Biological Conservation 114 (2003) 371–376 www.elsevier.com/locate/biocon
Genetic depletion in Swiss populations of Rana latastei: conservation implications Trenton W.J. Garner*, Sonia Angelone, Peter B. Pearman Zoologisches Institut, Universita¨t Zu¨rich, Winterthurerstrasse 190, CH-8057 Zu¨rich, Switzerland Received 28 August 2002; received in revised form 22 January 2003; accepted 5 February 2003
Abstract Rana latastei, the Italian Agile Frog, is found over a restricted range in Central Europe, with populations in Switzerland (Tessin) occurring at the very margin of the species distribution. We used microsatellite DNA polymorphisms to study the genetic diversity of Swiss populations of Rana latastei and compared them with several Italian populations located in the central and western parts of the species’ range. On average, Swiss populations showed a much lower level of within-population genetic diversity relative to their Italian counterparts. Existing Swiss conservation efforts do not include genetic considerations and our results show that any attempt to increase genetic diversity within Switzerland would require a collaborative effort with Italian wildlife agencies. # 2003 Elsevier Ltd. All rights reserved. Keywords: Rana latastei; Peripheral populations; Microsatellites; Genetic diversity
1. Introduction The conservation value of peripheral populations is a matter of contention. Peripheral populations are traditionally viewed as more vulnerable to extinction due to isolation (Richter-Dyn and Goel, 1972; Brown and Kodric-Brown, 1977; Brown, 1984; Gaston, 1990; Vrijenhoek, 1994; Brown et al., 1995). Accordingly, peripheral populations of endangered species are likely doomed and merit little conservation effort on demographic grounds (e.g. Pearl, 1992), which bodes ill for species with restricted ranges. Other work, to the contrary, suggests that the demographic status of populations with respect to proximity to range edge has little to do with extinction probability (Channell and Lomolino, 2000a). Because factors other than proximity to range edge determine likelihood of extinction (Channell and Lomolino, 2000b), the implication is that peripheral populations can play an important role in the conservation of species through the maintenance of range size. Peripheral populations often occur in a different country than that of the majority of the species distribution (e.g. Hydromantes ambrosii populations in southeast France, Swedish populations of Rana lessonae, Gasc et * Corresponding author. E-mail address:
[email protected] (T.W.J. Garner). 0006-3207/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0006-3207(03)00065-X
al., 1997). As conservation and management policies do vary widely among countries, national borders must be taken into account when managing populations marginalized by both species distribution and political boundaries. Intuitively, it makes little sense to pursue a conservation program that, due to political factors, considers only a small subset of a species overall range. An example of the lack of integrated conservation of peripheral populations across national borders is the case of the Tiger Salamander, Ambystoma tigrinum. Populations of this species found in British Columbia, Canada are of the subspecies melanostictum (Irschick and Shaffer, 1997), are considered as endangered (COSEWIC, http://www.cosewic.gc.cal) and have been red-listed for years (Wildlife Branch and Habitat Protection Branch, B.C. Ministry of Environment, Lands and Parks, 1995). Nonthreatened populations of this subspecies and one other (Ambystoma tigrinum diaboli) occur in provinces east of B.C., but B.C. populations are part of a disjunct range of melanostictum that are related, geographically and evolutionarily, to unprotected Washington State salamander populations (Stebbins, 1985). Recruitment into B.C. populations could thus be seriously hampered by the lack of conservation of Washington State A. t. melanstictum populations and this may render meaningless Canadian conservation efforts.
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The role of peripheral populations in conservation planning, when considered from a genetic perspective, is similarly unclear. Reduced genetic diversity is often detected in peripheral populations (Lesica and Allendorf, 1992, 1995) and, overall, neutral genetic variation is less pronounced in species with restricted ranges, even when phylogenetic biases are accounted for (Karron, 1987; Hamrick and Godt, 1989). These observations have led some workers to suggest that such populations are of poor evolutionary or conservation ‘value’ (discussed in Lammi et al., 1999; Pearman, 2001). When adaptive genetic variation is considered, though, peripheral populations may be subjected to selective regimes that may favour local adaptation and maintain adaptive genetic variation among populations (Lesica and Allendorf, 1995; Garcia-Ramos and Kirkpatrick, 1997). Thus, peripheral populations can act as repositories of functionally important intraspecific genetic diversity that contributes to the maintanence of species ranges. Conservation of genetic diversity within species is a major conservation issue (Hughes et al., 1997), and is one of three levels of diversity prioritized by the IUCN (McNeely et al., 1990). This suggests that general patterns of conservation value of peripheral population need to be determined. The Italian Agile frog, Rana latastei (Boulenger), inhabits a geographically restricted range limited primarily to northern Italy. Marginal populations exist in southern Switzerland, and in western Slovenia and Croatia (Grossenbacher, 1982; Capula et al., 1991). Southern Switzerland populations are restricted to a small (12 km8 km) geographic area and represent the northern limit of the western portion of the species’ range. Rana latastei is ‘nearly threatened’ globally (IUCN-SSC, 2000), but in Switzerland it is a red list species threatened with local extinction (Grossenbacher, 1994). Swiss populations are well-protected and ongoing conservation efforts improve and maintain existing breeding and adjacent habitat (Grossenbacher, 1997a), while Italian conservation efforts are not as extensive. Exactly what is being conserved within the borders of Switzerland at the genetic level has yet to be determined. In this study, we assess genetic variation within populations of R. latastei in Switzerland. We also determine whether Swiss frogs differ at the molecular level from Italian counterparts. We use seven microsatellite loci (Gamer and Tomio, 2001) to analyse genetic relationships and patterns of genetic diversity in twelve Swiss and seven Italian populations.
