Scottish Natural HeritageCommissioned Report No. 570

Nativeness of great crested newts (Triturus cristatus) in the Scottish Highlands

C O M M I S S I O N E D R E P O R T

Commissioned Report No. 570

Nativeness of great crested newts (Triturus

cristatus) in the Scottish Highlands

For further information on this report please contact:

David O’Brien Scottish Natural Heritage Great Glen House INVERNESS IV3 8NW Telephone: 01463 725186 E-mail: david.obrien@snh.gov.uk

This report should be quoted as: Jehle, R. Orchard, D. & Barratt, C. 2013. Nativeness of great crested newts (Triturus cristatus) in the Scottish Highlands. Scottish Natural Heritage Commissioned Report No. 570.

This report, or any part of it, should not be reproduced without the permission of Scottish Natural Heritage. This permission will not be withheld unreasonably. The views expressed by the author(s) of this report should not be taken as the views and policies of Scottish Natural Heritage.

© Scottish Natural Heritage 2013.

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Nativeness of great crested newts (Triturus

cristatus) in the Scottish Highlands

Commissioned Report No.: 570 Project no: 13417 Contractor: Robert Jehle, University of Salford Year of publication: 2013 Background

Populations of great crested newt (Triturus cristatus) in the Scottish Highlands (Inverness area) are separated by >80 km of unfavourable habitat from their main distribution, and were thus in the past largely regarded as stemming from introductions. However, an alternative biogeographic scenario recently formulated by O’Brien and Hall (2012) suggests that these populations might actually be native. Given the national and international protection status of great crested newts, native populations would require significantly more conservation attention than individuals stemming from introductions. The aim of the project was to use genetic markers to address the question of nativeness of great crested newts in the Scottish Highlands. To achieve this, eight populations from the Highlands were genetically characterised using standard DNA fingerprinting techniques (microsatellite markers), and compared with two reference populations from the northern limits of their more continuous UK distribution in Central Scotland.

Main findings

The studied populations were characterised by (i) low amounts of neutral genetic variation, and (ii) high degrees of spatial genetic differentiation (including the frequent occurrence of ‘private’ alleles unique to single populations) both within Highland populations as well as between populations from the Highlands and Central Scotland. There was no genetic signature for population bottlenecks in the last decades, which would be expected if the current populations stem from a small number of introduced founder individuals. Taken together, these results suggest that the great crested newt is indeed a species native to the Scottish Highlands.

For further information on this project contact: David O’Brien, Scottish Natural Heritage, Great Glen House, Inverness, IV3 8NW.

Tel: 01463 725186 For further information on the SNH Research & Technical Support Programme contact:

Knowledge & Information Unit, Scottish Natural Heritage, Great Glen House, Inverness, IV3 8NW. Tel: 01463 725000 or research@snh.gov.uk

COMMISSIONED REPORT

Summary

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Table of Contents Page

1.  INTRODUCTION 1 1.1  The great crested newt (Triturus cristatus) and its local distribution 1 1.2  Genetic means to determine the origin of crested newt populations 3 1.3  Study aim 3 

2.  METHODS 4 2.1  Field work 4 2.2  Laboratory work and data analyses 5 

3.  RESULTS 7 

4.  DISCUSSION 9 

5.  CONCLUSIONS 11 

6.  NEXT STEPS 11 

7.  REFERENCES 12 

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Acknowledgements Sophie Ward (Nuffield Foundation student) helped during the laboratory work. Peter Leach, David Bell, Àlex Miró and Katie and Danny O’Brien helped with field work. The present project represents a collaboration between SNH (who initiated the project and funded the field work as well as project logistics) and the School of Environment and Life Sciences at the University of Salford (who funded the laboratory work through internal sources). SNH and the University of Salford would also like to thank Mrs Kay Burton, Mr Andrew Howard (Moray estates), Mr Angus McNicholl (Cawdor Trusts), Mr Frank Law (Seafield Estates), Mr Sandy Aird (Forres Golf Club) and Mr Derry Gunn, Mr Colin Leslie and Mr Giles Brockman (Forestry Commission Scotland) for their cooperation in arranging the pond visits. Finally we would like to acknowledge the amphibian records contributed by members of Highland Biological Recording Group, Ferintosh Environment Group and Amphibian and Reptile Conservation Trust, without which this project would have been impossible.

