Population Genetic Diversity of the Endemic Sardinian Newt Euproctus Platycephalus: Implications for Conservation

BIOLOGICAL CONSERVATION

Biological Conservation 119 (2004) 263–270 www.elsevier.com/locate/biocon

Population genetic diversity of the endemic Sardinian newt Euproctus platycephalus: implications for conservation

Roberta Lecis a,*, Ken Norris b a Lab.Genetica, Istituto Nazionale Fauna Selvatica, Via Ca Fornacetta 9, 40064 Ozzano dellÕEmilia (Bo), Italy, Via Cagna n.66, 09126, Cagliari, Italy b School of Animal and Microbial Sciences, University of Reading, Whiteknights, P.O. Box 228, Reading RG6 6AJ, UK

Received 27 June 2003; received in revised form 20 November 2003; accepted 21 November 2003

Abstract

The Sardinian mountain newt Euproctus platycephalus, endemic to the island of Sardinia, (Italy), is considered a rare and threatened species and is classed as critically endangered by IUCN. It inhabits streams, small lakes and pools on the main mountain systems of the island. Threats from climatic and anthropogenic factors have raised concerns for the long-term survival of newt populations on the island. MtDNA sequencing was used to investigate the genetic population structure and phylogeography of this endemic species. Patterns of genetic variation were assessed by sequencing the complete Dloop region and part of the 12SrRNA, from 74 individuals representing four different populations. Analyses of molecular variance suggest that populations are signifi- cantly differentiated, and the distribution of haplotypes across the island shows strong geographical structuring. However, phylogenetic analyses also suggest that the Sardinian population consists of two distinct mtDNA groups, which may reflect ancient isolation and expansion events. Population structure, evolutionary history of the species and implications for the conservation of newt populations are discussed. Ó 2003 Elsevier Ltd. All rights reserved.

Keywords: Control region; Critically endangered; Management units; Phylogeography; Sardinian brook salamander

1. Introduction some degree of short-term demographic independence (Sherwin et al., 2000). Characterizing genetic diversity at the molecular level Amplification and direct sequencing of highly poly- has been applied to a wide range of species conservation morphic regions of the mitochondrial genome provide a problems (Hoelzel and Dover, 1994). One of the main potentially rich source of variation for investigating the practical applications of conservation genetics is the molecular population structure within species and the identification of Evolutionary Significant Units (ESUs) phylogeny of intraspecific lineages (Wenink et al., 1993). and Management Units (MUs) within species and among There are numerous recent examples of mtDNA studies populations. As defined by Moritz (1994), ESUs are applied to the conservation of endangered amphibians, geographically discrete populations which have evolved through investigations of genetic variability and popu- separately for a substantial period of time, being recip- lation structure (Murphy et al., 2000; Shaffer et al., rocally monophyletic at mitochondrial DNA, and 2000), patterns of gene flow (Barber, 1999) and phy- showing significant frequency differences of nuclear al- logeography (McGuigan et al., 1998; Bos and Sites, leles. MUs are appropriate units for implementing short- 2001). We report here an intraspecific investigation term conservation measures, being populations with based on the nucleotide sequences of Sardinian newtsÕ significant divergence of allele frequencies at nuclear control region. or mitochondrial loci (Moritz, 1994), which indicates The mountain newt Euproctus platycephalus (Urodela, Amphibia), endemic to the island of Sardi- nia, Italy, is listed in Appendix II/Annex II of the Bern * Corresponding author. Tel.: +39-0516512257/3282779966. Convention (1998) and is classed as critically endan- E-mail address: [email protected] (R. Lecis). gered by IUCN (2000). It is also protected by the

