# An extended mtDNA phylogeography for the alpine newt illuminates the provenance of introduced populations

Jody Robbemont, Sam van Veldhuijzen, Steven J.R. Allain, Johanna Ambu, Ryan Boyle, Daniele Canestrelli, Éinne Ó Cathasaigh, Chris Cathrine, Andrea Chiocchio, Dan Cogalniceanu, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Jody Robbemont, Sam van Veldhuijzen, Steven J.R. Allain, Johanna Ambu, Ryan Boyle, et al.. An extended mtDNA phylogeography for the alpine newt illuminates the provenance of introduced populations. Amphibia-Reptilia, 2023, 44 (3), pp.347-361. 10.1163/15685381-bja10144. hal-04548431

HAL Id: hal-04548431

https://hal.science/hal-04548431

Submitted on 16 Apr 2024

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
# An extended mtDNA phylogeography for the alpine newt illuminates the provenance of introduced populations

Jody Robbemont, Sam van Veldhuijzen, Steven J.R. Allain, Johanna Ambu, Ryan Boyle, Daniele Canestrelli, Éinne Ó Cathasaigh, Chris Cathrine, Andrea Chiocchio, Dan Cogalniceanu, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Jody Robbemont, Sam van Veldhuijzen, Steven J.R. Allain, Johanna Ambu, Ryan Boyle, et al.. An extended mtDNA phylogeography for the alpine newt illuminates the provenance of introduced populations. Amphibia-Reptilia, 2023, 44 (3), pp.347-361. 10.1163/15685381-bja10144. hal-04548431

HAL Id: hal-04548431

https://hal.science/hal-04548431v1

Submitted on 16 Apr 2024

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

An extended mtDNA phylogeography for the alpine newt illuminates
the provenance of introduced populations

Jody Robbebont¹,²,*, Sam van Veldhuijzen¹,²,*, Steven J.R. Allain³, Johanna Ambu⁴,
Ryan Boyle⁵, Daniele Canestrelli⁶, Éinne Ó Cathasaigh⁷, Chris Cathrine⁸, Andrea Chiocchio⁶,
Dan Cogalniceanu⁹, Milena Cvijanović¹⁰, Christophe Dufresnes⁴, Collie Ennis¹¹, Rob Gandola¹¹,
Daniel Jablonski¹², Angela Julian¹³, Daria Kranželić¹⁴, Simeon Lukanov¹⁵,
Iñigo Martínez-Solano¹⁶, Ryan Montgomery⁵,¹³, Borislav Naumov¹⁵, Matthew O'Neill¹⁷,
Alexandra North¹⁸,¹⁹, Maciej Pabijan²⁰, Robert Pushendorf¹⁸, Daniele Salvi²¹, Bruno Schmidt¹⁴,
Konstantinos Sotiropoulos²², Florina Stanescu⁹,²³, David Stanković²⁴, Sarah Stapleton²⁵,
Emina Šunje²⁶,²⁷,²⁸, Márton Szabolcs²⁹, Emiliya Vacheva¹⁵, David Willis³⁰, Adnan Zimić²⁷,³¹,
James France¹,², Willem R.M. Meilink¹,², Tariq Stark³², Richard P.J.H. Struijk³²,
Anagnostis Theodoropoulos¹,², Manon C. de Visser¹,², Ben Wielstra¹,²,**

1 - Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, The Netherlands

2 - Institute of Biology Leiden, Leiden University, P.O. Box 9505, 2300 RA Leiden, The Netherlands

3 - Durrell Institute of Conservation and Ecology, School of Anthropology and Conservation, University of Kent, Canterbury, CT2 7NR, UK

4 - Laboratory for Amphibian Systematic and Evolutionary Research (LASER), College of Biology & Environment, Nanjing Forestry University, Nanjing, China

5 - Northern Ireland Amphibian and Reptile Group, Belfast, UK

6 - Department of Ecological and Biological Sciences, Tuscia University, Largo dell'Università snc, 01100, Viterbo, Italy

7 - Herpetological Society of Ireland, Munster, Ireland

8 - Caledonian Conservation Ltd, Office 2 and 3, Craigarnhall, Bridge of Allan, Stirling, FK9 4NG, Scotland, UK

9 - Faculty of Natural Sciences and Agricultural Sciences, Ovidius University Constanța, Al. Universității 1, 900470 Constanța, Romania

10 - Institute for Biological Research “Siniša Stanković”, National Institute of Republic of Serbia, University of Belgrade, Bul. Despota Stefana 142, 11060 Belgrade, Serbia

11 - Herpetological Society of Ireland, Wicklow, Ireland

12 - Department of Zoology, Comenius University in Bratislava, Ilkovičova 6, 842 15 Bratislava, Slovakia

13 - Amphibian and Reptile Groups of UK (ARG UK), 82 Gidley Way, Horspath, Oxford, OX33 1TG, UK

14 - Association Hyla, Lipovac I no. 7, 10000 Zagreb, Croatia

15 - Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street, 1113 Sofia, Bulgaria

16 - Museo Nacional de Ciencias Naturales (MNCN-CSIC), c/ José Gutiérrez Abascal, 2, 28006 Madrid, Spain

17 - Atlantic Technological University, Freshwater and Marine Biology Program, Old Dublin Rd, H91 T8NW Galway, Ireland

18 - School of Biological and Marine Science, University of Plymouth, Plymouth PL4 8AA, UK

19 - Institute of Zoology, Zoological Society London, London, NW1 4RY, UK
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

An extended mtDNA phylogeography for the alpine newt illuminates
the provenance of introduced populations

Jody Robbebont¹,²,*, Sam van Veldhuijzen¹,²,*, Steven J.R. Allain³, Johanna Ambu⁴,
Ryan Boyle⁵, Daniele Canestrelli⁶, Éinne Ó Cathasaigh⁷, Chris Cathrine⁸, Andrea Chiocchio⁶,
Dan Cogalniceanu⁹, Milena Cvijanović¹⁰, Christophe Dufresnes⁴, Collie Ennis¹¹, Rob Gandola¹¹,
Daniel Jablonski¹², Angela Julian¹³, Daria Kranželić¹⁴, Simeon Lukanov¹⁵,
Iñigo Martínez-Solano¹⁶, Ryan Montgomery⁵,¹³, Borislav Naumov¹⁵, Matthew O'Neill¹⁷,
Alexandra North¹⁸,¹⁹, Maciej Pabijan²⁰, Robert Pushendorf¹⁸, Daniele Salvi²¹, Bruno Schmidt¹⁴,
Konstantinos Sotiropoulos²², Florina Stanescu⁹,²³, David Stanković²⁴, Sarah Stapleton²⁵,
Emina Šunje²⁶,²⁷,²⁸, Márton Szabolcs²⁹, Emiliya Vacheva¹⁵, David Willis³⁰, Adnan Zimić²⁷,³¹,
James France¹,², Willem R.M. Meilink¹,², Tariq Stark³², Richard P.J.H. Struijk³²,
Anagnostis Theodoropoulos¹,², Manon C. de Visser¹,², Ben Wielstra¹,²,**

1 - Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, The Netherlands

2 - Institute of Biology Leiden, Leiden University, P.O. Box 9505, 2300 RA Leiden, The Netherlands

3 - Durrell Institute of Conservation and Ecology, School of Anthropology and Conservation, University of Kent, Canterbury, CT2 7NR, UK

4 - Laboratory for Amphibian Systematic and Evolutionary Research (LASER), College of Biology & Environment, Nanjing Forestry University, Nanjing, China

5 - Northern Ireland Amphibian and Reptile Group, Belfast, UK

6 - Department of Ecological and Biological Sciences, Tuscia University, Largo dell'Università snc, 01100, Viterbo, Italy

7 - Herpetological Society of Ireland, Munster, Ireland

8 - Caledonian Conservation Ltd, Office 2 and 3, Craigarnhall, Bridge of Allan, Stirling, FK9 4NG, Scotland, UK

9 - Faculty of Natural Sciences and Agricultural Sciences, Ovidius University Constanța, Al. Universității 1, 900470 Constanța, Romania

10 - Institute for Biological Research “Siniša Stanković”, National Institute of Republic of Serbia, University of Belgrade, Bul. Despota Stefana 142, 11060 Belgrade, Serbia

11 - Herpetological Society of Ireland, Wicklow, Ireland

12 - Department of Zoology, Comenius University in Bratislava, Ilkovičova 6, 842 15 Bratislava, Slovakia

13 - Amphibian and Reptile Groups of UK (ARG UK), 82 Gidley Way, Horspath, Oxford, OX33 1TG, UK

14 - Association Hyla, Lipovac I no. 7, 10000 Zagreb, Croatia

15 - Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street, 1113 Sofia, Bulgaria

16 - Museo Nacional de Ciencias Naturales (MNCN-CSIC), c/ José Gutiérrez Abascal, 2, 28006 Madrid, Spain

17 - Atlantic Technological University, Freshwater and Marine Biology Program, Old Dublin Rd, H91 T8NW Galway, Ireland

18 - School of Biological and Marine Science, University of Plymouth, Plymouth PL4 8AA, UK

