Molecular Ecology (2010) 19, 886–897 doi: 10.1111/j.1365-294X.2010.04539.x Restricted dispersal and genetic diversity in populations of an endangered montane lizard (Eulamprus leuraensis, Scincidae) S. DUBEY and R. SHINE School of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia Abstract Many alpine species are under threat from global climate change, as their geographic ranges become increasingly fragmented and unsuitable. Understanding rates and deter- minants of gene flow among such fragmented populations, over historical as well as recent timescales, can help to identify populations under threat. It is also important to clarify the degree to which loss of local populations reduces overall genetic diversity within the taxon. The endangered Blue Mountains Water Skink (Eulamprus leuraensis) is restricted to <40 small swamps in montane south-eastern Australia. Our analyses of seven microsatellite loci of 241 animals from 13 populations show strong geographic structure, with major genetic divergence even between populations separated by <0.5 km. Dispersal between populations is scarce, and appears to involve mostly males. Our analyses suggest potential recent bottleneck events in all the identified populations, and lower genetic diversity and population size parameter at lower-elevation sites than at higher-elevation sites. Manage- ment of this endangered taxon thus needs to treat most populations separately, because of their genetic distinctiveness and low rates of genetic exchange. Keywords: altitudinal gradient, dispersal, microsatellites, reptiles, spatial structure Received 1 October 2009; revision accepted 29 December 2009 tion than by increases in temperature; the impact of Introduction reduced water availability will be particularly impor- Processes such as climate change and habitat degrada- tant in areas that are already under hydrological tion can result in fragmentation of previously continu- stress (Arau´ jo et al. 2006). Already rare organisms ous populations. Fragmentation poses a major threat restricted to montane habitat types, as well as those to population viability, because it not only reduces confined to the tops of low mountains, are under population size within any given habitat fragment, imminent threat from global warming (for studies on but also decreases rates of migration (and hence, gene amphibians and reptiles, see Raxworthy et al. 2008; flow) among previously connected populations Pounds et al. 1999; plants, Theurillat & Guisan 2001; (e.g. Frankham et al. 2002; Cushman 2006), leading to insects, Scalercio 2009). a loss of genetic diversity within fragments (e.g. Moreover, we need to identify genetic units that war- Hitchings & Beebee 1997). Climate change may well rant separate conservation and management, due to induce fragmentation of currently continuous popula- their independent evolution and resulting local adapta- tions, via shifts in both precipitation and thermal tion (see e.g. King 2009). Understanding dispersal and regimes. Modelling of future climates in Europe has population fragmentation can also allow us to estimate predicted that populations of reptiles and amphibians the potential of a species to colonize new habitat, a criti- will be more affected by the reductions in precipita- cal determinant of its chance of long-term survival in a context of global climate change (e.g. Almany et al. Correspondence: Sylvain Dubey, Fax: +61-2-93515609; 2009; Gaston 2009; Wilson et al. 2009). The ability of E-mail: [email protected] reptiles and amphibians to perform rapid large-scale Ó 2010 Blackwell Publishing Ltd GENE FLOW IN AN ENDANGERED LIZARD SPECIES 887 migrations remains controversial (e.g. Arau´ jo & Pearson entire distribution of the species. Our sampling 2005; Smith & Green 2005; Arau´ jo et al. 2006). included ten populations in the Blue Mountains (BH3, The spatial distribution of gene frequencies within a BH4, BH5, KT1, MH4, MRP1, WF1, WF5, WFL) and taxon has important consequences not only for issues three in the Newnes Plateau (NP4, PNP1, XFC1; Fig. 1). such as inbreeding rates and opportunities for local The animals were captured with funnel traps and pit- adaptation, but also can provide insights into mating fall traps, sexed by manual eversion of hemipenis (as in systems and movement patterns (e.g. DeWoody & Harlow 1996), and measured with callipers. Individuals Avise 2001; Dalerum et al. 2007; Keogh et al. 2007; Du- of all size classes and both sexes were caught, but the bey et al. 2008). If individuals are highly sedentary, or sex ratio was slightly skewed towards females return to natal sites to breed, strong spatial structure in (126F ⁄ 106M ⁄ 9 undetermined). gene frequencies is expected; that is, local populations Total cellular DNA was isolated