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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 a,* , Ken Norris b 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 significantly 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 Characterizing genetic diversity at the molecular level has been applied to a wide range of species conservation problems (Hoelzel and Dover, 1994). One of the main practical applications of conservation genetics is the identification of Evolutionary Significant Units (ESUs) and Management Units (MUs) within species and among populations. As defined by Moritz (1994), ESUs are geographically discrete populations which have evolved separately for a substantial period of time, being reciprocally monophyletic at mitochondrial DNA, and showing significant frequency differences of nuclear alleles. MUs are appropriate units for implementing shortterm conservation measures, being populations with significant divergence of allele frequencies at nuclear or mitochondrial loci (Moritz, 1994), which indicates * Corresponding author. Tel.: +39-0516512257/3282779966. E-mail address: [email protected] (R. Lecis). 0006-3207/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocon.2003.11.011 some degree of short-term demographic independence (Sherwin et al., 2000). Amplification and direct sequencing of highly polymorphic regions of the mitochondrial genome provide a potentially rich source of variation for investigating the molecular population structure within species and the phylogeny of intraspecific lineages (Wenink et al., 1993). There are numerous recent examples of mtDNA studies applied to the conservation of endangered amphibians, through investigations of genetic variability and population structure (Murphy et al., 2000; Shaffer et al., 2000), patterns of gene flow (Barber, 1999) and phylogeography (McGuigan et al., 1998; Bos and Sites, 2001). We report here an intraspecific investigation based on the nucleotide sequences of Sardinian newtsÕ control region. The mountain newt Euproctus platycephalus (Urodela, Amphibia), endemic to the island of Sardinia, Italy, is listed in Appendix II/Annex II of the Bern Convention (1998) and is classed as critically endangered by IUCN (2000). It is also protected by the 264 R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270 Regional Law n 23/1998, but should receive special conservation status in Sardinia and nationally, being probably the rarest and most threatened of all European salamanders (Grossenbacher cited by Andreone and Luiselli, 2000). The other two species belonging to the genus, Euproctus montanus and Euproctus asper, are also endemics, inhabiting, respectively, the Corsican and the Pyrenean mountains. A mtDNA study investigating phylogenetic relationships between the three species of the genus Euproctus, found the Sardinian and Corsican newts more closely related to each other than to the Pyrenean newt (Caccone et al., 1997). Populations of E. platycephalus are found in all major mountain systems of Sardinia: Sette Fratelli, Gennargentu and Limbara. Prior to our studies (Lecis and Norris, 2004a,b), scarce information existed on the geographic distribution and habitat ecology of the species, which is usually described as a fully aquatic newt inhabiting streams, pools and small lakes on the eastern side of the island (Rimpp, 1998). The range of Sardinian newts has been shrinking and the population size declining in the past two decades (Puddu et al., 1988; Colomo, 1999). This decline could be due to loss and fragmentation of newt habitat, caused primarily by a prolonged climatic drought which has involved the whole island (Regione Sardegna, 2000). An understanding of the genetic variation of Sardinian newt populations is crucial for formulating conservation strategies and management proposals. The geographical isolation and small size of some populations of E. platycephalus and its aquatic lifestyle (Colomo, 1999; Voesenek and van Rooy, 1984) make genetically structured populations very likely. Recent work with other Urodeles has shown substantial genetic divergence and geographical structuring among salamander populations (Alexandrino et al., 2000; Murphy et al., 2000). This paper reports on a mtDNA sequence-based study designed to assess the population structure and phylogenetic relationships of Sardinian newt populations across their entire range. The aim of the study is to investigate patterns of genetic variation in E. platycephalus in order to evaluate implications for its conservation. 