Regional Law n 23/1998, but should receive special 2. Materials and methods conservation status in Sardinia and nationally, being probably the rarest and most threatened of all Euro- 2.1. Study sites and sample collection pean salamanders (Grossenbacher cited by Andreone and Luiselli, 2000). The other two species belonging to During 1999 and 2000, a total of 74 newts were the genus, Euproctus montanus and Euproctus asper, sampled from 8 localities across the species known are also endemics, inhabiting, respectively, the Corsi- range, the purpose being to get samples from the three can and the Pyrenean mountains. A mtDNA study major mountain systems on the island (Table 1). investigating phylogenetic relationships between the Streams and pools inhabited by newts were selected three species of the genus Euproctus, found the Sar- through field surveys. As shown in Fig. 1, a total of 11 dinian and Corsican newts more closely related to each individuals were sampled in the north of Sardinia, 27 other than to the Pyrenean newt (Caccone et al., 1997). were sampled in the centre east (Supramonte mountains, Populations of E. platycephalus are found in all major eastern ridges of the Gennargentu), 18 in the central mountain systems of Sardinia: Sette Fratelli, Gennar- mountains of Gennargentu, and 18 individuals in the gentu and Limbara. Prior to our studies (Lecis and south of the island, Sette Fratelli mountains. Table 1 Norris, 2004a,b), scarce information existed on the shows in detail study sites and number of samples col- geographic distribution and habitat ecology of the spe- lected. Samples within each population originated from cies, which is usually described as a fully aquatic newt the same river catchments. inhabiting streams, pools and small lakes on the eastern All individuals were sampled using either a toe-clip- side of the island (Rimpp, 1998). The range of Sardinian ping technique (Sutherland, 1996) or by clipping a tiny newts has been shrinking and the population size de- bit of the tail. In both cases, the digit or the tail tip re- clining in the past two decades (Puddu et al., 1988; grows in a short period of time (Griffiths, 1996). Sex, Colomo, 1999). This decline could be due to loss and total and snout-vent length of individuals, description of fragmentation of newt habitat, caused primarily by a sampling sites and environmental parameters (water prolonged climatic drought which has involved the temperature, relative humidity, water pH) were recorded whole island (Regione Sardegna, 2000). (Lecis and Norris, 2004b). Tissue samples were taken An understanding of the genetic variation of Sardinian from newts caught by hand or using fishing nets in newt populations is crucial for formulating conservation streams and pools. Animals were released into the pool strategies and management proposals. The geographical as close as possible to where they were found after the isolation and small size of some populations of E. data were collected and the tissue sample taken. These platycephalus and its aquatic lifestyle (Colomo, 1999; were preserved in 95% ethanol and stored at 4 °C until Voesenek and van Rooy, 1984) make genetically struc- their use for DNA extraction. tured populations very likely. Recent work with other Urodeles has shown substantial genetic divergence and 2.2. DNA extraction and primer selection geographical structuring among salamander populations (Alexandrino et al., 2000; Murphy et al., 2000). This Total DNA was extracted from tissue samples using a paper reports on a mtDNA sequence-based study de- standard phenol/chlorophorm method and Proteinase K signed to assess the population structure and phyloge- (10 mg/ml; Kirby, 1990). Four primers were selected netic relationships of Sardinian newt populations across from Steinfartz et al. (2000), and one from a set of their entire range. The aim of the study is to investigate universal primers designed primarily for mitochondrial patterns of genetic variation in E. platycephalus in order DNA (Kocher et al., 1989). In 2000, three new specific to evaluate implications for its conservation. primers were designed directly on the Dloop sequence of

Table 1 E. platycephalus study sites and number of samples collected N Site Locality No. samples 1999 No. samples 2000 1 Rio Suergiu Mannu M.te Sette Fratelli (south) 1 2 Rio Monte Gattu M.te Sette Fratelli (south) 4 10 3 Rio Guventu M.te Sette Fratelli (south) 3 4 Roa Paolinu Gennargentu (centre) 7 5 5 Rio Lardai Gennargentu (centre) 6 6 Pischina Ortaddala Gennargentu, Supramonte (centre.east) 20 7 7 Rio Pisciaroni M.te Limbara (north) 2 8 8 Lettodiﬁca Gallura (north) 1 N refers to the localities in Fig. 1. R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270 265

2.3. Polymerase chain reaction (PCR) protocols

The PCR profile was defined according to the Tm of the primers and the length of the expected PCR products (Hillis et al., 1997), as follows: first denaturation at 94 °C for 5 min, 30 cycles of denaturation at 94 °C (1 min), annealing at 50–55 °C (1 min) and extension at 72 °C(1 min), final extension at 72 °C for 5 min. The reactions were performed in 10 ll volumes with: 5 lM F Primer 0.5 ll, 5 lM R Primer 0.5 ll, 1Mm dNTPs 1 ll, 50 Mm MgCl2 0.3 ll, 10 Taq buffer 1 ll, distilled water 4.6 ll (variable), Taq DNA polymerase (5 U/ll) 0.1 ll, DNA 2 ll. When required, PCR was optimised adjusting the final concentration of MgCl2. PCR reactions were performed by a Hybaid PCRExpress thermal cycler. BioTaqTM DNA Polymerase was used at the required concentration in each reaction. Before sequencing, PCR products were checked by electrophoresis in 0.8% aga- rose in TBE buffer with ethidium bromide, and the bands visualized using ultraviolet illumination at 360 nm.

