declined drastically during the lastcentury. These disturbances commonly resultin population size reduction and decrease thepossibility of migration and subsequent geneflow between populations (e.g. Frankham etal., 2002; Cushman, 2006). The genetic conse-quences of population fragmentation are com-plex and critically depend upon levels of gene
1 - Department of Ecology and Evolution, Laboratory forConservation Biology, Biophore, CH-1015 Lausanne,Switzerland
2 - Heydon-Laurence Bld, A08, Science Road, School ofBiological Sciences, University of Sydney, Sydney,NSW 2006, Australia
3 - Department of Environmental Sciences, Section of Con-servation Biology, University of Basel, St. Johanns-Vorstadt 10, CH-4056 Basel, Switzerland
4 - A. Maibach Sàrl, CP99, Ch. de la Poya 10, 1610 CH-Oron-la-Ville, Switzerland∗Corresponding author; e-mail:[email protected]
flow between fragments. When it is restricted,population fragmentation is expected to reducewithin-population genetic polymorphism andincrease genetic differentiation among popula-tions, typically leading to a loss of genetic diver-sity within fragments (e.g. Hitchings and Bee-bee, 1997). Consequently, understanding the ef-fects of population fragmentation is crucial inconservation biology, since extinction risks aremore elevated in fragmented populations withlow levels of genetic variability (Frankham etal., 2002; Frankham, 2005).
The studied species, The European tree frog(Hyla arborea), is a pond-breeding specieswhich possess a distribution area extendingfrom Portugal to southern Sweden, and fromthe Balkans to west Asia (Gasc et al., 1997).As in several other amphibian species (Wake,1991), a strong decline across its entire dis-tribution range has been recorded during thelast decades (Gasc et al., 1997). In Switzerland,once widespread across this country, the specieshas been reduced to less than one dozen sec-
tors, which undergo local extinctions (Grossen-bacher, 1988, 1994). This severe decline isthought to be mainly due to anthropogenic ac-tivities. Studies carried out on the Europeantree frog in Sweden (Carlson and Edenhamn,2000), as well as in Switzerland (Pellet et al.,2006), highlighted regular extinction and re-colonisation events characteristic of a metapop-ulation dynamics (Hanski and Gilpin, 1997).Extensive theory has been developed to modelgenetic processes within metapopulation struc-tures. Due to frequent extinctions and bottle-necks during recolonisations, metapopulationsare likely to suffer more rapidly from inbreed-ing and fitness reduction than single large popu-
lations with the same total size (Gilpin, 1991;Hanski and Gilpin, 1997).
In this study we sampled the two rem-nant metapopulations of the western portion ofSwitzerland (Dubey et al., 2006): (i) the firstlies on the northern shore of the lake of Geneva(LG metapopulation, fig. 1), a region charac-terised by a very heterogeneous and mixed agri-cultural and urbanized landscape, and present alow number of occupied ponds (less than 25;Pellet et al., 2002); (ii) the second is locatedon the southern bank of the lake of Neuchâtel(LN metapopulation), which on the opposite isan uninterrupted landscape of suitable habitats,including marshes and wet meadows, as well
Figure 1. Localisation of demes within the northern shore of the lake of Geneva (LG) and the southern bank of the lakeof Neuchâtel (LN), with arrows corresponding to pairwise Fst values between demes within metapopulations (all valuessignificant at 0.05 level are indicated; the thickness of arrows indicates the level of gene flow).
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as woodland bordering the lake in a continuousway. In the year 2000, 40 calling ponds weredetected, totalling several thousand frogs (Pel-let and Neet, 2001). This area is considered asone of the largest metapopulation in Switzer-land (Grossenbacher, 1988).
Information concerning the extent of popu-lation fragmentation is critical to determinewhether a species requires proactive manage-ment plans to reduce extinction risks associ-ated with genetic stochasticity. Hence, to es-timate the effect of habitat fragmentation onthe genetic structure and variability of tree frogpopulations, we investigated the two isolatedmetapopulations in western Switzerland withsix microsatellite loci. We hypothesized that inmetapopulations embedded in continuous habi-tats genetic structure would be lower, while ge-netic variability and gene flow higher comparedto metapopulations characterized by lower den-sities and where heterogeneous habitat may actas a barrier to gene flow.