19 - Institute of Zoology, Zoological Society London, London, NW1 4RY, UK
20 - Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, Gronostajowa
9, 30-387 Kraków, Poland

21 - Department of Health, Life and Environmental Sciences, University of L'Aquila, Via Vetoio, 67100
Coppito, L'Aquila, Italy

22 - Department of Biological Applications and Technology, University of Ioannina, 45110 Ioannina, Greece

23 - Black Sea Institute for Development and Security Studies (BSIDSS) and Center for Morphological and Genetic Studies of Malignant Pathology (CEDMOG), Ovidius University Constanța, Al. Universității 1, 900470 Constanța, Romania

24 - National Institute of Biology (NIB), Department of Organisms and Ecosystems Research, Večna pot 111, 1000 Ljubljana, Slovenia

25 - National Parks and Wildlife Service, Dublin, D07 N7CV, Ireland

26 - Evolutionary Ecology Group, Faculty of Science, University of Sarajevo, Zmaja od Bosne 33-35, Sarajevo, Bosnia and Herzegovina

27 - Herpetological Association in Bosnia and Herzegovina - ATRA, Urijan Dedina 137, 71 000 Sarajevo, Bosnia and Herzegovina

28 - Laboratory of Functional Morphology, University of Antwerpen, Universiteitsplein 1, 2610 Wilrijk, Belgium

29 - Department of Tisza Research, Institute of Aquatic Ecology, Centre for Ecological Research, Bem tér 18/C, 4026 Debrecen, Hungary

30 - Hertfordshire Amphibian and Reptile Group, Hetfield, UK

31 - National Museum of Bosnia and Herzegovina, Zmaja od Bosne 3, 71 000 Sarajevo, Bosnia and Herzegovina

32 - Reptile, Amphibian and Fish Conservation Netherlands (RAVON), P.O. Box 1413, 6501 BK Nijmegen, The Netherlands

*These authors contributed equally to this work.

**Corresponding author; e-mail: ben.wielstra@naturalis.nl

ORCID iDs: Robbebont: 0009-0000-7111-5110; van Veldhuijzen: 0000-0002-3641-071X; Allain: 0000-0002-5669-0149; Ambu: 0000-0002-8072-7677; Canestrelli: 0000-0001-9351-4972; Cathasaigh: 0000-0001-8027-0905; Cathrine: 0009-0009-2069-7320; Chiocchio: 0000-0002-0067-7025; Cogalniceanu: 0000-0003-2959-014X; Dufresnes: 0000-0002-8497-8908; Ennis: 0000-0002-8714-9793; Gandola: 0000-0002-0550-1707; Jablonski: 0000-0002-5394-0114; Kranželić: 0000-0001-6291-0593; Lukanov: 0000-0002-2546-6590; Martínez-Solano: 0000-0002-2260-226X; North: 0000-0002-2942-3467; Pabijan: 0000-0001-5557-2338; Pushendorf: 0000-0002-8105-171X; Salvi: 0000-0002-3804-2690; Schmidt: 0000-0003-3544-4739; Sotiropoulos: 0000-0002-3622-7907; Stanescu: 0000-0001-6872-664X; Stanković: 0000-0002-3312-382X; Šunje: 0000-0003-3050-8545; Vacheva: 0000-0002-3008-6648; Meilink: 0009-0006-4451-1779; Theodoropoulos: 0009-0003-0272-6358; de Visser: 0000-0003-3681-6151; Wielstra: 0000-0002-7112-5965

Received 10 February 2023; final revision received 6 June 2023; accepted 6 June 2023;
published online 1 August 2023; published in print 8 August 2023
Associate Editor: Sylvain Ursenbacher

**Abstract.** Many herpetofauna species have been introduced outside of their native range. MtDNA barcoding is regularly used to determine the provenance of such populations. The alpine newt has been introduced across the Netherlands, the United Kingdom and Ireland. However, geographical mtDNA structure across the natural range of the alpine newt is still incompletely understood and certain regions are severely undersampled. We collect mtDNA sequence data of over seven hundred individuals, from both the native and the introduced range. The main new insights from our extended mtDNA phylogeography are that 1) haplotypes from Spain do not form a reciprocally monophyletic clade, but are nested inside the mtDNA clade that covers western and eastern Europe; and 2) haplotypes from the northwest Balkans form a monophyletic clade together with those from the Southern Carpathians and Apuseni Mountains. We also home in on the regions where the distinct mtDNA clades meet in nature. We show that four out of the seven distinct mtDNA clades that comprise the alpine newt are implicated in the introductions in the Netherlands, United Kingdom and Ireland. In several introduced localities,
20 - Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, Gronostajowa
9, 30-387 Kraków, Poland

21 - Department of Health, Life and Environmental Sciences, University of L'Aquila, Via Vetoio, 67100
Coppito, L'Aquila, Italy

22 - Department of Biological Applications and Technology, University of Ioannina, 45110 Ioannina, Greece

23 - Black Sea Institute for Development and Security Studies (BSIDSS) and Center for Morphological and Genetic Studies of Malignant Pathology (CEDMOG), Ovidius University Constanța, Al. Universității 1, 900470 Constanța, Romania

24 - National Institute of Biology (NIB), Department of Organisms and Ecosystems Research, Večna pot 111, 1000 Ljubljana, Slovenia

25 - National Parks and Wildlife Service, Dublin, D07 N7CV, Ireland

26 - Evolutionary Ecology Group, Faculty of Science, University of Sarajevo, Zmaja od Bosne 33-35, Sarajevo, Bosnia and Herzegovina

27 - Herpetological Association in Bosnia and Herzegovina – ATRA, Urijan Dedina 137, 71 000 Sarajevo, Bosnia and Herzegovina

28 - Laboratory of Functional Morphology, University of Antwerpen, Universiteitsplein 1, 2610 Wilrijk, Belgium

29 - Department of Tisza Research, Institute of Aquatic Ecology, Centre for Ecological Research, Bem tér 18/C, 4026 Debrecen, Hungary

30 - Hertfordshire Amphibian and Reptile Group, Hetfield, UK

31 - National Museum of Bosnia and Herzegovina, Zmaja od Bosne 3, 71 000 Sarajevo, Bosnia and Herzegovina

32 - Reptile, Amphibian and Fish Conservation Netherlands (RAVON), P.O. Box 1413, 6501 BK Nijmegen, The Netherlands

*These authors contributed equally to this work.

**Corresponding author; e-mail: ben.wielstra@naturalis.nl

ORCID iDs: Robbebont: 0009-0000-7111-5110; van Veldhuijzen: 0000-0002-3641-071X;

Allain: 0000-0002-5669-0149; Ambu: 0000-0002-8072-7677; Canestrelli: 0000-0001-9351-4972;

Cathasaigh: 0000-0001-8027-0905; Cathrine: 0009-0009-2069-7320; Chiocchio: 0000-0002-0067-7025;

Cogalniceanu: 0000-0003-2959-014X; Dufresnes: 0000-0002-8497-8908; Ennis: 0000-0002-8714-9793;

Gandola: 0000-0002-0550-1707; Jablonski: 0000-0002-5394-0114; Kranželić: 0000-0001-6291-0593;

Lukanov: 0000-0002-2546-6590; Martínez-Solano: 0000-0002-2260-226X; North: 0000-0002-2942-3467;

Pabijan: 0000-0001-5557-2338; Pushendorf: 0000-0002-8105-171X; Salvi: 0000-0002-3804-2690;

Schmidt: 0000-0003-3544-4739; Sotiropoulos: 0000-0002-3622-7907; Stanescu: 0000-0001-6872-664X;

Stanković: 0000-0002-3312-382X; Šunje: 0000-0003-3050-8545; Vacheva: 0000-0002-3008-6648;

Meilink: 0009-0006-4451-1779; Theodoropoulos: 0009-0003-0272-6358; de Visser: 0000-0003-3681-6151;

Wielstra: 0000-0002-7112-5965

Received 10 February 2023; final revision received 6 June 2023; accepted 6 June 2023;

published online 1 August 2023; published in print 8 August 2023

Associate Editor: Sylvain Ursenbacher

**Abstract.** Many herpetofauna species have been introduced outside of their native range. MtDNA barcoding is regularly used to determine the provenance of such populations. The alpine newt has been introduced across the Netherlands, the United Kingdom and Ireland. However, geographical mtDNA structure across the natural range of the alpine newt is still incompletely understood and certain regions are severely undersampled. We collect mtDNA sequence data of over seven hundred individuals, from both the native and the introduced range. The main new insights from our extended mtDNA phylogeography are that 1) haplotypes from Spain do not form a reciprocally monophyletic clade, but are nested inside the mtDNA clade that covers western and eastern Europe; and 2) haplotypes from the northwest Balkans form a monophyletic clade together with those from the Southern Carpathians and Apuseni Mountains. We also home in on the regions where the distinct mtDNA clades meet in nature. We show that four out of the seven distinct mtDNA clades that comprise the alpine newt are implicated in the introductions in the Netherlands, United Kingdom and Ireland. In several introduced localities,
two distinct mtDNA clades co-occur. As these mtDNA clades presumably represent cryptic species, we urge that the extent of genetic admixture between them is assessed from genome-wide nuclear DNA markers. We mobilized a large number of citizen scientists in this project to support the collection of DNA samples by skin swabbing and underscore the effectiveness of this sampling technique for mtDNA barcoding.