2. Materials and methods 2.1. Field sampling and molecular methods In the spring of 2000, we surveyed previously detected Rana latastei breeding sites in the Tessin, Switzerland
for both adults and egg masses. We sampled egg masses from 12 of these sites (Fig. 1) and removed embryos from a maximum of 20 egg masses per site. Seven Italian populations were sampled in the Spring of 2001 as well (Fig. 1). Sampling eggs reduced our impact on sites as early egg mortality is already high (Dueliman and Trueb, 1986) and avoided the potentially harmful effects associated with adult toe clipping (Clarke, 1972). Eggs were reared to hatching and a single larva per egg mass was used for DNA extraction. A total of 318 (181 Swiss, 137 Italian) larvae were genotyped at 7 dinucleotide microsatellite loci following Gamer and Tomio (2001). All PCR products were electrophoresed using the SEA 2000TM advanced submerged gel electrophoresis apparatus (Elchrom Scientific AG, Switzerland) and run out on SpreadexTM EL 300 gels (Elchrom Scientific AG, Switzerland) at 100 V for 90 min. We visually scored all alleles against the M3 Marker ladder (Elchrom Scientific AG, Switzerland).
Fig. 1. Locations for sample populations of Rana lasterei. Lower panels shows locations of six of seven Italian populations, excluding Moreggi Pedrinate. Abbreviations are as found in Table 3. Upper panel shows locations of populations sampled in Switzerland and Italian population Moreggi Pedriante (MP). Numbers correlate with Swiss populations as follows (abbreviations in Table 3): 1=CSCH, 2=PCCH, 3=PBPCH, 4=FCH, 5=DLLCH, 6=PCCGCH, 7=CCH, 8=PACH, 9=MCH, 10=VBCH, 11=PMCH, 12=DCBCH.
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2.2. Data analysis We tested all loci for deviations from Hardy–Weinberg equilibrium using FSTAT (Goudet, 1995, 2001) and global tests with 1000 permutations, as suggested when the number of loci analysed is less than 10. Tests for deviations from Hardy–Weinberg proved significant for loci RlatCa18, Rt2Ca9, RlatCa2l and RlatCa4l at the 0.05 nominal confidence level, after Bonferroni adjustment. Therefore, population pairwise and locus by locus tests for population differentiation were carried out using FSTAT and not assuming Hardy–Weinberg equilibrium (Goudet et al., 1996). We compared allelic richness (Goudet, 2001), and gene diversity (Hs, Goudet, 2001) among groups using FSTAT, also without assuming Hardy–Weinberg equilibrium. For all randomization tests we conducted 1000 permutations. We calculated an unbiased estimate of mean heterozygosity per locus (Nei, 1978) for each population using Biosys-1 (Swofford and Selander, 1981). We tested for differences Table 1 Number of alleles detected in Switzerland versus Italy Locus
N (ltaly)
N (Switz.)
S
RlatCa18 RlatCa27 Rt2Ca9 RlatCa9 RlatCa21 RlatCa17 RlatCa41
13 9 6 10 2 4 5
5 6 1 2 2 1 4
5 6 1 2 2 1 3
N=number of alleles detected in either Italy or Switzerland, S=number of alleles shared between the two regions.
in mean heterozygosity among Swiss and Italian populations using a t-test implemented using the Satterthwaite method to account for unequal sample sizes and unequal variances (SAS Institute, 1999). Among-population relationships were determined by generating an unrooted neighbour-joining tree (Saitou and Nei, 1987) using the program package PHYLIP 3.5 (Felsenstein, 1989). Allele frequency data were first generated using Genepop version 3.1c (Raymond and Rousset, 1995). Allele frequency data were bootstrapped 1000 times and Cavalli-Sforza and Edward’s chord distances (1967) were generated for each bootstrapped data set. Unrooted neighbour-joining trees were generated for each distance matrix and a single consensus tree was generated using the CONSENSE component of PHYLIP 3.5.