During spring 2002 and 2003, a total of 235 samplesof Hyla arborea (tadpoles and eggs) were collected in 8ponds in the two studied areas (fig. 1). Five ponds weresampled within the LG metapopulation (Allaman, CampRomain, Lavigny, Mossières, Vaudalle) and three pondsin the LN metapopulation (Chabrey, Gletterens, Trouville).When eggs were collected, only one egg per clutch was usedfor genetic analyses.
DNA extraction from tissues and egg samples was car-ried out using the QIAamp DNA Mini Kit (QIAGEN), orwith a CTAB protocol (Milligan, 1992). Six microsatelliteloci isolated and characterized for Hyla arborea (Wha1-9, Wha1-20, Wha1-25, Wha1-103, Wha1-104, Wha1-140;Arens et al., 2000) were amplified and scored (see Arens etal. (2000) for the specific PCR profiles). Amplified productswere genotyped with an ABI PRISM 377 DNA Sequencerusing genescan analysis 2.1 software (Applied Biosystems).
Gene diversities comprising observed (Ho), expected(Hs) within-deme (the populations will be treated as demesin the different sections of the manuscript) and expectedoverall heterozygosities (Ht) were estimated following (Neiand Chesser, 1983). Genotypic disequilibrium betweenloci in each sample and deviations from Hardy-Weinbergequilibrium (HWE) within samples were tested based on2400 permutations and 10 000 randomizations, respectively.Wright’s fixation indices for within-deme deviation fromrandom mating (FIS), as well as pairwise deme differen-tiation (FST ), were estimated following Weir and Cocker-ham (1984). Deviation from random mating within demes(FIS) per locus and sample were computed with a boot-strap procedure (2000 randomizations). Statistical support
for pairwise deme differentiation was obtained through ex-act G-tests on allelic frequencies as described by Goudet etal. (1996) with 2000 randomizations. All summary statisticsand tests mentioned above have been computed using FSTAT
Version 2.9.3.2 (Goudet, 1995). Permutation tests were car-ried out in order to detect significant differences in allelicrichness, expected (Hs) and observed (Ho) heterozygositiesand FST indices among the two studied metapopulations.
Genetic isolation by distance at the metapopulationallevel and overall was tested by using a partial Mantel test(Mantel, 1967); p-value were given after 10 000 randomiza-tions.
A Bayesian model-based clustering method (Pritchardet al., 2000) for inferring population structure and assign-ing individuals to populations was used as implemented instructure version 2.1 (Falush et al., 2003). Based on allelefrequencies, individuals are assigned, through the use of aMarkov chain Monte Carlo (MCMC) simulation, a member-ship coefficient for each of K populations. We performed 10runs of 6 ·105 iterations (the first 105 considered as burn-in)for K = 1 to K = 10 (i) including all the populations and(ii) within the two metapopulations. The number of popu-lations best fitting our data set was defined as described inEvanno et al. (2005). The latter statistics compares the rateof change in the log probability of data between successiveK and the corresponding variance of log probabilities.
We used the software migrate 2.0.6 (Beerli and Felsen-stein, 2001; Beerli, 2004) to estimate the scaled migrationrate (M) between demes within metapopulations. This soft-ware is based on a coalescence model with mutation andmigration, and estimates a measure of effective populationsize, θ , defined as 4Neμ, where μ denotes mutation rate,and migration M , defined as m/μ, where m denotes migra-tion rate. We assumed a stepwise mutation model and basedestimates on 15 short [104 Markov Chain Monte Carlo(MCMC) steps] and five long (105 MCMC steps) chains.To ensure convergence, we used the ‘adaptive heating’ op-tion with one ‘cold’ and three ‘hot’ chains.