Keywords: citizen science, *Ichthyosaura alpestris*, invasive species, mtDNA barcoding, skin swabbing.

Introduction

Invasive species are species that have estab-
lished outside of their natural distribution and
have a negative environmental impact at the
expense of native biodiversity (Simberloff,
2013; Bellard et al., 2016; Pyšek et al., 2020;
Diagne et al., 2021). Therefore, mapping and
monitoring of invasive species is crucial when
contemplating conservation action, and genetic
tools play an important role in this process
(Allendorf et al., 2010). MtDNA typically dis-
plays strong geographical genetic structuring,
allowing closely related species as well as
intraspecific lineages to be distinguished (Avise,
2000).

For most European amphibians, comprehen-
sive mtDNA phylogeographies have already
been published (e.g., Babik et al., 2005; Fijar-
czyk et al., 2011; Vences et al., 2013; Wielstra
et al., 2013; Dufresnes et al., 2020; Ambu et
al., 2023). MtDNA barcoding involves the com-
parison of sequences for a particular mtDNA
marker across many individuals that belong to
e.g., different species or structured populations
(Hebert et al., 2003; Mir et al., 2021). Match-
ing mtDNA barcodes from introduced amphib-
ian populations with published phylogeographic
datasets can be used to determine from where
within the natural distribution range these popu-
lations originate.

Sample collection for a project on mtDNA
barcoding of invasive amphibian species lends
itself well to ‘citizen science’ initiatives, where
members of the public team up with profes-
sional scientists to collect data (Devictor et al.,
2010; Bonney et al., 2016). A network of citizen
scientists would allow comprehensive sampling
to be achieved efficiently – particularly when
the focal species is present in the volunteers’
private garden ponds. For the purpose of citizen

science, any DNA sample collection method-
ology needs to be simple and easy to follow,
minimize the impact on animals and involve as
little bureaucracy as possible.

Collecting amphibian tissue requires training
and ethical approval and can be controversial
with the public (Zemanova, 2019). While tak-
ing tail tips of salamanders has been proven
not to negatively affect survival (Arntzen et
al., 1999; Polich et al., 2013), amputating toes,
as often conducted on frogs, may be harm-
ful (McCarthy and Parris, 2004; Funk et al.,
2005). Swabbing is considered a ‘minimally
invasive’ alternative to tissue sampling (Bro-
quet et al., 2007; Ringler, 2018; Ambu and
Dufresnes, 2023). While one could argue about
what would constitute ‘minimally invasive’,
swabbing is in practice not considered an animal
experiment in most instances, and is therefore
exempt from approval by an animal ethics com-
mittee. Accordingly, the required permissions to
take swabs from animals in their natural envi-
ronment are relatively easy to obtain for a large
group of people, particularly if they are already
involved in monitoring schemes (i.e., with ter-
rain access and permissions/licenses to handle
animals already in place).

While buccal swabbing is a suitable source of
DNA, it requires physically opening the mouth
of the animal (Pidancier et al., 2003; Poschadel
and Möller, 2004; Broquet et al., 2007). Buc-
cal swabbing could risk injury if the person
taking the sample is unexperienced and/or the
mouth of the animal being sampled is small rel-
ative to the swab (Poschadel and Möller, 2004;
Müller et al., 2013; Ringler, 2018). Skin swab-
bing has been used as an alternative for buc-
cal swabbing for over a decade now (Prunier et
al., 2012; Ward et al., 2019). While skin swab-
bing performs poorly in relatively dry skinned

Downloaded from Brill.com 04/16/2024, 12:28:35PM

Terms of use for this license

of the CC BY 4.0 license.
two distinct mtDNA clades co-occur. As these mtDNA clades presumably represent cryptic species, we urge that the extent of genetic admixture between them is assessed from genome-wide nuclear DNA markers. We mobilized a large number of citizen scientists in this project to support the collection of DNA samples by skin swabbing and underscore the effectiveness of this sampling technique for mtDNA barcoding.

Keywords: citizen science, *Ichthyosaura alpestris*, invasive species, mtDNA barcoding, skin swabbing.

Introduction

Invasive species are species that have estab-
lished outside of their natural distribution and
have a negative environmental impact at the
expense of native biodiversity (Simberloff,
2013; Bellard et al., 2016; Pyšek et al., 2020;
Diagne et al., 2021). Therefore, mapping and
monitoring of invasive species is crucial when
contemplating conservation action, and genetic
tools play an important role in this process
(Allendorf et al., 2010). MtDNA typically dis-
plays strong geographical genetic structuring,
allowing closely related species as well as
intraspecific lineages to be distinguished (Avise,
2000).

For most European amphibians, comprehen-
sive mtDNA phylogeographies have already
been published (e.g., Babik et al., 2005; Fijar-
czyk et al., 2011; Vences et al., 2013; Wielstra
et al., 2013; Dufresnes et al., 2020; Ambu et
al., 2023). MtDNA barcoding involves the com-
parison of sequences for a particular mtDNA
marker across many individuals that belong to
e.g., different species or structured populations
(Hebert et al., 2003; Mir et al., 2021). Match-
ing mtDNA barcodes from introduced amphib-
ian populations with published phylogeographic
datasets can be used to determine from where
within the natural distribution range these popu-
lations originate.

Sample collection for a project on mtDNA
barcoding of invasive amphibian species lends
itself well to ‘citizen science’ initiatives, where
members of the public team up with profes-
sional scientists to collect data (Devictor et al.,
2010; Bonney et al., 2016). A network of citizen
scientists would allow comprehensive sampling
to be achieved efficiently – particularly when
the focal species is present in the volunteers’
private garden ponds. For the purpose of citizen

science, any DNA sample collection method-
ology needs to be simple and easy to follow,
minimize the impact on animals and involve as
little bureaucracy as possible.

Collecting amphibian tissue requires training
and ethical approval and can be controversial
with the public (Zemanova, 2019). While tak-
ing tail tips of salamanders has been proven
not to negatively affect survival (Arntzen et
al., 1999; Polich et al., 2013), amputating toes,
as often conducted on frogs, may be harm-
ful (McCarthy and Parris, 2004; Funk et al.,
2005). Swabbing is considered a ‘minimally
invasive’ alternative to tissue sampling (Bro-
quet et al., 2007; Ringler, 2018; Ambu and
Dufresnes, 2023). While one could argue about
what would constitute ‘minimally invasive’,
swabbing is in practice not considered an animal
experiment in most instances, and is therefore
exempt from approval by an animal ethics com-
mittee. Accordingly, the required permissions to
take swabs from animals in their natural envi-
ronment are relatively easy to obtain for a large
group of people, particularly if they are already
involved in monitoring schemes (i.e., with ter-
rain access and permissions/licenses to handle
animals already in place).

While buccal swabbing is a suitable source of
DNA, it requires physically opening the mouth
of the animal (Pidancier et al., 2003; Poschadel
and Möller, 2004; Broquet et al., 2007). Buc-
cal swabbing could risk injury if the person
taking the sample is unexperienced and/or the
mouth of the animal being sampled is small rel-
ative to the swab (Poschadel and Möller, 2004;
Müller et al., 2013; Ringler, 2018). Skin swab-
bing has been used as an alternative for buc-
cal swabbing for over a decade now (Prunier et
al., 2012; Ward et al., 2019). While skin swab-
bing performs poorly in relatively dry skinned

Downloaded from Brill.com 04/16/2024, 12:28:35PM

of the CC BY 4.0 license.
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Distribution map for the alpine newt (*Ichthyosaura alpestris*). Background colour reflects the rough natural distribution of the mtDNA clades and symbols reflect sampled localities. The inset shows the Netherlands in more detail (lighter shading reflects uncertainty about the status of populations in the northeast of the country). Numbers refer to localities/regions mentioned in the text: 1 = Oudeland, 2 = Veluwe and 3 = Holterberg. Sampling information in supplementary table S1.</figcaption></figure>

frog species (Ringler, 2018), it has been shown to work well in species with a relatively moist skin, such as newts (Prunier et al., 2012). Skin swabbing is arguably simpler and quicker than buccal swabbing and less impactful on the sampled animals, making it particularly suitable in citizen science projects.

The alpine newt (*Ichthyosaura alpestris*) occurs throughout most of Europe (fig. 1). The taxon harbours multiple highly distinct mtDNA clades (Sotiropoulos et al., 2007; Recuero et al., 2014) and is suspected to encompass multiple ‘cryptic species’ (Raffaëlli, 2018; Speybroeck et al., 2020; Frost, 2021). Unfortunately, previous studies have used different mtDNA markers, which hampers the combination of datasets (Sotiropoulos et al., 2007; Recuero et al., 2014; Chiocchio et al., 2017). Furthermore, despite multiple studies, sampling gaps remain. Most mtDNA clades meet in the Balkans. However, the distribution of these lineages and the extent of comingling between them are not yet well understood. Finally, the postglacially colonized range, such as Eastern and Western Europe,

is genetically relatively homogeneous and has been poorly sampled.