3. Results Two of the seven loci used were fixed for a single allele across the Tessin (Rt2Ca9 and RlatCa17), while much higher levels of polymorphism were detected at almost all loci in the Italian sample (Table 1). Locus RlatCa21 was nearly fixed across Switzerland, as only a single variant allele was detected in one population (Prato Grande, Table 1). Tests for locus by locus differences not assuming Hardy–Weinberg showed that all loci were significantly different across the whole sample (G=0.001 in all cases). Pairwise population differentiation was, for the most part, nonsignificant among Swiss sites (high gene flow), while comparisons of Swiss versus Italian sites, or among Italian sites, were mostly significant (low gene flow, Table 2).
Table 2 Results of pairwise tests for population differentiation, not assuming Hardy–Weinberg Pop. DCBCH DLLCH FCH PBPCH CSCH PGCH MCH CCH PCCH PACH VBCH PMCH MP MO SS BF CR TC
DLLCH FCH PBPCH CSCH PGCH MCH CCH PCCH PACH VBCH PMCH MP MO SS BF CR TC CS NS
NS NS
NS * NS
NS * * NS
NS NS NS NS *
NS NS NS NS * NS
NS NS NS NS * NS NS
NS NS NS NS NS NS NS NS
NS NS NS NS * NS NS NS NS
* * NS * * NS * * NS NS
NS * NS * * NS NS NS NS NS NS
* * * * * * * * * * * *
* * * NS * * * * * * * * *
* * * * * * * * * * * * * *
* * * * * * * * * * * * * * *
NS * NS * * NS * * * * * * * * * NS
* * * * * * * * * * * * * * * * *
NS * * * * * * * * * * * * * * * * *
Statistic is log-likelihood G (Goudet et al., 1996). Significance was assessed after strict Bonferroni adjustment. * Indicates significant difference at the 5% nominal level, NS indicates non-significance. Population abbreviations as in Table 1.
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We found no evidence for differences between Swiss and Italian populations in mean heterozygosity (t-statistic 1.70, d.f. 6.66, P=0.l35). Gene diversity was marginally different among Swiss and Italian populations (FSTAT, P=0.067). Allelic richness, on the other hand, differed significantly among the Swiss and Italian samples (FSTAT, P=0.011). Italian populations clustered separately, for the most part, from Swiss populations (Fig. 2). Campagna Seseglio fell between the more centrally located and the most westerly located Italian populations, but this, and many other, relationships were poorly supported (Fig. 2). Pozza Bosco Penz also clustered within the Italian clade, and grouped with the most northerly located Italian population (Moreggi Pedrinate, Fig. 2).
Table 3 Sample size (N) and unbiased estimate of mean heterozygosity per locus (HE with SE. in parentheses) Population
N
He (S.E.)
Swiss Campagna Seseglio (CSCH) Pra Coltello (PCCH) Pozza Bosco Penz (PBPCH) Fomace (FCH) Dighe di Legno Loi (DLLCH) Prato Grande (PGCH) Colombera (CCH) Ponte Autostrada (PACH) Molino (MCH) Vigna Besazio (VBCH) Pre Murin (PMCH) Discarica Ca Boscat (DCBCH)
16 10 20 13 17 15 17 18 19 18 13 5
0.223 0.252 0.307 0.294 0.317 0.265 0.295 0.298 0.309 0.206 0.213 0.222
Average for Switzerland
4. Discussion Overall, Swiss populations of Rana latastei exhibit lower genetic diversity than do Italian populations. At least one Italian population, though, exhibited more severe genetic depletion than that detected in Switzerland (TC, see Fig. 1). This population is peripherally located as well, and other peripheral Italian populations (MP, MO and CR, Fig. 1) also exhibited lower diversity values relative to the more centrally located Italian populations (i.e. CS, SS, BF, Fig. 1, Table 3). A post hoc analysis including peripheral Italian sites within the Swiss sample showed that allelic richness differed significantly among the two new groups (FSTAT, P=0.001), as did gene diversity (FSTAT, P=0.002) and mean heterozygosity (t-statistic 4.09, d.f. 2.27,
Fig. 2. Unrooted consensus neighbour-joining tree of Rana latastei populations included in this study. Numbers are bootstrap (1000 replicates) support for tree nodes. See Table 3 for population name abbreviations.