from small tail clips. will diverge, and inbreeding will be common. In con- Tissues were placed in 200 lLof5% Chelex containing trast, little spatial genetic structure is expected in highly 0.2 mg ⁄ mL of proteinase K, incubated overnight at mobile, widely dispersing animals. The sexes often dif- 56°C, boiled at 100°C for 10 min and centrifuged at fer in their degree of dispersal and, hence, in their con- 13 300 g for 10 min. Then, the supernatant, containing tribution to spatial genetic structure both within and purified DNA, was removed and stored at )20°C. among populations (Handley & Perrin 2007). The Blue Mountains water skink (Eulamprus leuraen- Microsatellite analysis sis) is a medium-sized (to 200 mm total length) vivipa- rous scincid lizard. It is restricted to montane areas Seven microsatellite loci isolated and characterized from west of Sydney in south-eastern Australia. The species Eulamprus kosciuskoi (Scott et al. 2001; EK8, EK23, EK37, is known from fewer than 40 isolated small swamps at EK39, EK100, EK107) and Gnypetoscincus queenslandiae 560–1060 m elevation, distributed in two distinct (Sumner et al. 2001; GQ20 ⁄ 21) were amplified and patches separated by about 20 km (in the Blue Moun- scored. PCR amplifications were performed in a 9800 tains and Newnes Plateau). This species is classified as Fast thermal cycler (Applied Biosystems) as 5 lL reac- ‘endangered’ under the Threatened Species Conserva- tions containing 0.075 U Taq Ti DNA polymerase (Bio- tion Act (1995) and the Environmental Protection and tech), 0.1 mM dNTPs, 0.4 mM of each primer, 20 mM Biodiversity Conservation Act (1999; see http:// Tris–HCl, pH 8.5, 50 mM KCl and 1.25 mM MgCl2. www.environment.nsw.gov.au/threatenedspecies/ for Cycling conditions included a hot start denaturation of both Acts), on the basis that it is an ecological specialist, 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, with severely reduced populations subject to substantial 60°C (55°C for EK23, GQ20 ⁄ 21 and EK37) annealing ongoing threats. Because the distribution of the species temperature for 30 s, 72°C for 30 s (1 min for EK23, is strictly limited to montane areas, it may be under sig- GQ20 ⁄ 21 and EK37) and a final extension of 72°C for nificant risk from global climate change. Models of cli- 30 min. Amplified products were genotyped with a matic variation predict higher temperatures and lower 3130-xl genetic analyzer (Applied Biosystems) using rainfall in the Blue Mountains (http://www.climate- GeneMapper software V3.7 (Applied Biosystems). changeinaustralia.gov.au). Such changes might affect both the skink’s habitat (e.g. reduced rainfall and thus F-statistics and genetic diversity parameters seepage might dry out the hanging swamps) and the lizard itself. Despite these major threats and its current Gene diversities comprising observed (Ho), expected classification as ‘endangered’, the ecology of E. leuraen- within-subpopulation (Hs) and expected overall hetero- sis is poorly known (Shea & Peterson 1985; LeBreton zygosities (HT) were estimated following Nei & Chesser 1996). The level of genetic differentiation of the popula- (1983). Genotypic disequilibria between loci in each tions, and the capacity for individuals of this species to sample and deviations from Hardy–Weinberg equilib- disperse among populations, remain unknown. An rium (HWE) within samples were tested based on 5460 understanding of these topics can facilitate the effective permutations and 10 000 randomizations respectively. management of these few remaining populations. Wright’s fixation indices for within-subpopulation devi- ation from random mating (FIS), as well as pair-wise subpopulation differentiation (F ), were estimated fol- Materials and methods ST lowing Weir & Cockerham (1984). The deviation from random mating within populations (F ) per locus and Tissue sampling and DNA extraction IS sample was computed with a bootstrap procedure (1820 Tissue samples from 241 Eulamprus leuraensis were col- randomizations). Statistical support for pair-wise popu- lected from November 2008 to April 2009 across the lation differentiation was obtained through exact G-tests Ó 2010 Blackwell Publishing Ltd 888 S. DUBEY and R. SHINE b Fig. 1 Distribution of sampled popula- a tions (a), in the Newnes Plateau (b) and Blue Mountains (c), and genetic clusters identified by STRUCTURE analysis (K = 10). c on allelic frequencies as described by Goudet et al. Significance values were corrected for multiple tests (1996) with 10 000 randomizations. Allelic richness was using the sequential Bonferroni procedure (Rice 1989). estimated using the rarefaction method of El Mousadik The detection of null alleles was tested according to & Petit (1996), in which genotype data are resampled