Alpine newts are popular pets and have regularly been introduced outside of their native range (Sillero et al., 2014). MtDNA barcoding has been applied to determine the origin of several incidental introductions. Italian alpine newts have been introduced on the other side of the globe, in New Zealand (Arntzen et al., 2016). Less extreme are the introduction of alpine newts from the Cantabrian Mountains of northern Spain into central Spain (Palomar et al., 2017), Cantabrian and Italian alpine newts into Catalonia (Fibla et al., 2015), and alpine newts from unspecified but distinct sources in Eastern/Western Europe into northern Poland (Jakóbik et al., 2019) and southern France (Arntzen et al., 2016). The alpine newt has been introduced on a massive scale in The Netherlands (Creemers and van Delft, 2009; https://www.ravon.nl/Soorten/Soortinformatie/alpenwatersalamander) as well as the United Kingdom and Ireland (Meehan, 2013; Allain and Lynn, 2021). The provenance of these introductions is currently unclear.
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Distribution map for the alpine newt (*Ichthyosaura alpestris*). Background colour reflects the rough natural distribution of the mtDNA clades and symbols reflect sampled localities. The inset shows the Netherlands in more detail (lighter shading reflects uncertainty about the status of populations in the northeast of the country). Numbers refer to localities/regions mentioned in the text: 1 = Oudeland, 2 = Veluwe and 3 = Holterberg. Sampling information in supplementary table S1.</figcaption></figure>

frog species (Ringler, 2018), it has been shown to work well in species with a relatively moist skin, such as newts (Prunier et al., 2012). Skin swabbing is arguably simpler and quicker than buccal swabbing and less impactful on the sampled animals, making it particularly suitable in citizen science projects.

The alpine newt (*Ichthyosaura alpestris*) occurs throughout most of Europe (fig. 1). The taxon harbours multiple highly distinct mtDNA clades (Sotiropoulos et al., 2007; Recuero et al., 2014) and is suspected to encompass multiple ‘cryptic species’ (Raffaëlli, 2018; Speybroeck et al., 2020; Frost, 2021). Unfortunately, previous studies have used different mtDNA markers, which hampers the combination of datasets (Sotiropoulos et al., 2007; Recuero et al., 2014; Chiocchio et al., 2017). Furthermore, despite multiple studies, sampling gaps remain. Most mtDNA clades meet in the Balkans. However, the distribution of these lineages and the extent of comingling between them are not yet well understood. Finally, the postglacially colonized range, such as Eastern and Western Europe,

is genetically relatively homogeneous and has been poorly sampled.

Alpine newts are popular pets and have regularly been introduced outside of their native range (Sillero et al., 2014). MtDNA barcoding has been applied to determine the origin of several incidental introductions. Italian alpine newts have been introduced on the other side of the globe, in New Zealand (Arntzen et al., 2016). Less extreme are the introduction of alpine newts from the Cantabrian Mountains of northern Spain into central Spain (Palomar et al., 2017), Cantabrian and Italian alpine newts into Catalonia (Fibla et al., 2015), and alpine newts from unspecified but distinct sources in Eastern/Western Europe into northern Poland (Jakóbik et al., 2019) and southern France (Arntzen et al., 2016). The alpine newt has been introduced on a massive scale in The Netherlands (Creemers and van Delft, 2009; https://www.ravon.nl/Soorten/Soortinformatie/alpenwatersalamander) as well as the United Kingdom and Ireland (Meehan, 2013; Allain and Lynn, 2021). The provenance of these introductions is currently unclear.

Downloaded from Brill.com 04/16/2024 12:28:35PM

of the CC BY 4.0 license.
The marked geographical genetic structure in mtDNA across the vast alpine newt range (Sotiropoulos et al., 2007; Recuero et al., 2014) facilitates determining the provenance of introduced alpine newt populations. However, the situation would benefit from the survey of a single mtDNA from individuals, throughout the natural distribution range. To this end, we employ mtDNA barcoding of a single mtDNA gene with the aim to: 1) expand the alpine newt mtDNA phylogeography and 2) determine the geographical origin of introduced alpine newt populations. Additionally, we 3) test the effectiveness of buccal versus skin swabs for mtDNA barcoding in alpine newts.

## Materials and methods

### Sampling and DNA extraction

We obtained new mtDNA data for 456 individuals from 234 localities (fig. 1, supplementary table S1). For 247 individuals samples consisted of skin swabs. The outer surface of the newt was swabbed (40 times in total, 10 times on the dorsal, ventral and each lateral side) with Copan 155C Rayon swabs. Swabs were stored dry and moved to -20°C as soon as feasible. For 18 individuals, buccal swabs were also taken, i.e., alongside skin swabs, to compare DNA yield (measured with a DropSense96 machine) and PCR performance (i.e., if a sequence could be produced). We also obtained tissue samples, either freshly collected or taken from museum specimens (Džukić et al., 2015). DNA was extracted using the Wizard® Genomic DNA purification kit (Promega). Samples from Belgium and Slovenia were provided as DNA extract.

### PCR and sequencing

For 411 individuals we amplified a 897 bp fragment of the ND4 (NADH dehydrogenase subunit 4) mtDNA gene and adjacent tRNA. We adjusted the ‘universal’ ND4 primers (ND4 and Leu) from Arévalo et al. (1994) to exactly match the sequence in the *Ichthyosaura alpestris* mitogenome published by Zhang et al. (2008), using Geneious Prime 2021.1.1. This resulted in the primers ND4-Ich (5'-CACCTCTGACTTCCAAAAGCCCACGTA GAGGC-3') and Leu-Ich (5'-CATACTTTTACTTGGAG TTGCACCA-3'). For 45 individuals for which we could not obtain a usable sequence, presumably due to DNA degradation, internal primers were designed in Geneious Prime to amplify a stretch of 241 bp, namely primer pairs ND4-Ich-i299vlasiF (5'-TGCTTCTAGCCCGCGGAACC-3') and Leu-Ich-i559vlasiR (5'-ATGGGTTTATCAGGCGA AGA-3') for the distinct Vlasina mtDNA clade or ND4-Ich.

i299F (5'-TACTCTTAGCCCGCGGAGTM-3') and Leu-Ich-i559R (5'-ATGGGTTTATCGGGYGAAGG-3') for the remaining mtDNA lineages.

PCRs were performed in 12 µl reactions, containing 0.06 µl of both forward and reverse primer (0.05 µM end concentration of each primer), 7.2 µl QIAGEN multiplex PCR master mix, 3.68 µl purified water and 1 µl of DNA extract. PCR conditions were: a hot start for 15 minutes at 95°C, followed by 35 cycles of denaturation for 30 seconds at 95°C, annealing for 1 minute at 55°C and extension for 1 minute at 72°C, and extension at 72°C of ten minutes. Sanger sequencing was outsourced to BaseClear B.V. and sequences were edited and trimmed in Geneious Prime.

### Compilation of mtDNA ND4 reference database

ND4 sequences were available from previous studies from across the natural range of *I. alpestris* (Pabijan and Babik, 2006; Recuero et al., 2014; Vörös et al., 2021). To be able to compare between studies, we focussed on a shared stretch of 651 bp and we trimmed our sequences accordingly. ND4 sequences of sufficient length were available from several introduced populations as well (Recuero et al., 2014; Arntzen et al., 2016). This database of 651 bp ND4 haplotypes provides a comprehensive overview of genetic variation across the natural range and allows us to link introduced populations to the part of the natural range that they originate from. We introduce a new naming system for these 651 bp ND4 haplotypes (supplementary tables S1 and S2).

Our 241 bp ND4 data, together with additional studies that focussed on either a different fragment of ND4 (i.e., not our target 651 bp) (Jakóbik et al., 2019) or different mtDNA markers (Sotiropoulos et al., 2007; Šunje et al., 2021), were incorporated to help further delineate the geographical distribution of mtDNA clades (supplementary table S1). The colour scheme we use to highlight the different mtDNA clades follows Recuero et al. (2014) and Arntzen et al. (2016).

### Genetic analyses

The Haplotype Collapsr function in FaBox (Villesen, 2007) was used to determine if the mtDNA haplotypes of the newly sequenced individuals match any of the previously identified 651 bp ND4 haplotypes in our database. To determine the phylogenetic position of newly identified haplotypes we conducted Bayesian phylogenetic inference with MrBayes v.3.2.6. (Ronquist et al., 2012). The IQ-TREE web server functionality ModelFinder (Trifinopoulos et al., 2016; Kalyaanamoorthy et al., 2017) was used to approximate the most appropriate model of sequence evolution for each codon position (HKY + G, GTR + G and GTR + I for position 1, 2 and 3, respectively). The northern crested newt *Triturus cristatus* (haplotype Tcri01, Genbank Code: GU982383, taken from Wielstra et al., 2013) was used as an outgroup. We ran two runs of four-chains for five million generations with a sampling frequency of 0.001 and a heating parameter of 0.2 in MrBayes and used a 25% burnin. Tracer v1.7 (Rambaut et al., 2018) was used to confirm that runs converged and that ESS was above the standard threshold of the CC BY 4.0 license.