2. Materials and methods 2.1. Study sites and sample collection During 1999 and 2000, a total of 74 newts were sampled from 8 localities across the species known range, the purpose being to get samples from the three major mountain systems on the island (Table 1). Streams and pools inhabited by newts were selected through field surveys. As shown in Fig. 1, a total of 11 individuals were sampled in the north of Sardinia, 27 were sampled in the centre east (Supramonte mountains, eastern ridges of the Gennargentu), 18 in the central mountains of Gennargentu, and 18 individuals in the south of the island, Sette Fratelli mountains. Table 1 shows in detail study sites and number of samples collected. Samples within each population originated from the same river catchments. All individuals were sampled using either a toe-clipping technique (Sutherland, 1996) or by clipping a tiny bit of the tail. In both cases, the digit or the tail tip regrows in a short period of time (Griffiths, 1996). Sex, total and snout-vent length of individuals, description of sampling sites and environmental parameters (water temperature, relative humidity, water pH) were recorded (Lecis and Norris, 2004b). Tissue samples were taken from newts caught by hand or using fishing nets in streams and pools. Animals were released into the pool as close as possible to where they were found after the data were collected and the tissue sample taken. These were preserved in 95% ethanol and stored at 4 °C until their use for DNA extraction. 2.2. DNA extraction and primer selection Total DNA was extracted from tissue samples using a standard phenol/chlorophorm method and Proteinase K (10 mg/ml; Kirby, 1990). Four primers were selected from Steinfartz et al. (2000), and one from a set of universal primers designed primarily for mitochondrial DNA (Kocher et al., 1989). In 2000, three new specific primers were designed directly on the Dloop sequence of Table 1 E. platycephalus study sites and number of samples collected N Site Locality 1 2 3 4 5 6 7 8 Rio Suergiu Mannu Rio Monte Gattu Rio Guventu Roa Paolinu Rio Lardai Pischina Ortaddala Rio Pisciaroni Lettodifica M.te Sette Fratelli (south) M.te Sette Fratelli (south) M.te Sette Fratelli (south) Gennargentu (centre) Gennargentu (centre) Gennargentu, Supramonte (centre.east) M.te Limbara (north) Gallura (north) N refers to the localities in Fig. 1. No. samples 1999 1 4 7 20 2 1 No. samples 2000 10 3 5 6 7 8 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% agarose in TBE buffer with ethidium bromide, and the bands visualized using ultraviolet illumination at 360 nm. 2.4. DNA sequencing Fig. 1. Sampling localities for E. platycephalus (for number of samples collected in each site, see Table 1): 1, Rio Suergiu Mannu; 2, Rio Monte Gattu; 3, Rio Guventu; 4, Roa Paolinu; 5, Rio Lardai; 6, Pischina Ortaddala; 7, Rio Pisciaroni; 8, Lettodifica. The big circles show the distribution of the 22 newt haplotypes across the island (haplotypes are named after the first individual falling into the group). Below, the number of individuals for each mtDNA type. E1 27 + 6, R3 3, M7 3, R2 2, M6 2, M5 1, E51 1, L4 4, L8 3, L11 1, Gu1 1 + 3, E46 1, E45 1, G3 4, E34 2, G15 2, G14 2, E21 1, E32 1, E38 1, G19 1, G12 1. E. platycephalus. One of these (SarEu1-H) in particular has been successfully used for both amplifying and sequencing the 50 -end of the control region. Table 2 lists all primers used in this study. PCR products were cycle sequenced using the ABI PrismÒ BigDyeTM Terminator Cycle Sequencing Ready Reaction Kits (PE Biosystems), following the protocol suggested by the manufacturer. The sequence reaction recipe and the sequencing profile are as follows: DNA template (PCR product) 2 ll, dilution buffer 3 ll, ready reaction mix 2 ll, primer 1 ll, water to 20 ll; 30 cycles of denaturation at 96 °C (10 s), annealing at 50 °C (5 s), extension at 60 °C (4 min), (change of T° ¼ 1 °C/sec.). Sequence reactions were precipitated by adding 2.5 volumes of 95% ethanol and 1/10 volume of sodium acetate (pH 4.6), centrifuging for 10 min. at 4 °C, removing the ethanol and repeating the procedure with 200 ll of 70% ethanol. Sequence products were stored at )20 °C, wrapped in foil, until their use for acrylamide electrophoresis. This was performed using an ABI Prism 377 Sequencer, by the Plant Science Sequence Service (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 H1478 L-pro-ML H12S1-ML E.platyc.-L E.platyc.-H SarEu-L SarEu1-H SarEu2-H Sequence 0 0 5 -tgactgcagagggtgacgggcggtgtgt-3 50 -ggcacccaaggccaaaattct-30 50 -caaggccaggaccaaaccttta-30 50 -ggcccatgatcaacagaact-30 50 -gctggcacgagatttaccaa-30 50 -gtcaaataacccaacaggag-30 50 -tcgtgtactgataagacgga-30 50 -ctgtcttagcattttcagtgc-30 Tm (°C) Position Source 72.4 59.8 60.3 57.3 57.3 58.5 58 59 12SrRNA tRNA-Pro 12SrRNA Dloop 12SrRNA Cyt b Dloop tRNA-Phe Kocher et al. (1989) Steinfartz et al. (2000) Steinfartz et al. (2000) Steinfartz et al. (2000) Steinfartz et al. (2000) This study This study This study 266 R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270 the amplified products were sequenced for all samples. Sequence electropherograms were edited using Chromas version 1.43 (http//www.technelysium.com.au/ chromas.html). 