2.4. DNA sequencing

PCR products were cycle sequenced using the ABI PrismÒ BigDyeTM Terminator Cycle Sequencing Ready Reaction Kits (PE Biosystems), following the protocol Fig. 1. Sampling localities for E. platycephalus (for number of samples suggested by the manufacturer. The sequence reaction collected in each site, see Table 1): 1, Rio Suergiu Mannu; 2, Rio recipe and the sequencing profile are as follows: DNA Monte Gattu; 3, Rio Guventu; 4, Roa Paolinu; 5, Rio Lardai; 6, template (PCR product) 2 ll, dilution buffer 3 ll, ready Pischina Ortaddala; 7, Rio Pisciaroni; 8, Lettodifica. The big circles show the distribution of the 22 newt haplotypes across the island reaction mix 2 ll, primer 1 ll, water to 20 ll; 30 cycles of (haplotypes are named after the first individual falling into the group). denaturation at 96 °C (10 s), annealing at 50 °C (5 s), Below, the number of individuals for each mtDNA type. E1 27 + 6, R3 extension at 60 °C (4 min), (change of T° ¼ 1 °C/sec.). 3, M7 3, R2 2, M6 2, M5 1, E51 1, L4 4, L8 3, L11 1, Gu1 1 + 3, E46 1, Sequence reactions were precipitated by adding 2.5 E45 1, G3 4, E34 2, G15 2, G14 2, E21 1, E32 1, E38 1, G19 1, G12 1. volumes of 95% ethanol and 1/10 volume of sodium acetate (pH 4.6), centrifuging for 10 min. at 4 °C, re- moving the ethanol and repeating the procedure with 200 ll of 70% ethanol. Sequence products were stored at E. platycephalus. One of these (SarEu1-H) in particular )20 °C, wrapped in foil, until their use for acrylamide has been successfully used for both amplifying and se- electrophoresis. This was performed using an ABI Prism quencing the 50-end of the control region. Table 2 lists 377 Sequencer, by the Plant Science Sequence Service all primers used in this study. (University of Reading). Both the L and H strands of

Table 2 List of primers tried and used (sequence in bold) during this study; their nucleotide sequence, melting Temperature (Tm), domain of the mtDNA in which their sequence falls (position) and source. Letters L and H refer to the light and heavy strands. All primers were obtained by MWG AG Biotech