Downloaded from www.fao.org by 07/16/2024 12:28:35PM
The marked geographical genetic structure in mtDNA across the vast alpine newt range (Sotiropoulos et al., 2007; Recuero et al., 2014) facilitates determining the provenance of introduced alpine newt populations. However, the situation would benefit from the survey of a single mtDNA from individuals, throughout the natural distribution range. To this end, we employ mtDNA barcoding of a single mtDNA gene with the aim to: 1) expand the alpine newt mtDNA phylogeography and 2) determine the geographical origin of introduced alpine newt populations. Additionally, we 3) test the effectiveness of buccal versus skin swabs for mtDNA barcoding in alpine newts.

## Materials and methods

### Sampling and DNA extraction

We obtained new mtDNA data for 456 individuals from 234 localities (fig. 1, supplementary table S1). For 247 individuals samples consisted of skin swabs. The outer surface of the newt was swabbed (40 times in total, 10 times on the dorsal, ventral and each lateral side) with Copan 155C Rayon swabs. Swabs were stored dry and moved to -20°C as soon as feasible. For 18 individuals, buccal swabs were also taken, i.e., alongside skin swabs, to compare DNA yield (measured with a DropSense96 machine) and PCR performance (i.e., if a sequence could be produced). We also obtained tissue samples, either freshly collected or taken from museum specimens (Džukić et al., 2015). DNA was extracted using the Wizard® Genomic DNA purification kit (Promega). Samples from Belgium and Slovenia were provided as DNA extract.

### PCR and sequencing

For 411 individuals we amplified a 897 bp fragment of the ND4 (NADH dehydrogenase subunit 4) mtDNA gene and adjacent tRNA. We adjusted the 'universal' ND4 primers (ND4 and Leu) from Arévalo et al. (1994) to exactly match the sequence in the *Ichthyosaura alpestris* mitogenome published by Zhang et al. (2008), using Geneious Prime 2021.1.1. This resulted in the primers ND4-Ich (5'-CACCTCTGACTTCCAAAAGCCCACGTA GAGGC-3') and Leu-Ich (5'-CATACTTTTACTTGGAG TTGCACCA-3'). For 45 individuals for which we could not obtain a usable sequence, presumably due to DNA degradation, internal primers were designed in Geneious Prime to amplify a stretch of 241 bp, namely primer pairs ND4-Ich-i299vlasiF (5'-TGCTTCTAGCCCGCGGAACC-3') and Leu-Ich-i559vlasiR (5'-ATGGGTTTATCAGGCGA AGA-3') for the distinct Vlasina mtDNA clade or ND4-Ich.

i299F (5'-TACTCTTAGCCCGCGGAGTM-3') and Leu-Ich-i559R (5'-ATGGGTTTATCGGGYGAAGG-3') for the remaining mtDNA lineages.

PCRs were performed in 12 µl reactions, containing 0.06 µl of both forward and reverse primer (0.05 µM end concentration of each primer), 7.2 µl QIAGEN multiplex PCR master mix, 3.68 µl purified water and 1 µl of DNA extract. PCR conditions were: a hot start for 15 minutes at 95°C, followed by 35 cycles of denaturation for 30 seconds at 95°C, annealing for 1 minute at 55°C and extension for 1 minute at 72°C, and extension at 72°C of ten minutes. Sanger sequencing was outsourced to BaseClear B.V. and sequences were edited and trimmed in Geneious Prime.

### Compilation of mtDNA ND4 reference database

ND4 sequences were available from previous studies from across the natural range of *I. alpestris* (Pabijan and Babik, 2006; Recuero et al., 2014; Vörös et al., 2021). To be able to compare between studies, we focussed on a shared stretch of 651 bp and we trimmed our sequences accordingly. ND4 sequences of sufficient length were available from several introduced populations as well (Recuero et al., 2014; Arntzen et al., 2016). This database of 651 bp ND4 haplotypes provides a comprehensive overview of genetic variation across the natural range and allows us to link introduced populations to the part of the natural range that they originate from. We introduce a new naming system for these 651 bp ND4 haplotypes (supplementary tables S1 and S2).

Our 241 bp ND4 data, together with additional studies that focussed on either a different fragment of ND4 (i.e., not our target 651 bp) (Jakóbik et al., 2019) or different mtDNA markers (Sotiropoulos et al., 2007; Šunje et al., 2021), were incorporated to help further delineate the geographical distribution of mtDNA clades (supplementary table S1). The colour scheme we use to highlight the different mtDNA clades follows Recuero et al. (2014) and Arntzen et al. (2016).

### Genetic analyses

The Haplotype Collapsr function in FaBox (Villesen, 2007) was used to determine if the mtDNA haplotypes of the newly sequenced individuals match any of the previously identified 651 bp ND4 haplotypes in our database. To determine the phylogenetic position of newly identified haplotypes we conducted Bayesian phylogenetic inference with MrBayes v.3.2.6. (Ronquist et al., 2012). The IQ-TREE web server functionality ModelFinder (Trifinopoulos et al., 2016; Kalyaanamoorthy et al., 2017) was used to approximate the most appropriate model of sequence evolution for each codon position (HKY + G, GTR + G and GTR + I for position 1, 2 and 3, respectively). The northern crested newt *Triturus cristatus* (haplotype Tcri01, Genbank Code: GU982383, taken from Wielstra et al., 2013) was used as an outgroup. We ran two runs of four-chains for five million generations with a sampling frequency of 0.001 and a heating parameter of 0.2 in MrBayes and used a 25% burnin. Tracer v1.7 (Rambaut et al., 2018) was used to confirm that runs converged and that ESS was above the standard threshold of the CC BY 4.0 license.

https://creativecommons.org/licenses/by/4.0/
old of 200. A median spanning haplotype network was constructed in PopArt 1.7 under default settings (Leigh and Bryant, 2015). We calculated nucleotide diversity ($\pi$) across the natural distribution range in DNAsp v6 (Rozas et al., 2017), at the level of the country and for each mtDNA clade separately (if more than one occurred in the same country).

## Results

Our database of 651 bp ND4 sequences contains 411 newly sequenced and 185 previously published individuals (from 273 localities). This dataset encompasses 100 haplotypes, including 62 not previously identified (figs. 1-3, supplementary tables S1 and S2). These haplotypes are used to conduct phylogenetic reconstruction and to tie introduced populations to the natural distribution range (see below). For another 45 individuals (covering 36 localities) an internal 241 bp ND4 fragment is available. Furthermore, another 80 individuals (covering 62 localities)

are included for which either a relatively short stretch of ND4 or a different mtDNA marker are available from previous studies. These data are only used to assign individuals to mtDNA clade to help delineate the distribution of mtDNA clades (fig. 1, supplementary table S1).

Our extended mtDNA phylogeography most-ly confirms the distinct mtDNA clades identi-fied in previous studies and the relationships among them (Sotiropoulos et al., 2007; Recuero et al., 2014) – for consistency between studies we stick to the previous division into mtDNA clades as much as possible. However, haplo-types from Spain are not reciprocally mono-phyletic from, but are nested within, a clade containing haplotypes from across Eastern and Western Europe.

While we do not improve much upon the known distribution of the mtDNA clades here referred to as the Italy, Southern Balkans and

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Majority rule consensus Bayesian phylogeny for the alpine newt (*Ichthyosaura alpestris*) based on 651 bp of the mtDNA gene ND4. The outgroup is not shown. Spanish haplotypes are highlighted with an asterisk. Haplotype details can be found in supplementary table S1 and a more detailed version of this phylogeny is available in supplementary fig. S1.</figcaption></figure>

Downloaded from britt.com 04/16/2021 12:28:35PM
old of 200. A median spanning haplotype network was constructed in PopArt 1.7 under default settings (Leigh and Bryant, 2015). We calculated nucleotide diversity ($\pi$) across the natural distribution range in DNAsp v6 (Rozas et al., 2017), at the level of the country and for each mtDNA clade separately (if more than one occurred in the same country).

## Results

Our database of 651 bp ND4 sequences contains 411 newly sequenced and 185 previously published individuals (from 273 localities). This dataset encompasses 100 haplotypes, including 62 not previously identified (figs. 1-3, supplementary tables S1 and S2). These haplotypes are used to conduct phylogenetic reconstruction and to tie introduced populations to the natural distribution range (see below). For another 45 individuals (covering 36 localities) an internal 241 bp ND4 fragment is available. Furthermore, another 80 individuals (covering 62 localities)

are included for which either a relatively short stretch of ND4 or a different mtDNA marker are available from previous studies. These data are only used to assign individuals to mtDNA clade to help delineate the distribution of mtDNA clades (fig. 1, supplementary table S1).

Our extended mtDNA phylogeography most-ly confirms the distinct mtDNA clades identi-fied in previous studies and the relationships among them (Sotiropoulos et al., 2007; Recuero et al., 2014) – for consistency between studies we stick to the previous division into mtDNA clades as much as possible. However, haplo-types from Spain are not reciprocally mono-phyletic from, but are nested within, a clade containing haplotypes from across Eastern and Western Europe.