Tests for HWE indicated that all loci testedwere at HWE and in genotypic equilibrium. Forthe six microsatellite loci, the number of allelesper locus ranged from 7 to 17 (average = 9.83),with a total of 59 alleles across 6 loci. The al-lelic richness within deme ranges from 4.41 to5.29, with an overall mean of 6.25 (table 1). Ex-pected heterozygosities per locus within demes(Hs) ranged from 0.16 to 0.90, with an av-erage of 0.58, whereas expected overall het-erozygosity (Ht) averaged 0.62 (range per lo-cus: 0.35-0.87). Observed heterozygosity (Ho)values varied from 0.44 to 0.68, with an aver-age of 0.51 (see table 1). There was a signifi-cant deviation from random mating in the ana-lyzed demes (overall FIS = 0.12, p > 0.001;
130 Short Notes
LN FIS = 0.06, LG FIS = 0.16), suggest-ing the occurence of a within-sample sub-structure. The genetic differentiation betweendemes (pairwise FST ) within each metapopu-lation was low, ranging from 0.01 to 0.07, allvalues being significant, except for the pairs ofdemes: Mossière-Allaman, Vaudalle-Lavigny,and Trouville-Chabrey (p > 0.05). Compara-ble overall FST values were found in the LN andLG metapopulations (respectively 0.046, 95%CI: 0.017-0.05 and 0.039, 95% CI: 0.025-0.079;
Table 1. Genetic diversities for eight Hyla arborea demesin two distinct metapopulations based on 6 microsatelliteloci. N = maximum number of singers in the population in2002 (Pellet et al., 2002); n = sample size; Ho = observedheterozygosity; Hs = expected heterozygosity; AR = allelicrichness.
p = 0.86; see fig. 1). The pairwise FST val-ues among demes from separated metapopula-tions varies from 0.086 to 0.142 (mean = 0.11),for geographical distances ranging from 59.2 to69.7 km.
No isolation by distance within the LGmetapopulation was detected (p = 0.40, r2 =0.09), indicating the absence of a pattern of ge-netic isolation with geographical distance (nottested on the three populations of LN). Signifi-cant differences between metapopulations wereobserved for Ho (LN Ho = 0.60, LG Ho =0.45, p = 0.019) and Hs (LN Hs = 0.64, LGHs = 0.54, p = 0.019), whereas allelic rich-ness (AR), FST and FIS were not significantlydifferent (respectively p = 0.43, p = 0.86 andp = 0.11; see table 1).
The analyses performed with structure, re-vealed that the number of populations best fit-ting our data set is K = 2. No substructure wasrevealed within the metapopulations.
Concerning the pairs of unidirectional migra-tion rates (M) estimated between demes withinmetapopulations, 11 of the 13 in total wereasymmetric (i.e. where 95% CI did not overlap;table 2). In addition, the analysis clearly showedthat recent migrations occurred between demeswithin both metapopulations.
Table 2. Gene flow between populations within demes (M = m/μ, with 95% confidence interval).
Polulation (i) Allaman C. romain Lavigny Mossières Vaudalle Chabrey Gletterens Trouville→ i → i → i → i → i → i → i → i
The main result stemming from this studyis the absence of substantial differences interms of genetic differentiation between the twometapopulations, despite an important contrastin habitat fragmentation and demes densities, asshown by (i) the low global structure observedwithin metapopulations (mean FST ≈ 0.04),with pair-wise FST ranging from 0.01 to 0.07;(ii) the absence of substructure within metapop-ulations, as revealed by clustering analyses and(iii) the unidirectional indices of migration (M)observed between demes within metapopula-tions, with some very high values observed (Mmax. for LN: 4.26 and for LG: 7.49). Therefore,our data suggest a high overall rate of disper-sal within both metapopulations. Though, thepairs of unidirectional indices of migration (M)were mostly asymmetric within both metapop-ulations (11 of 13 pairs), with values varyingfrom 0.42 to 7.49, revealing that the demes con-tributed differently to the low structure observedbetween them. Therefore, the genetic diversityof demes could be reduced by the effect ofasymmetric gene flow.
In contrast with the low structure observedwithin both areas, the analyses revealed signif-icant (p = 0.019) lower gene diversity (Hs)and observed heterozygosity (Ho), within LGmetapopulation, as well as a higher, but not sig-nificantly different, substructure (FIS).
Consequently, the results pointed out that thelandscape of LG is less suitable for the Euro-pean tree frog than that of LN and as a result,dispersal is less effective to maintain gene flowamong local demes, and as consequence geneticdiversity within demes. This pattern is illus-trated within LG by a strong variability of pair-wise FST values between spatially closer demesdistant of 1.5 and 1.8 km, with FST varyingfrom 0.01 to 0.06, respectively. Thus, it con-firmed that other independent factors than dis-tance alone must be taken into account in thedispersion of the tree frog, such as roads, urbanareas, as well as natural obstacles and historicalfactors (Pellet et al., 2004).