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1. INTRODUCTION

1.1 The great crested newt (Triturus cristatus) and its local distribution

The great crested newt (Triturus cristatus) is the rarest of three newt species native to Britain. It is listed in Annex II and Annex IVa of the European Habitats Directive, and therefore requires strict legal protection. It has a widespread distribution across much of lowland England, extending northwards into the central belt of Scotland (e.g. Jehle et al., 2011, Fig. 1).

Figure 1. The distribution of great crested newts (Triturus cristatus) in the UK (from Jehle et al., 2011).

A remarkable occurrence of great crested newts in Scotland is represented by six groups of ponds in the Highlands around Inverness (O’Brien & Hall 2012, Fig. 2). Given that the distance of these populations from the more continuous distribution range exceeds 80 km, it was assumed that these records stem from introductions (Langton & Beckett, 1995), as do other isolated occurrences, e.g. on the Isle of Skye (see Fig. 1). A scenario recently proposed by O’Brien & Hall (2012), however, outlined a possible colonisation route from the South 3000 to 7000 years ago when climate and habitats were more favourable. Together

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with a local distribution which is difficult to explain based on a single (or very few) introduction(s), O’Brien & Hall (2012) argue that the occurrences of great crested newts around Inverness might indeed represent native populations.

Figure 2. The occurrence of great crested newts (Triturus cristatus) in the Scottish Highlands around Inverness, including barriers to movement between six groups of ponds (from O’Brien & Hall, 2012).

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1.2 Genetic methods of determining the origin of crested newt populations

In conservation, genetic information can be used to measure dispersal and migration between populations, or to identify whether a specific population has suffered from size reductions in the recent past (for an overview on studies on amphibians see Jehle & Arntzen, 2002). Highly variable DNA markers (‘microsatellites’) from which individual-specific fingerprints are identified have previously been characterised for great crested newts (Krupa et al., 2002), and were used to study the connectivity between populations at different landscape scales across the species’ European range (e.g., Jehle et al., 2005; Meyer & Grosse 2007, Maletzky et al., 2009, Schön et al., 2011). Besides obtaining ecological information, the genetic reconstruction of spatio-temporal population processes can also enable the ancestry of local crested newt occurrences to be traced, given that specific assumptions are available to formalise a hypothesis-testing framework. For example, genetic markers were able to tentatively identify the source of a known introduction of another species of crested newt (T. carnifex) which hybridises locally with native T. cristatus populations at the border between France and Switzerland (Arntzen & Thorpe, 1999). In cases when the origin of the populations under consideration is unknown (‘cryptogenic’ occurrences), potential introductions can furthermore be disentangled from native occurrences. Putatively introduced populations are expected to bear the genetic signature of source populations rather than resembling their neighbouring populations as would be expected in isolation-by-distance scenarios. Based on the assumption that founder numbers are small, introductions should also result in past population bottlenecks and subsequent expansions which can be revealed through signatures in the genetic data (‘allele frequency distortions’, Luikart et al., 1998). Such spatial and temporal considerations were recently applied to a case study to determine the origin of several great crested newt populations about 20 km outside a more continuous distribution in France (Arntzen et al., 2010). Contrary to previous assumptions, these populations were identified as native using genetic methods. 1.3 Study aim

The aim of the present work was to use genetic methods to identify whether great crested newt occurrences in the Scottish Highlands represent native or introduced populations.

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2. METHODS

2.1 Field work

Fieldwork took place between 23rd May and 29th May 2012. DNA sampling was based on the collection and immediate storage of eggs in ethanol in the field (approximately 30 individuals per site, whenever possible). Egg sampling is a standard method to retrieve DNA for newts (e.g. Jehle et al., 2005), avoiding pond perturbation through trapping and netting (in many cases eggs can be collected from the pond shore) and the sampling of DNA from adults. Since each female lays about 200 – 400 eggs per season at high natural mortality rates (e.g. Jehle et al., 2011), egg sampling has minimal effects on population dynamic processes. Eggs were identified through visual searches of the vegetation (newts individually wrap their eggs into leaves, Fig. 3). Field work and sample collecting was conducted under licences issued by Scottish Natural Heritage (Licence 13374).

Figure 3. A female great crested newt (Triturus cristatus) from the Scottish Highlands (left); females individually wrap their eggs into leaves of aquatic plants which can be visually detected during surveys (right) and which can serve as a source for DNA. ©David O’Brien.