While we do not improve much upon the known distribution of the mtDNA clades here referred to as the Italy, Southern Balkans and

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Majority rule consensus Bayesian phylogeny for the alpine newt (*Ichthyosaura alpestris*) based on 651 bp of the mtDNA gene ND4. The outgroup is not shown. Spanish haplotypes are highlighted with an asterisk. Haplotype details can be found in supplementary table S1 and a more detailed version of this phylogeny is available in supplementary fig. S1.</figcaption></figure>

Downloaded from britt.com 04/16/2021 12:28:35PM
<figure><img src="image_7.png" /><figcaption>**Figure 3.** Haplotype network for the alpine newt (*Ichthyosaura alpestris*) based on 651 bp of the mtDNA gene ND4. Pie size reflects haplotype frequency and bars reflect the number of substitutions. White pie slices represent introduced localities. Spanish haplotypes are highlighted with an asterisk. Haplotype details can be found in supplementary table S1.</figcaption></figure>

Vlasina mtDNA clades, we clarify the distributions of the other mtDNA clades (fig. 1). Haplotypes from the Southern Carpathians and the Apuseni Mountains in Romania and the north-west Balkans (northern Croatia and adjacent Slovenia and Bosnia and Herzegovina) compose a monophyletic clade that we refer to as the Northern Balkans & Romania mtDNA clade (figs 1 and 2, supplementary fig. S1). The Central Balkans mtDNA clade stretches from Bulgaria (and just a bit of Greece), through Serbia to Bosnia and Herzegovina, and slightly extends into Montenegro and Croatia (fig. 1). The Montenegro mtDNA clade is mainly found in Montenegro but slightly protrudes into neighbouring Bosnia and Herzegovina, Serbia and Albania (fig. 1). We show that the Eastern/Western Europe & Spain mtDNA clade extends as far south as the Southern Carpathians and the Slovenian/Croatian border (fig. 1). Most genetic diversity in this mtDNA clade is found at the southern fringe of its distribution, just north of the Balkan Peninsula (fig. 4, supplementary table S3).

Most introduced localities in the Netherlands contain one particular haplotype from the Eastern/Western Europe & Spain mtDNA clade, namely EWES_1, with the exception of introduced locality Holterberg, where we find EWES_21 (figs 1 and 3, supplementary table S1). Additional haplotypes from distinct mtDNA clades are present in several introduced localities, sometimes in syntopy (fig. 1). Namely, introduced locality Oudeland features haplotype IT_7 (Italy mtDNA clade) and haplotype EWES_1. In the Veluwe region, haplotype NBR_12 (Northern Balkans & Romania mtDNA clade) is widespread, while haplotype CB_6 (Central Balkans mtDNA clade) is restricted to a single locality (close to NBR_12). We also find the EWES_1 haplotype in two localities in this region; in one it is the only haplotype identified, in the other it co-occurs with NBR_12. In both Ireland and Northern Ireland, the only haplotype found is EWES_38 of the Eastern/Western Europe & Spain mtDNA clade. Scotland and Wales host haplotype EWES_1. In one locality from England we find EWES_1.

via Open Access.

downloaded from Brill.com 04/16/2021 12:28:35PM

In one locality from England we find EWES_1
of the CC BY 4.0 license.
<figure><img src="image_7.png" /><figcaption>**Figure 3.** Haplotype network for the alpine newt (*Ichthyosaura alpestris*) based on 651 bp of the mtDNA gene ND4. Pie size reflects haplotype frequency and bars reflect the number of substitutions. White pie slices represent introduced localities. Spanish haplotypes are highlighted with an asterisk. Haplotype details can be found in supplementary table S1.</figcaption></figure>

Vlasina mtDNA clades, we clarify the distributions of the other mtDNA clades (fig. 1). Haplotypes from the Southern Carpathians and the Apuseni Mountains in Romania and the north-west Balkans (northern Croatia and adjacent Slovenia and Bosnia and Herzegovina) compose a monophyletic clade that we refer to as the Northern Balkans & Romania mtDNA clade (figs 1 and 2, supplementary fig. S1). The Central Balkans mtDNA clade stretches from Bulgaria (and just a bit of Greece), through Serbia to Bosnia and Herzegovina, and slightly extends into Montenegro and Croatia (fig. 1). The Montenegro mtDNA clade is mainly found in Montenegro but slightly protrudes into neighbouring Bosnia and Herzegovina, Serbia and Albania (fig. 1). We show that the Eastern/Western Europe & Spain mtDNA clade extends as far south as the Southern Carpathians and the Slovenian/Croatian border (fig. 1). Most genetic diversity in this mtDNA clade is found at the southern fringe of its distribution, just north of the Balkan Peninsula (fig. 4, supplementary table S3).

Most introduced localities in the Netherlands contain one particular haplotype from the Eastern/Western Europe & Spain mtDNA clade, namely EWES_1, with the exception of introduced locality Holterberg, where we find EWES_21 (figs 1 and 3, supplementary table S1). Additional haplotypes from distinct mtDNA clades are present in several introduced localities, sometimes in syntopy (fig. 1). Namely, introduced locality Oudeland features haplotype IT_7 (Italy mtDNA clade) and haplotype EWES_1. In the Veluwe region, haplotype NBR_12 (Northern Balkans & Romania mtDNA clade) is widespread, while haplotype CB_6 (Central Balkans mtDNA clade) is restricted to a single locality (close to NBR_12). We also find the EWES_1 haplotype in two localities in this region; in one it is the only haplotype identified, in the other it co-occurs with NBR_12. In both Ireland and Northern Ireland, the only haplotype found is EWES_38 of the Eastern/Western Europe & Spain mtDNA clade. Scotland and Wales host haplotype EWES_1. In one locality from England we find EWES_1.

via Open Access.

downloaded from Brill.com 04/16/2021 11:28:35PM

Terms of the CC BY 4.0 license.
<figure><img src="image_8.png" /><figcaption>**Figure 4.** Nucleotide diversity ($\pi$) across the natural distribution range of the alpine newt (*Ichthyosaura alpestris*) for the main mtDNA clades and at country-level, based on 651 bp of the mtDNA gene ND4. N/a means $\pi$ could not be established because only a single individual was available. Background colour reflects the rough natural distribution of the mtDNA clades. Further details can be found in supplementary table S3.</figcaption></figure>

again, another features haplotype IT_7 of the Italy mtDNA clade, and a third contains both EWES_1 and IT_7.

We have obtained sequence data for 247 swab samples. Another 32 swabs (c. 11%) did not produce a sequence. For the 18 individuals that have been both buccal- and skin-swabbed we obtain sequences from both approaches for 16 individuals (for one individual the buccal swab failed and for another individual the skin swab failed). There is accordingly no difference in performance between buccal and skin-swabbing (Chi-squared test, $p > 0.05$, df = 1, n = 36). The two sequences per individual based on buccal

cal and skin swabs are identical. Even though success rate in terms of obtaining a sequence is similar for buccal and skin swabs, DNA yield is consistently lower for skin swabs (supplementary table S4).

## Discussion

*New insights from extended mtDNA phylogeography*

While the alpine newt has received acute phylo-geographic interest, the sampling schemes and markers employed in previous studies differ.

Downloaded from Brilt.com 04/16/2024 28:35PM
<figure><img src="image_8.png" /><figcaption>**Figure 4.** Nucleotide diversity ($\pi$) across the natural distribution range of the alpine newt (*Ichthyosaura alpestris*) for the main mtDNA clades and at country-level, based on 651 bp of the mtDNA gene ND4. N/a means $\pi$ could not be established because only a single individual was available. Background colour reflects the rough natural distribution of the mtDNA clades. Further details can be found in supplementary table S3.</figcaption></figure>

again, another features haplotype IT_7 of the Italy mtDNA clade, and a third contains both EWES_1 and IT_7.

We have obtained sequence data for 247 swab samples. Another 32 swabs (c. 11%) did not produce a sequence. For the 18 individuals that have been both buccal- and skin-swabbed we obtain sequences from both approaches for 16 individuals (for one individual the buccal swab failed and for another individual the skin swab failed). There is accordingly no difference in performance between buccal and skin-swabbing (Chi-squared test, $p > 0.05$, df = 1, n = 36). The two sequences per individual based on buccal

cal and skin swabs are identical. Even though success rate in terms of obtaining a sequence is similar for buccal and skin swabs, DNA yield is consistently lower for skin swabs (supplementary table S4).

## Discussion

*New insights from extended mtDNA phylogeography*

While the alpine newt has received acute phylo-geographic interest, the sampling schemes and markers employed in previous studies differ.

Downloaded from Brill.com 04/16/2024 28:35PM

of the CC BY 4.0 license.
and sampling gaps remain (Sotiropoulos et al., 2007; Recuero et al., 2014; Chiocchio et al., 2017; Šunje et al., 2021; Vörös et al., 2021). Our range-wide mtDNA phylogeography, based on a single marker, allows us to clarify the distribution of the main mtDNA clades and locate (potential) contact zones between them.

