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, phy- logenetic 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 0006-3207/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocon.2003.11.011 264 R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270 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 Â 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 fi- nal concentration of MgCl2. PCR reactions were per- formed by a Hybaid PCRExpress thermal cycler.