2.5. Sequence analysis Sequences were aligned and consenses were produced using Clustal X (http://www.igbmc.u-strasbg.fr/). Sequence ambiguities were resolved by comparing complementary strands. The identity of the consensus sequences was investigated and confirmed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). Genetic structure and variation were investigated using ARLEQUIN version 2.000 (Schneider et al., 2000; http://anthro.unige. ch/arlequin), used to compute haplotype diversity (H), nucleotide diversity ðpÞ, and infer population genetic structure by analysis of variance (AMOVA), calculating F-statistics (Wright, 1965). Levels of significance of Fstatistics were determined through 1023 random permutation replicates. The AMOVA approach in Arlequin takes into account the number of mutations between molecular haplotypes (Excoffier et al., 1992). Phylogeny was investigated using PAUP 4.0 beta version (Swofford, 1999; http://www.sinauer.com). Parsimony and distance methods were used to infer phylogenetic relationships of haplotypes from the Dloop, tRNA-Phe and 12SrRNA combined data set. Parsimony trees were constructed and the strict consensus tree generated from 100 MP trees. Distances were estimated using the Kimura-2-parameter distance method (Kimura, 1980). The robustness of each phylogeny was assessed by implementing bootstrap analysis consisting of 1000 replicates. 3. Results The 50 end of the sequenced region show a greater number of variable sites, while the less variability was found in conserved blocks between pair position 300 and 420 (Dloop) and 680 and 860 (30 -end of Dloop and 12SrRNA). Estimates of haplotypic diversity (gene diversity) was ranging from 0 (centre-east) to 0.9216 ± 0.0391 (south) across the populations, with a value of 0.7927 ± 0.0479 for the entire data set. A test of neutrality (Tajima, 1989) indicated no evidence of a departure from a standard neutral genealogy in a panmictic population ðD ¼ 0:73377; P > 0:1Þ. 3.2. Population genetic structure Overall, there is a highly significant geographical structuring in E. platycephalus haplotype distribution. The centre-east fixed mitochondrial type is also found in the centre population, where other six distinct and unique haplotypes are represented. One haplotype in the south population is shared with one individual from the north, while the remaining nine south and five north mtDNA types are all unique to the geographical area where they are found (Fig. 1). All AMOVA analyses resulted in significant structure. The analysis of variance yielded highly significant values of Fst (0.37517), revealing that a relevant proportion of the sequence variation was distributed among populations. Significant structuring was also observed in the pairwise comparisons between populations (Table 3). The least divergence (Fst ¼ 0.10492) was observed between north and south populations, while the genetically most distant populations (Fst ¼ 0.72253) were the north 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 probably 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 north, centre, centre-east and south of the island. The sequence obtained consists of approximately 700 bp of the control region, 70 bp of tRNA-Phe and 145 bp belonging to 12SrRNA. Phylogenetic and population genetic structure analyses were performed on the combined data set (Dloop, tRNA-Phe and part of 12SrRNA). Sequence analysis revealed 22 unique haplotypes, based on 19 polymorphic sites. Variable sites include 14 transitions, 2 transversions and 3 indels. Sequence divergence ranged from 0.11% to 1.85%. Nucleotide diversity was 0.0045 ± 0.0025 (0.45%) for the whole set of sequences. On average, base composition was A 32%, T 31.6%, C 20.8% and G 15.5%. In order to compare all the sequences under a phylogeographic perspective, a phylogenetic tree was constructed using the neighbour-joining algorithm and the Kimura 2-parameter distance (Fig. 2). Parsimony analysis gave a similar result (observation of a consensus from Table 3 Population pairwise FSTs Centre-east Centre North South Computing conventional F-statistics from haplotype frequencies Centre-east 0.00000 Centre 0.42656 0.00000 North 0.72253 0.15651 0.00000 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 haplotypes, constructed using Kimura 2-parameter distances and midpoint rooting. Haplotypes are coded with letters corresponding to geographic areas (N, north; S, south; C, centre; CE, centre east). 100 equally parsimonious trees, Fig. 3). From both trees, it is clear that Sardinian newtsÕ haplotypes fall into two strongly supported groups (1000 bootstrap replicates in the MP tree). Each of these clades comprises haplotypes found in the north, centre and south of the island. Despite the highly significant geographical structuring in E. platycephalus haplotype distribution, the presence of the two clades suggests a more complicated phylogeographic picture. North, south and centre haplotypes appear quite clustered in clade B, while clade A is characterized by a less distinct inner structure (Fig. 2). Haplotype E1, fixed for the centre-east population and also present in the centre populations, is found in clade A, genetically very close to other centre and south haplotypes (M6, M7, E51, E32). Most of the north haplotypes belong to clade B, apart from L4 and the sample L3, which represents the shared haplotypes between north and south (Gu1). Based on the mtDNA clock of 0.8% sequence divergence per million year for the Dloop of salamanders (Steinfartz et al., 2000), it is possible to construct an approximate time frame for the splitting of the different groups identified. Using the estimated substitution rate, 4. Discussion 4.1. Dloop in salamanders and population structuring As discussed by Steinfartz et al. (2000), the control region (the most variable part of the mitochondrial