Ten sites were sampled in total (Table 1), eight in the Highlands and two in Central Scotland. The latter were selected as control sites where populations are known to be long-established and believed to harbour native populations. The Highland sites included at least one site from each of O’Brien & Hall’s (2012) proposed sub-populations (Fig. 4). Table 1: Details of sample sites.

Pond name Abbreviation Grid reference Highland Blackmuir Wood BW NH480572 Cinquefoil Loch CL NH593538 Dunain D NH632416 Forres Golf Club FGC NJ049587 Muir of Ord MO NH530501 Ness Side NS NH643426 Piper Hill PH NH865503 Loch Vaa LV NH910177 Central Scotland Pitmedden Forest PF NO195136 Pumpherston P NT065702

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Figure 4. Location of sampling sites.

2.2 Laboratory work and data analyses

The laboratory work took place at the University of Salford and followed existing protocols. DNA extractions were performed using standard phenol–chloroform procedures (Bruford et al., 1998). Six polymorphic microsatellite loci isolated for T. cristatus by Krupa et al., (2002) were characterised. Polymerase Chain Reactions (PCRs) were carried out in 10 μL reaction volumes under the following conditions: 1 or 1.5 mM MgCl2, 0.1 mM dNTPs, 0.5 U polymerase (GoTaq) and 0.1 mM of each primer in the manufacturer’s buffer. Thermal profiles for Tcri27, Tcri35, Tcri36 and Tcri43 were 39 cycles of 30 seconds at 93°C, 30 seconds at the primer-specific annealing temperature, 45 seconds at 72°C; for Tcri29 and Tcri46 we used a ‘touchdown’ thermal profile (for more details see Krupa et al., 2002). Primers were labelled with fluorochromes TET, HEX, and 6FAM. PCR products were separated on an ABI3130 Genetic Analyser (Applied Biosystems) and scored using PEAKSCANNER v1.0 (Applied Biosystems) and TANDEM v1.08 (Matschiner & Salzburger, 2009). Samples have been retained at Salford University. Allele frequencies, Hardy–Weinberg equilibria, and measures of genetic differentiation (FST) were calculated using GENEPOP 4.0 (Rousset, 2008). The possible existence of past population bottlenecks was investigated with the software BOTTLENECK (Luikart et al., 1998), using a mixed model of stepwise and infinite allelic mutations of 95% and 5%, respectively (as determined previously for T. cristatus microsatellites; Jehle & Arntzen, 2002). The methods implemented in BOTTLENECK are based on the premise that bottlenecking temporarily results in a large amount of genetic drift, during which the probability for given alleles to become lost depends on the initial allele frequency before the bottleneck (i.e. rare alleles have a higher risk of becoming lost than common alleles). This leads to a distortion of rare and common allele frequencies away from expected mutation-drift equilibrium

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distributions after a bottleneck has taken place (i.e. common alleles are observed more often than expected), which in turn gives rise to a detectable excess of overall heterozygotes in the sample1. The Wilcoxon sign rank test was applied, which has the highest power of all methods implemented in BOTTLENECK, to determine if the magnitude of gene diversity excess on average across loci is significantly larger than expected under equilibrium. Because multiple tests were conducted across the 10 study sites, Bonferroni-adjusted significance levels of 0.0052 were applied.

1 For example, two alleles of 50% frequencies each would result in more heterozygotes for a pool of individuals than two alleles with frequencies of 90% and 10%, respectively. 2 I.e. 0.05/10. When multiple tests are conducted for a single purpose, then the conventional significance threshold of 0.05 is arguably not applicable as there is a high probability that one or several tests fall below the threshold without rejection of the overall null hypothesis.

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3. RESULTS

In total, 235 individuals were successfully amplified for at least two loci; the overall PCR success rate was about 50% (total sample sizes per site and locus are given in Table 2). Six out of 10 populations showed significant heterozygote deficiencies (Blackmuir Wood BW, Dunain D, Muir of Ord MO, Ness-side NS, Loch Vaa LV, Pumpherston PF). As these deviations from Hardy-Weinberg equilibrium are not based on specific loci (detailed data not shown), the possibility that the observed heterozygote deficiencies are caused by locus-specific non-amplifying alleles (“null alleles”, a regular occurrence in microsatellite datasets) can be excluded. The observed deviations from Hardy-Weinberg equilibrium are likely to have been caused by deviations from random mating due to small effective population sizes. The number of alleles found for each population was low overall, and ranged between one and five depending on locus and site (range of mean: 2.7-3.6 depending on the locus). In several instances, otherwise polymorphic loci were observed as fixed in specific populations (e.g. locus Tcri27 for population Blackmuir woods, BW, and locus Tcri36 for population Dunain, D). The populations from Central Scotland showed allelic diversities within the range of the Highland populations (Table 2). Table 2 - Number of alleles for each microsatellite, with numbers of private alleles for each site given after the comma, if applicable.