Overall and despite significant differencesobserved in term of genetic diversity betweenour two metapopulations, these values are com-parable to genetic variation at microsatellite locifound in other amphibian species, e.g. fromArens et al. (2007) on Rana arvalis, or fromScribner et al. (2001) on Bufo bufo. In addition,our estimates of genetic variability Hs, vary-ing from 0.49 to 0.66, are slightly in contrastwith the data from Andersen et al. (2004; 0.35-0.53) and Arens et al. (2006; 0.39-0.59) on H.arborea metapopulations in Denmark and theNetherlands, respectively, or e.g. from Hitch-ings and Beebee (1997) on Rana temporariaand Newman and Squire (2001) on Rana syl-vatica, where heterozygosity levels are lower.
Mostly based on the same microsatellitemarkers, genetic differentiation is in opposi-tion substantially higher within the Danish andDutch metapopulations (Andersen et al., 2004,Arens et al., 2006), characterized by FST val-ues ranging from 0.03 to 0.33 (overall FST =0.22) and from 0 to 0.35 (overall = 0.19), re-spectively. Several mutually non-exclusive hy-potheses could explain the substantial diffe-rence in genetic differentiation between our re-sults and the two available studies on H. ar-borea: (i) in contrast with the area studied byAndersen et al. (2004) and Arens et al. (2006),some natural structuring elements such as ripar-ian ecosystem exist in the LG metapopulationlandscape, and such complementary terrestrialhabitats could act as potential dispersal corri-dors for H. arborea; (ii) geographical scale issmaller in our study (groups of ponds in Ander-sen et al. (2004) are for instance separated byseveral km); (iii) the Danish and Dutch stud-ied areas being situated in the northern mar-gins of the distribution of the species, phenom-ena as local drift and local extinction could bemore significant, leading to an increase of ge-netic structure; (iv) when sampling tadpoles, po-tential sampling of siblings (individuals fromthe same clutches) may upwardly bias FST es-timates. Although the occurrence of closely re-lated tadpoles could not be excluded, our sam-
132 Short Notes
pling was performed in order to minimize theirpresence. On the contrary, the Danish samplingpossibly included related individuals, as fourdifferent tadpoles per clutch were analyzed (noinformation was given concerning the Dutchsampling strategy).
Overall, our results suggest that even ifthe dispersal of H. arborea among contiguousponds is high in areas of heterogeneous land-scape, as shown in LG, a loss of genetic diver-sity can occur. Although no signs of inbreed-ing depression have been highlighted withinthe fragmented LG metapopulation of H. ar-borea, the small size of the demes, coupledwith a lower genetic diversity compared to LNmetapopulation, might increase the risk of de-mographic and genetic stochasticity. Thus it islikely that the size and the level of connectiv-ity among demes is a crucial factor for the sur-vival of the metapopulations of Hyla arborea.Consequently, in order to protect the Europeantree frog populations in an efficient way, prior-ity should be given to conserving areas of suit-able habitat to promote population connectivityand maintain genetic diversity and evolutionarypotential. In particular, conservation manage-ment actions should be focused on: (i) increas-ing the metapopulation sizes and the number ofdemes; (ii) encourage the connectivity withinthe metapopulations. In this context, the rela-tively high migration potential of the Europeantree frog should permit a rapid natural coloni-sation of newly created ponds. Although thetwo metapopulations have been separated foronly 20 years, they are genetically well differ-entiated (mean FST between the two metapop-ulations 0.11). Consequently, environment im-provements between them might be undertakenin order to reconnect each other and to recre-ate gene flow between both regions. Since theEuropean tree frog is a mobile species, the con-nection between the two metapopulations couldprobably be realised with the creation of onlya limited group of ponds with favourable sur-rounding habitats.
Acknowledgements. This work was partly funded by theOFEV (Office Fédéral de l’Environnement; E. Kohli). Weacknowledge N. Duvoisin and N. di Marco for help in thelaboratory. The samples were collected with the autorisa-tion of the “Conservation de la Faune du Canton de Vaud”(C. Neet) and the KARCH (Centre de Coordination pourla protection des Amphibiens et des Reptiles de Suisse;S. Zumbach and J.-C. Monney). We also thank J.-M. Fi-vat, J. Goudet, S. Trouvé and two anonymous referees forhelpful comments.
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