genome in many taxa) is found to be comparatively slow evolving in Urodeles. This is probably due to the lack of some hypervariable segments which are apparently lost in salamander Dloop, resulting overall much shorter than, for example, mammalian Dloop (Steinfartz et al., 2000). Nevertheless, variation in the mtDNA control region has revealed remarkable levels of genetic structuring in the endemic newt E. platycephalus and is useful to describe the relationships between newt populations and discuss conservation implications. The distribution of the newts haplotypes revealed by sequence analysis shows a high geographical structuring across Sardinia. The analysis of variance indicates that 37.5% of sequence variation is distributed among newt populations, and 62.5% within them. The monomorphy of the centre-east population brings as a consequence a general increase in the pairwise Fsts values (Table 3) between this population and the others. Pairwise Fsts among north, centre and south populations appear lower than expected, due to this effect. In general, the geographical structure found in the 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 not appear particularly supported by AMOVA pairwise results and by the phylogenetic trees, where individuals from the north, centre and south of the island do not cluster as independently as expected (Figs. 2 and 3). As discussed by Neigel and Avise (1986) and Avise (2000), the observed polyphyletic pattern of maternal genealogies and the relatively high number of haplotypes found in all but one population (Supramonte) would be expected – under neutrality assumption – when the effective population size is much greater than the number of generations since founding. In the case of E. platycephalus, this could suggest that either foundings are recent events or effective population sizes are large. 4.2. Phylogeographic relationships and molecular clock Few studies exist on the intraspecific phylogenetic structure and its association with geography over the native range of newts and salamanders (Moritz et al., 1992; Phillips, 1994; Alexandrino et al., 2000; Tarkhnishvili et al., 2000). An understanding of the biogeography of the Sardinian mountain newt throughout its range has very important implications for the conservation of the species. The estimated sequence divergence for salamander Dloop suggests an approximate time frame for the events originating the detected haplotype clades (Fig. 2). However, further genetic studies using both mtDNA and nuclear markers (such as microsatellite loci), with greater sampling throughout the distribution of E. platycephalus, are needed to confirm any hypothesis. Following a molecular clock and using clock calibrations for the Dloop in salamanders (0.8% sequence divergence pMY; Steinfartz et al., 2000), the distinction of two clades observed in E. platycephalus was found corresponding to a genetic isolation of approximately one to two million years ago. Given the time frame involved, the major climatic and environmental changes that occurred during the Pleistocene (1.8 million years to 11,000 years ago) appear to have determined the evolutionary history of Sardinian newts. In the Northern Hemisphere, Pleistocene glaciations have had a major influence on the evolutionary history of most species (Hewitt, 1996). The biogeography of many species in Europe suggests that their population structure was influenced by the quaternary climatic oscillations that have lead to glaciation events (Taberlet et al., 1998; Steinfartz et al., 2000). The apparent split of the two clades in E. platycephalus could have been caused by adverse climatic conditions during the Pleistocene, such as cold and dry glacial periods. These might have contributed to a south-north separation of populations (or generally to the isolation of populations in two refugia), originating the two major groups of haplotypes. A subsequent period of better climatic and hydrological conditions, typical of warm and humid interglacial periods, could have promoted a secondary wide range expansion with migration of individuals, various re-colonization events and the consequent presence of haplotypes from both clades all over the island. Following this hypothesis, the actual phylogeographic picture within the species might have been caused by a repeated process of population contractions and expansions, originating from two opposite climatic scenarios. Founder effect or strong population bottlenecking, followed by isolation, might have originated the fixed haplotype characteristic of the centre-east population. 