Total sample sizes for each population, as well as locus-specific sample sizes (indicating PCR success rates for each population) are given in brackets.

Populations above the line are from the Highlands, populations below the line are from central Scotland.

P: significance levels for past population bottlenecks (Wilcoxon test, for details see text)

Site (sample size) Tcri27 Tcri29 Tcri35 Tcri36 Tcri43 Tcri46 P

BW (n = 28) 1 (28) 2 (16) 2 (24) 4 (17) 3 (22) 5, 1 (19) 0.156

CL (n = 24) 3 (22) 3 (8) 3 (21) 5 (5) 3 (20) 4 (14) 0.016

D (n = 33) 3 (29) 3 (12) 3 (15) 1 (16) 2, 1 (29) 3 (20) 0.625

FGC (n = 6) 1 (4) 1 (2) 3 (6) 1 (2) (0) 2 (5) 0.250

MO (n = 30) 4 (29) 4, 1 (26) 4, 1 (18) 3 (7) 3 (29) 5 (16) 0.109

NS (n = 28) 3 (18) 4 (11) 4, 1 (21) 2 (11) 4 (22) 4, 1 (18) 0.687

PH (n = 23) 4, 1 (18) 1 (10) 2 (10) 2 (10) 4 (20) 3, 1 (15) 0.093

LV (n = 29) 4 (25) 1 (13) 3 (15) 6, 1 (9) 3 (23) 2 (16) 0.219

PF (n = 20) 3, 1 (9) 5, 2 (10) 4, 2 (10) 4, 2 (9) 4, 1 (16) 5, 2 (17) 0.016

P (n = 14) 2 (8) 3, 1 (10) 2 (12) 3, 1 (6) 4 (8) 3 (13) 0.562

MEAN 2.8 2.7 3 3.1 3.3 3.6

There was no consistent signature of past population bottlenecks across the study populations, and there were no noticeable differences between populations from the Highlands and from Central Scotland with regard to the BOTTLENECK analysis (Table 2). Two populations (Cinquefoil Loch CL: Highlands, Pumpherston PF: Central Scotland) had p-values below 0.05, which is non-significant when applying a Bonferroni-adjusted significance threshold (0.005). As evidenced by the regular occurrence of alleles unique to specific populations (‘private alleles’, Table 2) as well as measures of pair-wise genetic differentiation (Table 3), most of

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the study populations were highly genetically differentiated from each other. FST values between Highland and Central Scotland sites were >0.22 throughout, whereas FST values between the two Central Scotland sites was 0.08; FST values within Highland sites had a wide range between 0.05 and 0.54. Table 3 - Pairwise genetic differentiation (FST values) across sampling sites. Populations above/left of the line are from the Highlands, populations below/right of the line are from central Scotland.