We find Spanish haplotypes nested within a clade that also contains haplotypes from Eastern and Western Europe. We suggests this pattern reflects our greatly increased sampling, rather than low phylogenetic resolution. We recommend a genomic study should test the phylogenetic position of Spanish alpine newts.

While, outside of Spain, the Eastern/Western Europe & Spain mtDNA clade is genetically homogeneous across most of its range, considerable genetic variation is found at its southern fringe, just north of the Balkan Peninsula, in the north-east of Italy, Slovenia and Hungary (fig. 3, supplementary table S3). Haplotype EWES_1 occurs most frequently and is widely distributed, including in the Italian eastern Alps, adjacent to the region that harbours most of the genetic diversity for the Eastern/Western Europe & Spain mtDNA clade. This pattern suggests that the eastern Alps were the point of origin for the postglacial recolonization of western and central Europe (Sotiropoulos et al., 2007).

Another new insight concerns to which mtDNA clade the haplotypes from the north-west Balkans (northern Croatia and adjacent Slovenia and Bosnia and Herzegovina) should be allocated (fig. 1). In an attempt to integrate data from a previous study (Sotiropoulos et al., 2007), concerning a different, relatively low-resolution mtDNA marker, these localities were misinterpreted as belonging to the Central Balkans mtDNA clade in Recuero et al. (2014). Rather, the haplotypes are part of an mtDNA clade that is further distributed in the Southern Carpathians and Apuseni Mountains in Romania (figs 1 and 2). Currently, these two range sections are allopatric (Sillero et al., 2014), being separated by hundreds of kilome-

tres of unsuitable lowland habitat. This disjunct distribution pattern suggests that alpine newts had a more extensive range in the past (presumably during glacial cycles), in line with montane relictual distribution patches observed in other parts of the range (Pabijan et al., 2009; Chiocchio et al., 2017; Vörös et al., 2021). A phylogenomic study should further explore the phylogenetic position of these geographical populations.

Our denser sampling allows us to delineate the distributions of the distinct mtDNA clades with more accuracy and informs upon where they (potentially) meet. In particular, we pinpoint the transition between the Central Balkans and Northern Balkans & Romania mtDNA clade, in the north-west of Bosnia and Herzegovina and just across the border in Croatia (fig. 1) (see also Šunje et al., 2021). Syntopy of the Central Balkans and Montenegro mtDNA clade is observed at the crossover between Serbia, Bosnia and Herzegovina and Montenegro. While we show that the Vlasina mtDNA clade extends slightly into Bulgaria (fig. 1), further sampling is required to determine if it is geographically isolated from the Central Balkans mtDNA clade. Additional sampling in Kosovo is required to assess whether the Southern Balkans mtDNA clade comes into contact with the Montenegro and Central Balkans mtDNA clades. We show that the Eastern/Western Europe & Spain mtDNA clade is distributed as far south as the Southern Carpathians and the border between Slovenia and Croatia, in close proximity to the Northern Balkans & Romania mtDNA clade.

While it has been well-established that the alpine newt harbours multiple, highly distinct mtDNA clades (Sotiropoulos et al., 2007; Recuero et al., 2014), only limited nuclear DNA data have been studied so far (Recuero et al., 2014). In some instances, the distinct mtDNA clades are associated with distinct nuclear DNA alleles, suggesting they probably represent cryptic species. However, the highly distinct Vlasina mtDNA clade might concern a

Downloaded from Brail.com 04/16/2024 12:28:35PM

of the CC BY 4.0 license.
and sampling gaps remain (Sotiropoulos et al., 2007; Recuero et al., 2014; Chiocchio et al., 2017; Šunje et al., 2021; Vörös et al., 2021). Our range-wide mtDNA phylogeography, based on a single marker, allows us to clarify the distribution of the main mtDNA clades and locate (potential) contact zones between them.

We find Spanish haplotypes nested within a clade that also contains haplotypes from Eastern and Western Europe. We suggests this pattern reflects our greatly increased sampling, rather than low phylogenetic resolution. We recommend a genomic study should test the phylogenetic position of Spanish alpine newts.

While, outside of Spain, the Eastern/Western Europe & Spain mtDNA clade is genetically homogeneous across most of its range, considerable genetic variation is found at its southern fringe, just north of the Balkan Peninsula, in the north-east of Italy, Slovenia and Hungary (fig. 3, supplementary table S3). Haplotype EWES_1 occurs most frequently and is widely distributed, including in the Italian eastern Alps, adjacent to the region that harbours most of the genetic diversity for the Eastern/Western Europe & Spain mtDNA clade. This pattern suggests that the eastern Alps were the point of origin for the postglacial recolonization of western and central Europe (Sotiropoulos et al., 2007).

Another new insight concerns to which mtDNA clade the haplotypes from the north-west Balkans (northern Croatia and adjacent Slovenia and Bosnia and Herzegovina) should be allocated (fig. 1). In an attempt to integrate data from a previous study (Sotiropoulos et al., 2007), concerning a different, relatively low-resolution mtDNA marker, these localities were misinterpreted as belonging to the Central Balkans mtDNA clade in Recuero et al. (2014). Rather, the haplotypes are part of an mtDNA clade that is further distributed in the Southern Carpathians and Apuseni Mountains in Romania (figs 1 and 2). Currently, these two range sections are allopatric (Sillero et al., 2014), being separated by hundreds of kilome-

tres of unsuitable lowland habitat. This disjunct distribution pattern suggests that alpine newts had a more extensive range in the past (presumably during glacial cycles), in line with montane relictual distribution patches observed in other parts of the range (Pabijan et al., 2009; Chiocchio et al., 2017; Vörös et al., 2021). A phylogenomic study should further explore the phylogenetic position of these geographical populations.

Our denser sampling allows us to delineate the distributions of the distinct mtDNA clades with more accuracy and informs upon where they (potentially) meet. In particular, we pinpoint the transition between the Central Balkans and Northern Balkans & Romania mtDNA clade, in the north-west of Bosnia and Herzegovina and just across the border in Croatia (fig. 1) (see also Šunje et al., 2021). Syntopy of the Central Balkans and Montenegro mtDNA clade is observed at the crossover between Serbia, Bosnia and Herzegovina and Montenegro. While we show that the Vlasina mtDNA clade extends slightly into Bulgaria (fig. 1), further sampling is required to determine if it is geographically isolated from the Central Balkans mtDNA clade. Additional sampling in Kosovo is required to assess whether the Southern Balkans mtDNA clade comes into contact with the Montenegro and Central Balkans mtDNA clades. We show that the Eastern/Western Europe & Spain mtDNA clade is distributed as far south as the Southern Carpathians and the border between Slovenia and Croatia, in close proximity to the Northern Balkans & Romania mtDNA clade.

While it has been well-established that the alpine newt harbours multiple, highly distinct mtDNA clades (Sotiropoulos et al., 2007; Recuero et al., 2014), only limited nuclear DNA data have been studied so far (Recuero et al., 2014). In some instances, the distinct mtDNA clades are associated with distinct nuclear DNA alleles, suggesting they probably represent cryptic species. However, the highly distinct Vlasina mtDNA clade might concern a

Downloaded from Brail.com 04/16/2024 12:28:35PM

of the CC BY 4.0 license.
‘ghost lineage’ that does not reflect a diverged
nuclear DNA gene pool (Recuero et al., 2014).
Mitochondrial surveys are known to be mis-
leading in amphibian phylogeography and ensu-
ing taxonomy (Dufresnes and Jablonski, 2022).
Introgression of mtDNA across species bound-
aries is regularly observed (Toews and Brels-
ford, 2012).

Genome-scale data are becoming readily
available for newts (Rancilhac et al., 2021)
and have already been successfully applied
for species delineation in related newt gene-
ra (Wielstra and Arntzen, 2016; Pabijan et al.,
2017; Wielstra et al., 2018), also in combina-
tion with exploring potential ecological (Wiel-
stra et al., 2012; van Riemsdijk et al., 2017)
and morphological differentiation (Gerlach and
Fahrbach, 2016; Üzüm et al., 2019). A phyloge-
nomic study is required to clarify the evolution-
ary significance of the tremendous mitochon-
drial diversity in the alpine newt. In this respect,
our improved mtDNA phylogeography helps to
home in on the contact zones between the poten-
tial cryptic species hiding within the alpine newt
(fig. 1).

*Provenance of introductions illuminated*

We focus on alpine newt introductions in the
Netherlands as well as the United Kingdom and
Ireland, where, compared to earlier studied inci-
dental introductions (Arntzen et al., 2016; Palo-
mar et al., 2017; Jakóbik et al., 2019), alpine
newt introduction has occurred on a massive
scale (Creemers and van Delft, 2009; Allain
and Lynn, 2021). While these newly established
populations are thriving and expanding, based
on both the rapidness with which new locali-
ties have been discovered, and the geographical
distances between them, tens if not hundreds
of introduction and/or translocation events must
have been involved.