Primer name Sequence Tm (°C) Position Source H1478 50-tgactgcagagggtgacgggcggtgtgt-30 72.4 12SrRNA Kocher et al. (1989) L-pro-ML 50-ggcacccaaggccaaaattct-30 59.8 tRNA-Pro Steinfartz et al. (2000) H12S1-ML 50-caaggccaggaccaaaccttta-30 60.3 12SrRNA Steinfartz et al. (2000) E.platyc.-L 50-ggcccatgatcaacagaact-30 57.3 Dloop Steinfartz et al. (2000) E.platyc.-H 50-gctggcacgagatttaccaa-30 57.3 12SrRNA Steinfartz et al. (2000) SarEu-L 50-gtcaaataacccaacaggag-30 58.5 Cyt b This study SarEu1-H 50-tcgtgtactgataagacgga-30 58 Dloop This study SarEu2-H 50-ctgtcttagcattttcagtgc-30 59 tRNA-Phe This study 266 R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270 the amplified products were sequenced for all sam- The 50 end of the sequenced region show a greater ples. Sequence electropherograms were edited using number of variable sites, while the less variability was Chromas version 1.43 (http//www.technelysium.com.au/ found in conserved blocks between pair position 300 and chromas.html). 420 (Dloop) and 680 and 860 (30-end of Dloop and 12SrRNA). Estimates of haplotypic diversity (gene di- 2.5. Sequence analysis versity) was ranging from 0 (centre-east) to 0.9216 ± 0.0391 (south) across the populations, with a value of Sequences were aligned and consenses were produced 0.7927 ± 0.0479 for the entire data set. A test of neutrality using Clustal X (http://www.igbmc.u-strasbg.fr/). Se- (Tajima, 1989) indicated no evidence of a departure from quence ambiguities were resolved by comparing com- a standard neutral genealogy in a panmictic population plementary strands. The identity of the consensus ðD ¼ 0:73377; P > 0:1Þ. sequences was investigated and confirmed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). Genetic struc- 3.2. Population genetic structure ture and variation were investigated using ARLEQUIN version 2.000 (Schneider et al., 2000; http://anthro.unige. Overall, there is a highly significant geographical ch/arlequin), used to compute haplotype diversity (H), structuring in E. platycephalus haplotype distribution. nucleotide diversity ðpÞ, and infer population genetic The centre-east fixed mitochondrial type is also found in structure by analysis of variance (AMOVA), calculating the centre population, where other six distinct and un- F-statistics (Wright, 1965). Levels of significance of F- ique haplotypes are represented. One haplotype in the statistics were determined through 1023 random permu- south population is shared with one individual from the tation replicates. The AMOVA approach in Arlequin north, while the remaining nine south and five north takes into account the number of mutations between mtDNA types are all unique to the geographical area molecular haplotypes (Excoffier et al., 1992). Phylogeny where they are found (Fig. 1). was investigated using PAUP 4.0 beta version (Swofford, All AMOVA analyses resulted in significant structure. 1999; http://www.sinauer.com). Parsimony and distance The analysis of variance yielded highly significant values methods were used to infer phylogenetic relationships of of Fst (0.37517), revealing that a relevant proportion of haplotypes from the Dloop, tRNA-Phe and 12SrRNA the sequence variation was distributed among popula- combined data set. Parsimony trees were constructed and tions. Significant structuring was also observed in the the strict consensus tree generated from 100 MP trees. pairwise comparisons between populations (Table 3). Distances were estimated using the Kimura-2-parameter The least divergence (Fst ¼ 0.10492) was observed be- distance method (Kimura, 1980). The robustness of each tween north and south populations, while the genetically phylogeny was assessed by implementing bootstrap most distant populations (Fst ¼ 0.72253) were the north analysis consisting of 1000 replicates. and the centre-east ones. As expected, pairwise values for the centre-east population, characterized by a fixed haplotype, are the highest in the table. The lack of differentiation between north and south populations prob- 3. Results ably results from the presence of one shared haplotype. 3.1. Sequence variation 3.3. Phylogeographic relationships A total of 915 bp was sequenced from 74 individuals, representing 4 populations of the Sardinian newt from In order to compare all the sequences under a phy- north, centre, centre-east and south of the island. The logeographic perspective, a phylogenetic tree was consequence obtained consists of approximately 700 bp of structed using the neighbour-joining algorithm and the the control region, 70 bp of tRNA-Phe and 145 bp be- Kimura 2-parameter distance (Fig. 2). Parsimony anal- longing to 12SrRNA. Phylogenetic and population ysis gave a similar result (observation of a consensus from genetic structure analyses were performed on the combined data set (Dloop, tRNA-Phe and part of 12SrRNA). Table 3 Sequence analysis revealed 22 unique haplotypes, Population pairwise FSTs based on 19 polymorphic sites. Variable sites include 14 Centre-east Centre North South transitions, 2 transversions and 3 indels. Sequence di- Computing conventional F-statistics from haplotype frequencies vergence ranged from 0.11% to 1.85%. Nucleotide di- Centre-east 0.00000 versity was 0.0045 ± 0.0025 (0.45%) for the whole set of Centre 0.42656 0.00000 sequences. On average, base composition was A 32%, T North 0.72253 0.15651 0.00000 31.6%, C 20.8% and G 15.5%. South 0.60063 0.11438 0.10492 0.00000 R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270 267

Fig. 3. Unrooted Maximum parsimony genealogical tree of 22 mtDNA Dloop haplotypes from E. platycephalus (Length ¼ 20, C.I. ¼ 0.80). Percent bootstrap replication scores >500 are indicated on each branch.

the mtDNA lineages of the two haplotype clades of E. platycephalus would have diverged between one and two million years ago, therefore sometimes during the Pleistocene.