BW CL D FGC MO NS PH LV PF

CL 0.18

D 0.50 0.35

FGC 0.54 0.22 0.26

MO 0.21 0.05 0.40 0.25

NS 0.42 0.23 0.03 0.14 0.28

PH 0.41 0.30 0.24 0.35 0.32 0.18

LV 0.31 0.10 0.37 0.18 0.10 0.24 0.35

PF 0.41 0.29 0.26 0.32 0.35 0.22 0.37 0.34

P 0.53 0.36 0.28 0.36 0.42 0.26 0.46 0.42 0.08

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4. DISCUSSION

The population genetic analyses revealed three main insights. Firstly, great crested newts in the Scottish Highlands as well as in Central Scotland are characterised by mean numbers of alleles across loci which are markedly below values elsewhere across the species’ range3. Triturus cristatus has its main glacial refugium in the Balkans region, from where it (re)colonised Europe during warmer periods; a low overall degree of genetic variation is therefore expected for the Scottish populations which are at the species’ North-Western distribution limit (see e.g. Babik et al., 2009 for the distribution of genetic variation across the range of T. cristatus). However, three out of six loci studied were fixed (monomorphic) for specific populations while polymorphic in other populations. This suggests that more local forces, such as genetic drift and isolation of specific demes over the last decades, are more important for the observed patterns of genetic variation than a general biogeographic setting which would have led to the fixation of identical loci for example during a post-glacial colonisation of the area. That several populations are out of Hardy-Weinberg equilibrium further suggests that their effective sizes are small, leading to high amounts of drift. Secondly, there was no evidence in most studied populations that they underwent a recent founder event followed by expansion. Apart from strong demographic population fluctuations, signatures of genetic bottlenecks are expected when small numbers of founding individuals colonise a vacant pond, either through immigration from neighbouring demes or through an active introduction based on a small number of individuals. Population bottlenecks generally lead to (i) a depletion of genetic variation, and (ii) a distortion of the proportions between rare and common alleles. The employed method focuses on the second mechanism, and is able to detect a bottleneck signature for up to about 10 generations after its occurrence (Luikart et al., 1998). Average generation times for T. cristatus have for example been estimated as 4.3 years for populations in Western France (Jehle et al., 2001), and due to colder climatic regimes are likely to be markedly higher in the Scottish Highlands. Thus, our approach covered a timescale of about 4-5 decades back in time (~1960s). That there were no noticeable differences between populations from the Highlands and known native populations from Central Scotland with regard to evidence for past bottlenecks further suggests that the two groups of populations underwent very similar recent demographic histories. Thirdly, the populations are overall characterised by a particularly high amount of genetic differentiation, suggesting that gene flow between populations is absent or low. The observed average pair-wise FST values (see Table 3) exceeded the values documented during other studies elsewhere in Europe (for example 0.07-0.11: Jehle et al., 2005, 0.074-0.141: Schön et al., 2011). The existence of private alleles for six out of eight populations further suggests a high degree of overall long-term isolation, leading to differential loss of alleles in some populations through the forces of genetic drift. The observed high degree of population isolation is likely to be a consequence of low local population density, combined with a terrestrial environment around ponds which is unfavourable for dispersal. It is noteworthy that the geographic settings of study ponds are well reflected in the genetic data. With an FST value of 0.03, sites Ness-side NS and Dunain D have the lowest genetic differentiation between them, and indeed are situated in close geographic proximity; a low FST between sites Muir of Ord MO and Cinquefoil Loch CL coincides with the fact that both ponds are within one cluster without significant migration barriers between them as outlined in O’Brien & Hall (2012).

3 E.g. 4-9 alleles/locus in France: Jehle et al., 2005, 7-18 alleles/locus in Austria: Maletzky et al., 2009, 3.86-6.57 mean alleles/locus/population in Belgium: Schön et al., 2011. Note that the sample sizes and overall study scales were not fully comparable.

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When investigating the origin of the great crested newt populations in the Scottish Highlands, a main confounding factor was the lack of comparative genotypic data from known introductions elsewhere in Scotland and/or further samples from central UK populations. This was, however, beyond the scope of the present research. Introductions are also significantly easier to identify with prior information of potential sources of introduced individuals (as was the case in similar studies on crested newts, Arntzen & Thorpe, 1999; Arntzen et al., 2010). Nevertheless the collected evidence is sufficient to firmly conclude that an introduction is highly unlikely. Low genetic variation may be the consequence of a past introduction, or might accumulate through long-term isolation of native populations. Based on the observed spatial genetic differentiation, the scenario that great crested newts in the Scottish Highlands are introduced is only possible if (i) an ancient introduction was followed by a spread of introduced individuals across the occupied areas and a subsequent interception of dispersal corridors, or (ii) a series of independent introductions took place at a timescale for which allele frequency distortions are not detectable any more (at least 4-5 decades ago).

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5. CONCLUSIONS

The aim of the study was to assess whether great crested newts in the Highlands are native or introduced. Although it was not possible to completely rule out that existing populations are the result of multiple and/or ancient introductions, the hypothesis that they stem from a single introduction which took place within the last century was clearly rejected (no bottlenecks within the last 4-5 decades, combined with high genetic differentiation and a large number of private alleles which cannot accumulate over short timescales). By far the most parsimonious explanation for the observed patterns of genetic variation is that the Scottish Highlands harbour native populations of great crested newts. 6. NEXT STEPS

Given the surprisingly high genetic differentiation observed among the study populations, SNH has already initiated a follow-up study to characterise the existing samples with regard to their mitochondrial (mt)DNA. Given that mtDNA is non-recombining and evolves slower than microsatellites, such inferences should provide further insights into the long-term origin and history of great crested newts from the Scottish Highlands.