4.3. Gene flow and population isolation The mountain systems where Sardinian newt populations were sampled (Limbara, Gennargentu and Sette Fratelli mountains) can be considered as three isolated patches in the range inhabited by E. platycephalus. Currently isolated montane areas may have been connected transiently in the past. Sardinia used to be covered by forests over most of its territory, and it is likely that in the recent past a more capillary and widespread network of mountainous streams were connected to each other and to the major rivers. The period between 5000 and 1000 years BC was characterized by a warm and humid climate with frequent precipitation, maintaining forest cover on most of the island territory (Serra, 1980). In the last 2500 years, the combination of fires, stock breeding, human-induced deforestation, and consequent desertification of the island, have gradually changed the landscape and the climate in Sardinia (Serra, 1980; Pungetti, 1995). More recently, the reduction in rainfall and the change in climate which have dramatically reduced the water flow on the island (Regione Sardegna, 2000), and the consequent drainage of many water courses could have increased the already existent isolation between mountainous areas. Given that E. platycephalus is never found far from water (Puddu et al., 1988), it is unlikely that newt populations are now interconnected by gene flow among sampled areas. As most amphibians exist in metapopulations (Alford and Richards, 1999; Marsh and Trenham, 2001), newt populations in localized mountain ranges (i.e., Sette Fratelli in the south) may comprise metapopulations that could be interconnected in wet years, but this needs to be documented. Finer genetic markers and a finer spatial scale would be required to investigate patterns of dispersal over a network of streams and to quantify current gene flow between newt populations. 4.4. Conservation and management of populations of E. platycephalus The maintenance of genetic variation is a major objective of most species conservation plans (Avise and R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270 Hamrick, 1996). From the results obtained, it is possible to draw a number of inferences on the conservation of Sardinian newts. Loss and fragmentation of suitable habitat, caused primarily by drought, and also by predation and competition with introduced species (such as trout) and anthropogenic pressure could have reduced populations of E. platycephalus in Sardinia (Colomo, 1999; Read, 1998; Rimpp, 1998). Therefore the longterm survival of this species requires the conservation of as many genetic stocks as feasible for management. Although there is no evidence of monophyly at the mitochondrial level, newt populations inhabiting streams on different mountain regions across the island appear recently genetically isolated and possess unique mtDNA types. This level of genetic structuring would justify a differential management of various stocks on 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 management units (MU; Moritz, 1994b). Given that the environment does not change drastically among mountain systems and that E. platycephalus is threatened by the same possible factors across all its range, distinct MUs could be overall subjected to similar conservation measures, although their genetic distinctiveness must be considered in any re-location or translocation of individuals. Conservation management of E. platycephalus should also aim to expand population ranges and patches of suitable habitat for Sardinian newts, and population numbers. Within each MU, interconnection and gene flow between different populations inhabiting neighbouring streams should be promoted, instead of aggravating the isolation already existent among populations (due to geological, hydrological and climatic conditions). Conservation planning should take into account the historical and current biogeographic structuring of populations in the two major clades and in the north, centre and south of the island. As individuals representing the two clades are found in each sampled location, genetic diversity and evolutionary history would be conserved by protecting each individual population. The mountain system isolate (Sette Fratelli, Gennargentu and Supramonte, Limbara) could be used as the appropriate geographical scale for population monitoring, and, within each one, multiple sites should be monitored. Although the mountain systems where Sardinian newts occur are all under some degree of natural protection (Monte Limbara and Sette Fratelli being Regional Parks and Gennargentu will be a National Park), only real implementation of conservation practices focused on this endemic species would be adequate to protect the major components of genetic diversity of E. platycephalus in the three areas. Practical conservation management measures should include long-term monitoring of a sample of populations (estimating popula- 269 tion size and abundance, ecological requirements and habitat correlates of distribution), protection of stream habitats (also through some degree of fishing and tourism control), creation (or implementation) of bioreserves around the main stream and river systems inhabited by newts (Lecis and Norris, in press). Given the distinctiveness and apparently low adaptive potential of the centre-east population, a conservation goal should be to manage this population in order to maintain its current apparent demographic health (Sotgiu, 1996), giving the whole area of Supramonte and Golfo di Orosei special conservation concern. This area has been already recommended for the creation of a biogenetic reserve by previous studies, on the basis of herpetological surveys (Voesenek and van Rooy, 1984; Voesenek et al., 1987). 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