Fig. 2. Unrooted neighbour-joining tree of E. platycephalus haplo- 4. Discussion types, constructed using Kimura 2-parameter distances and midpoint rooting. Haplotypes are coded with letters corresponding to geo- 4.1. Dloop in salamanders and population structuring graphic areas (N, north; S, south; C, centre; CE, centre east). As discussed by Steinfartz et al. (2000), the control 100 equally parsimonious trees, Fig. 3). From both trees, region (the most variable part of the mitochondrial ge- it is clear that Sardinian newtsÕ haplotypes fall into two nome in many taxa) is found to be comparatively slow strongly supported groups (1000 bootstrap replicates in evolving in Urodeles. This is probably due to the lack of the MP tree). Each of these clades comprises haplotypes some hypervariable segments which are apparently lost found in the north, centre and south of the island. Despite in salamander Dloop, resulting overall much shorter the highly significant geographical structuring in E. than, for example, mammalian Dloop (Steinfartz et al., platycephalus haplotype distribution, the presence of the 2000). Nevertheless, variation in the mtDNA control two clades suggests a more complicated phylogeographic region has revealed remarkable levels of genetic struc- picture. turing in the endemic newt E. platycephalus and is useful North, south and centre haplotypes appear quite to describe the relationships between newt populations clustered in clade B, while clade A is characterized by a and discuss conservation implications. less distinct inner structure (Fig. 2). Haplotype E1, fixed The distribution of the newts haplotypes revealed by for the centre-east population and also present in the sequence analysis shows a high geographical structuring centre populations, is found in clade A, genetically very across Sardinia. The analysis of variance indicates that close to other centre and south haplotypes (M6, M7, 37.5% of sequence variation is distributed among newt E51, E32). Most of the north haplotypes belong to clade populations, and 62.5% within them. The monomorphy B, apart from L4 and the sample L3, which represents of the centre-east population brings as a consequence a the shared haplotypes between north and south (Gu1). general increase in the pairwise Fsts values (Table 3) Based on the mtDNA clock of 0.8% sequence diver- between this population and the others. Pairwise Fsts gence per million year for the Dloop of salamanders among north, centre and south populations appear (Steinfartz et al., 2000), it is possible to construct an lower than expected, due to this effect. approximate time frame for the splitting of the different In general, the geographical structure found in the groups identified. Using the estimated substitution rate, haplotype distribution (implying a degree of isolation 268 R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270 among populations from north, centre and south), does humid interglacial periods, could have promoted a sec- not appear particularly supported by AMOVA pairwise ondary wide range expansion with migration of individ- results and by the phylogenetic trees, where individuals uals, various re-colonization events and the consequent from the north, centre and south of the island do not presence of haplotypes from both clades all over the is- cluster as independently as expected (Figs. 2 and 3). As land. Following this hypothesis, the actual phylogeo- discussed by Neigel and Avise (1986) and Avise (2000), graphic picture within the species might have been caused the observed polyphyletic pattern of maternal genealo- by a repeated process of population contractions and ex- gies and the relatively high number of haplotypes found pansions, originating from two opposite climatic scenar- in all but one population (Supramonte) would be ex- ios. Founder effect or strong population bottlenecking, pected – under neutrality assumption – when the effec- followed by isolation, might have originated the fixed tive population size is much greater than the number of haplotype characteristic of the centre-east population. generations since founding. In the case of E. platycephalus, this could suggest that either foundings are recent 4.3. Gene flow and population isolation events or effective population sizes are large. The mountain systems where Sardinian newt popu- 4.2. Phylogeographic relationships and molecular clock lations were sampled (Limbara, Gennargentu and Sette Fratelli mountains) can be considered as three isolated Few studies exist on the intraspecific phylogenetic patches in the range inhabited by E. platycephalus. structure and its association with geography over the Currently isolated montane areas may have been con- native range of newts and salamanders (Moritz et al., nected transiently in the past. Sardinia used to be covered 1992; Phillips, 1994; Alexandrino et al., 2000; Tarkh- by forests over most of its territory, and it is likely that in nishvili et al., 2000). An understanding of the biogeog- the recent past a more capillary and widespread network raphy of the Sardinian mountain newt throughout its of mountainous streams were connected to each other range has very important implications for the conserva- and to the major rivers. The period between 5000 and tion of the species. The estimated sequence divergence for 1000 years BC was characterized by a warm and humid salamander Dloop suggests an approximate time frame climate with frequent precipitation, maintaining forest for the events originating the detected haplotype clades cover on most of the island territory (Serra, 1980). (Fig. 2). However, further genetic studies using both In the last 2500 years, the combination of fires, stock mtDNA and nuclear markers (such as microsatellite breeding, human-induced deforestation, and consequent loci), with greater sampling throughout the distribution desertification of the island, have gradually changed the of E. platycephalus, are needed to confirm any hypothesis. landscape and the climate in Sardinia (Serra, 1980; Following a molecular clock and using clock cali- Pungetti, 1995). More recently, the reduction in rainfall brations for the Dloop in salamanders (0.8% sequence and the change in climate which have dramatically re- divergence pMY; Steinfartz et al., 2000), the distinction duced the water flow on the island (Regione Sardegna, of two clades observed in E. platycephalus was found 2000), and the consequent drainage of many water corresponding to a genetic isolation of approximately courses could have increased the already existent isola- one to two million years ago. Given the time frame in- tion between mountainous areas. volved, the major climatic and environmental changes Given that E. platycephalus is never found far from that occurred during the Pleistocene (1.8 million years to water (Puddu et al., 1988), it is unlikely that newt pop- 11,000 years ago) appear to have determined the evo- ulations are now interconnected by gene flow among lutionary history of Sardinian newts. sampled areas. As most amphibians exist in metapopu- In the Northern Hemisphere, Pleistocene glaciations lations (Alford and Richards, 1999; Marsh and Tren- have had a major influence on the evolutionary history ham, 2001), newt populations in localized mountain of most species (Hewitt, 1996). The biogeography of ranges (i.e., Sette Fratelli in the south) may comprise many species in Europe suggests that their population metapopulations that could be interconnected in wet structure was influenced by the quaternary climatic os- years, but this needs to be documented. Finer genetic cillations that have lead to glaciation events (Taberlet markers and a finer spatial scale would be required to et al., 1998; Steinfartz et al., 2000). investigate patterns of dispersal over a network of The apparent split of the two clades in E. platycephalus streams and to quantify current gene flow between newt could have been caused by adverse climatic conditions populations. during the Pleistocene, such as cold and dry glacial periods. These might have contributed to a south-north sep- 4.4. Conservation and management of populations of E. aration of populations (or generally to the isolation of platycephalus populations in two refugia), originating the two major groups of haplotypes. A subsequent period of better cli- The maintenance of genetic variation is a major ob- matic and hydrological conditions, typical of warm and jective of most species conservation plans (Avise and R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270 269