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7. REFERENCES

Arntzen, J. W. & Thorpe R. 1999. Italian crested newts in the basin of Geneva: distribution and genetic interaction with autochthonous species. Herpetologica, 55, 423-433.

Arntzen, J. W., Burke, T. & Jehle, R. 2010. Estimating the propagule size of a cryptogenic crested newt population. Animal Conservation, 13 (Suppl. 1), 74-81.

Babik, W., Pabijan, M., Arntzen, J. W., Cogalniceanu, D., Durka, W. & Radwan, J. 2009. Long-term survival of a urodele amphibian despite depleted major histocompatibility complex variation. Molecular Ecology, 18, 769-781.

Bruford, M. W., Hanotte, O., Brookfield, J. F. Y. & Burke, T. 1998. Multilocus and single-locus DNA fingerprinting. In: Molecular Genetic Analysis of Populations: A Practical Approach, 2nd edn (ed. Hoelzel, A. R.), pp. 287–336. Oxford: IRL Press.

Jehle, R., Arntzen, J. W., Burke, T. A., Krupa, A. & Hödl, W. 2001. The annual number of breeding adults and the effective population size of syntopic newts (Triturus cristatus, T. marmoratus). Molecular Ecology, 10, 839-850.

Jehle, R. & Arntzen, J. W. 2002. Review: Microsatellite markers in amphibian conservation genetics. Herpetological Journal, 12, 1-9.

Jehle, R., Wilson, G. A., Arntzen, J. W. & Burke, T. 2005. Contemporary gene flow and the spatio-temporal genetic structure of subdivided newt populations (Triturus cristatus, T. marmoratus). Journal of Evolutionary Biology, 18, 619-628.

Jehle, R., Thiesmeier, B. & Foster, J. 2011. The Crested Newt: A Dwindling Pond Dweller. Bielefeld: Laurenti.

Krupa, A. P., Jehle, R., Dawson, D. A., Gentle, L. K., Gibbs, M., Arntzen, J. W. & Burke, T. 2002. Microsatellite loci in the crested newt (Triturus cristatus) and their utility in other newt taxa. Conservation Genetics, 3, 87-89.

Langton, T. E. S., & Beckett, C. L. 1995. A preliminary review of the distribution and status of great crested newts Triturus cristatus records in Scotland. Halesworth, Suffolk: HCI/ SNH.

Luikart, G., Allendorf, F. W., Cornuet, J. M., & Sherwin, W. B. 1998. Distortion of allele frequency distributions provides a test for recent population bottlenecks. Journal of Heredity, 89, 238-247.

Maletzky, A., Kaiser, R. & Mikulicek, P. 2009. Conservation genetics of crested newt species Triturus cristatus and T. carnifex within a contact zone in a central Europe: impact of interspecific introgression and gene flow. Diversity, 2, 28-46.

Matschiner, M. & Salzburger, W. 2009. TANDEM: integrating automated allele binning into genetics and genomics workflows. Bioinformatics, 25, 1982-1983.

Meyer, S. & Grosse, W.-R. 2007. Populationsgröße, Altersstruktur und genetische Diversität einer Metapopulation des Kammmolches (Triturus cristatus) in der Kulturlandschaft Sachsen-Anhalts. Zeitschrift für Feldherpetologie, 14, 9-24.

O’Brien, C. D. & Hall, J.E. 2012. A hypothesis to explain the distribution of the great crested newt Triturus cristatus in the Highlands of Scotland. Herpetological Bulletin, 119, 9-14.

Rousset, F. 2008. Genepop’007: a complete reimplementation of the Genepop software for Windows and Linux. Molecular Ecology Resources, 8, 103-106.

Schön, I., Raepsaet, A., Goddeeris, B., Bauwens, D., Mergeay, J., Vanoverbeke, J. & Martens, K. 2011. High genetic diversity but limited gene flow in Flemish populations of the crested newt, Triturus cristatus. Belgian Journal of Zoology, 141, 3-13.

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