Italian Moreggi Pedrinate (MP) Montevecchia (MO) Sorgenti del Sile (SS) Bosco Fontana (BF) Chiavica Rossi (CR) Tenuta Castagnolo (TC) Cascina Stella (CS) Average for Italy
(0.081) (0.120) (0.113) (0.121) (0.127) (0.114) (0.110) (0.123) (0.121) (0.111) (0.105) (0.088)
0.267
18 20 20 20 20 20 19
0.275 0.300 0.553 0.456 0.339 0.149 0.397
(0.111) (0.108) (0.05 1) (0.073) (0.109) (0.083) (0.087)
0.353
P=0.04). This suggests that marginal populations located in the western part of the species range are genetically depauperate. We cannot say for certain that Rana latastei as a species exhibits genetic depletion at range edges until sampling across the entire species range is completed. Alternative explanations for our observed patterns of diversity exist: for example, depletion may be distributed across an east-to-west gradient, rather than a central-to-periphery gradient. Low genetic diversity in peripheral populations has been previously detected in anuran amphibians. Rowe et al. (1999) found that range edge populations of natterjack toads (Bufo calamita) exhibited genetic depletion irrespective of their size. Sjo¨gren (1991) showed that isolated northern populations of Rana lessonae exhibit extremely low polymorphism with respect to more southern populations of R. lessonae located in Western and Central Europe. Zeisset and Beebee (2001) concluded that the low genetic diversity and phylogeographic clustering patterns of Norwegian, Swedish and British pool frogs (R. lessonae) reflect a postglacial recolonization bottleneck. A notable difference between these studies and the results reported here is the geographical scale at which genetic diversity varied. Zeisset and Beebee (2001) and Sjo¨gren (1991) compared genetic diversity in populations from across Western Europe, while Rowe et al. (1999) compared populations that were up to 500 km apart (as estimated from Fig. 1, Rowe et
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al., 1999). In this study, population genetic diversity differed significantly among populations separated by as little as 60 km. No matter the geographic scale, though, low population-level genetic diversity has been shown to negatively affect fitness in anurans, in particular common toads (Bufo bufo, Hitchings and Beebee, 1998), grass frogs (Rana temporaria, Hitchings and Beebee, 1997), natterjack toads (Bufo calamita, Rowe et al., 1999), green tree frogs (Hyla cinerea, McAlpine, 1992) and western toads (Bufo boreas, Samollow and Soule´, 1983). Although Swiss populations are actively protected both at the species and habitat level and are the subject of a habitat enhancement project (Grossenbacher, 1997a), the Tessin is a heavily disturbed area. Perhaps more importantly, the species is declining in Italy. Deciduous forests in the Padano Venetian Plain, important habitat for Rana latastei and other brown frog species (Dolce et al., 1985; Ildos and Ancona, 1994) have been heavily altered, leaving small, isolated relicts of this habitat type along some smaller rivers within the Po River drainage area (Grossenbacher, 1997b). Less than 100 known breeding sites are known, and water pollution threatens many of these sites (Grossenbacher, 1997b). Italy does protect amphibian habitat on paper, but breeding habitat enjoys protection only when it falls within previously established conservation areas, such as national parks (N. Bressi, personal communication). Although the Lombardy region of northern Italy, one of two regions bordering the Swiss Rana latastei populations, is protected over 21.27% of it’s area, the other adjacent region, Piedmont, protects only 7.11% (Gruppo di studio sulle Aree Protette, GRAP, http:// pegaso.bio.uniromal.it/gsap/ilista.htm). Only 1.78% of the total area is nationally protected park land, with the majority of the protected area enjoying only regional protection (Gruppo di studio sulle Aree Protette, GRAP, http://pegaso.bio.uniromal.itlgsap/ilista.htm). In Lombardy, only 2.52% of the total area is national park land. Within Switzerland, the focus on habitat protection, although a necessary component of species conservation, will do nothing to protect already depleted genetic variation. Instead, if genetic diversity is to be increased within Switzerland (e.g. ‘genetic rescue’, Stockwell et al., 1996; Westemeier et al., 1998; Madsen et al., 1999; Richards, 2000), it is likely frogs from more centrally located populations would need to be introduced. Populations adjacent to Swiss sites, both geographically and genetically (Fig. 2), also exhibit depletion, and habitat degradation in both the Tessin and the two nearby Italian regions would likely inhibit natural recruitment into Swiss populations. Anecdotally, Swiss populations of Rana latastei are not exhibiting obvious signs of inbreeding depression (K. Grossenbacher, unpublished data), but this needs to be investigated systematically.
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Acknowledgements This project benefited greatly from the assistance and unpublished data of K. Grossenbacher. N. Bressi graciously provided larval samples from Italian populations. S. Ro¨thlisberger aided the completion of the laboratory component of this study. W. Babek assisted with data analysis. Valuable comments on an earlier version of this manuscript were provided by H.-U. Reyer, T.J.C Beebee, W. Babek, R. Jehie and three anonymous reviewers. This project was supported by a Swiss National Foundation grant to H.-U. Reyer (SNF 31-40688.94).
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