Hamrick, 1996). From the results obtained, it is possible tion size and abundance, ecological requirements and to draw a number of inferences on the conservation of habitat correlates of distribution), protection of stream Sardinian newts. Loss and fragmentation of suitable habitats (also through some degree of fishing and habitat, caused primarily by drought, and also by pre- tourism control), creation (or implementation) of bio- dation and competition with introduced species (such as reserves around the main stream and river systems in- trout) and anthropogenic pressure could have reduced habited by newts (Lecis and Norris, in press). populations of E. platycephalus in Sardinia (Colomo, Given the distinctiveness and apparently low adaptive 1999; Read, 1998; Rimpp, 1998). Therefore the long- potential of the centre-east population, a conservation term survival of this species requires the conservation of goal should be to manage this population in order to as many genetic stocks as feasible for management. maintain its current apparent demographic health Although there is no evidence of monophyly at (Sotgiu, 1996), giving the whole area of Supramonte and the mitochondrial level, newt populations inhabiting Golfo di Orosei special conservation concern. This area streams on different mountain regions across the island has been already recommended for the creation of a appear recently genetically isolated and possess unique biogenetic reserve by previous studies, on the basis of mtDNA types. This level of genetic structuring would herpetological surveys (Voesenek and van Rooy, 1984; justify a differential management of various stocks on Voesenek et al., 1987). Sardinia. Based on the evidence of different allele frequencies, the populations in the north, centre and south of the island could be considered as three distinct Acknowledgements management units (MU; Moritz, 1994b). Given that the environment does not change drastically among moun- We thank Ettore Randi and Massimo Pierpaoli from tain systems and that E. platycephalus is threatened by Istituto Nazionale per la Fauna Selvatica (INFS, Bolo- the same possible factors across all its range, distinct gna, Italy) for helping throughout the molecular analy- MUs could be overall subjected to similar conservation ses, and Sebastian Steinfartz (University of Cologne), measures, although their genetic distinctiveness must for providing primer sequences at the beginning of the be considered in any re-location or translocation of project. individuals. Conservation management of E. platycephalus should also aim to expand population ranges and patches of suitable habitat for Sardinian newts, and population References numbers. Within each MU, interconnection and gene flow between different populations inhabiting neigh- Alexandrino, J., Froufe, E., Arntzen, J.W., Ferrand, N., 2000. Genetic bouring streams should be promoted, instead of aggra- subdivision, glacial refugia and postglacial recolonization in the golden-striped salamander, Chioglossa lusitanica (Amphibia, Uro- vating the isolation already existent among populations dela). Molecular Ecology 9, 771–781. (due to geological, hydrological and climatic conditions). Alford, R.A., Richards, S.J., 1999. Global amphibian declines: a Conservation planning should take into account the problem in applied ecology. 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