In the Netherlands, the Veluwe region has
been rapidly colonized since c. 1995 (Creemers
and van Delft, 2009). Haplotypes from three
distinct mtDNA clades are recovered in this
area. One (EWES_1) is naturally widespread

across Europe and also occurs natively in the
Netherlands, while the other two are indige-
nous to the Balkan Peninsula (fig. 1). The
most widespread of the two Balkan haplotypes
(NBR_12) is also present in a locality from
western Croatia. Another newt species that has
been introduced in the Veluwe region, the Ital-
ian crested newt *Triturus carnifex*, could also
be linked to a locality from western Croatia
(less than thirty kilometres away) based on
mtDNA barcoding (Meilink et al., 2015). Yet,
we also find a haplotype (CB_6) in a single
alpine newt that is naturally only known from
a single locality in Bosnia and Herzegovina, c.
135 kilometres further eastwards. We suggest
that these two distinct Balkan haplotypes may
have a wider natural distribution and may occur
in syntopy in nature, implying that the introduc-
tion could have involved a single Balkan stock.
However, we acknowledge that the difference
in frequency is striking. For example, two dis-
tinct mtDNA haplotypes also segregate in an
introduced population of *Pelobates* toads in the
Netherlands, but here haplotype frequency is
considerably more equal (Koster et al., 2023).
Nonetheless, the presence of an additional hap-
lotype, natively distributed throughout Eastern
and Western Europe, suggests that at least two
stocks must have been involved.

In Oudeland we observe a haplotype that nat-
urally occurs in Central Italy (IT_7). The same
haplotype has also been found in the intro-
duced population in New Zealand (Arntzen et
al., 2016). However, this area (and practically
all other introduced populations of the Nether-
lands) is also inhabited by newts that feature
a widespread European and even Dutch native
haplotype. There is one exception, Holterberg,
where a distinct haplotype from the same East-
ern/Western Europe & Spain mtDNA clade
(EWES_21) is found, but because we did not
sequence this haplotype across the natural range
we cannot map its geographical origin.

We now have a situation in the Netherlands
where genetically distinct mtDNA clades, pre-
sumably representing different cryptic species.

Downloaded from www.biolink.com 04/16/2024 12:28:35PM

Terms
of the CC BY 4.0 license.
‘ghost lineage’ that does not reflect a diverged
nuclear DNA gene pool (Recuero et al., 2014).
Mitochondrial surveys are known to be mis-
leading in amphibian phylogeography and ensu-
ing taxonomy (Dufresnes and Jablonski, 2022).
Introgression of mtDNA across species bound-
aries is regularly observed (Toews and Brels-
ford, 2012).

Genome-scale data are becoming readily
available for newts (Rancilhac et al., 2021)
and have already been successfully applied
for species delineation in related newt gene-
ra (Wielstra and Arntzen, 2016; Pabijan et al.,
2017; Wielstra et al., 2018), also in combina-
tion with exploring potential ecological (Wiel-
stra et al., 2012; van Riemsdijk et al., 2017)
and morphological differentiation (Gerlach and
Fahrbach, 2016; Üzüm et al., 2019). A phyloge-
nomic study is required to clarify the evolution-
ary significance of the tremendous mitochon-
drial diversity in the alpine newt. In this respect,
our improved mtDNA phylogeography helps to
home in on the contact zones between the poten-
tial cryptic species hiding within the alpine newt
(fig. 1).

Provenance of introductions illuminated

We focus on alpine newt introductions in the
Netherlands as well as the United Kingdom and
Ireland, where, compared to earlier studied inci-
dental introductions (Arntzen et al., 2016; Palo-
mar et al., 2017; Jakóbik et al., 2019), alpine
newt introduction has occurred on a massive
scale (Creemers and van Delft, 2009; Allain
and Lynn, 2021). While these newly established
populations are thriving and expanding, based
on both the rapidness with which new locali-
ties have been discovered, and the geographical
distances between them, tens if not hundreds
of introduction and/or translocation events must
have been involved.

In the Netherlands, the Veluwe region has
been rapidly colonized since c. 1995 (Creemers
and van Delft, 2009). Haplotypes from three
distinct mtDNA clades are recovered in this
area. One (EWES_1) is naturally widespread

across Europe and also occurs natively in the
Netherlands, while the other two are indige-
nous to the Balkan Peninsula (fig. 1). The
most widespread of the two Balkan haplotypes
(NBR_12) is also present in a locality from
western Croatia. Another newt species that has
been introduced in the Veluwe region, the Ital-
ian crested newt *Triturus carnifex*, could also
be linked to a locality from western Croatia
(less than thirty kilometres away) based on
mtDNA barcoding (Meilink et al., 2015). Yet,
we also find a haplotype (CB_6) in a single
alpine newt that is naturally only known from
a single locality in Bosnia and Herzegovina, c.
135 kilometres further eastwards. We suggest
that these two distinct Balkan haplotypes may
have a wider natural distribution and may occur
in syntopy in nature, implying that the introduc-
tion could have involved a single Balkan stock.
However, we acknowledge that the difference
in frequency is striking. For example, two dis-
tinct mtDNA haplotypes also segregate in an
introduced population of *Pelobates* toads in the
Netherlands, but here haplotype frequency is
considerably more equal (Koster et al., 2023).
Nonetheless, the presence of an additional hap-
lotype, natively distributed throughout Eastern
and Western Europe, suggests that at least two
stocks must have been involved.

In Oudeland we observe a haplotype that nat-
urally occurs in Central Italy (IT_7). The same
haplotype has also been found in the intro-
duced population in New Zealand (Arntzen et
al., 2016). However, this area (and practically
all other introduced populations of the Nether-
lands) is also inhabited by newts that feature
a widespread European and even Dutch native
haplotype. There is one exception, Holterberg,
where a distinct haplotype from the same East-
ern/Western Europe & Spain mtDNA clade
(EWES_21) is found, but because we did not
sequence this haplotype across the natural range
we cannot map its geographical origin.

We now have a situation in the Netherlands
where genetically distinct mtDNA clades, pre-
sumably representing different cryptic species.

Downloaded from www.biolchem.com at 04/16/2024 12:28:35PM

Terms
of the CC BY 4.0 license.
occur in syntopy. A similar situation has previ-
ously been observed in e.g., Spain (van Riems-
dijk et al., 2018) and the Netherlands (Kuijt et
al., 2023), where the mtDNA of two recently
recognized species of *Ommatotriton* newts and
*Hyla* treefrogs were found together in intro-
duced populations. Such a situation may stem
from the fact that cryptic species are typically
mixed in the pet trade, particularly before they
have been characterized as such in major taxo-
nomic lists. According to current taxonomy, the
alpine newt is regarded a single species, despite
the presumed presence of cryptic species (Spey-
broeck et al., 2020). We urge for an evalua-
tion of the extent of genetic admixture between
the potential cryptic species within the intro-
duced alpine newt populations based on many
unlinked nuclear DNA markers.

In Ireland and Northern Ireland we find
a single haplotype (EWES_38) that was pre-
viously reported from an introduced popula-
tion in Larzac, southern France (Arntzen et
al., 2016). This haplotype clearly belongs to
the Eastern/Western Europe & Spain mtDNA
clade. However, despite our increased sam-
pling, which spans the previously hypothe-
sized source in western Switzerland (Arntzen
et al., 2016), we could not locate this haplo-
type in the natural distribution range. Given
the apparently restricted geographical distribu-
tion of this haplotype in the wild, it is likely
that the ancestors of these geographically dis-
tinct introduced populations derive from the
same source. Note that Arntzen et al. (2016)
identified a haplotype in two individuals from
Larzac, France (GenBank Accession numbers
KR107546 and KR107549) that is quite dis-
tinct from any of the haplotypes known through-
out the natural range – also when considering
the enhanced sampling of the current study –
and appeared ‘intermediate’ between Spanish
and Eastern/Western European haplotypes. We
noticed that this haplotype is identical to mul-
tiple haplotypes found throughout Eastern and
Western Europe for our 241 internal primer
stretch, while carrying three fixed differences.

compared to Spanish haplotypes. Yet, in the
remainder of the sequence, the opposite pattern
is occasionally found. Therefore, we considered
it likely that this haplotype represents a chimeric
sequence and therefore opted to exclude it from
our dataset.

Wales, Scotland and two out of three local-
ities from England harbour a different haplo-
type (EWES_1) of the Eastern/Western Europe
& Spain mtDNA clade; a haplotype which is
widely distributed across the natural range of
this mtDNA clade and is also the most com-
mon haplotype observed in the introduced local-
ities in the Netherlands. A genome-scale dataset
would be required to try and provide further
insight on the provenance of populations bear-
ing this naturally widespread haplotype.

In one of those two England localities where
the Eastern/Western Europe & Spain mtDNA
clade is present, we also find the same Italian
mtDNA clade haplotype (IT_7) that has also
been introduced in Oudeland in the Netherlands
(see above) as well as in New Zealand (Arntzen
et al., 2016). Again this raises a question about
the extent of genetic admixture between two
potential cryptic alpine newt species. The same
Italian mtDNA clade haplotype is the sole hap-
lotype present in the third locality from Eng-
land we studied. Many more introduced alpine
newt localities, in large part revealed by cit-
izen science records, are known from Eng-
land (Allain and Lynn, 2021) and Scotland
(C. Cathrine, unpublished data); additional sam-
pling is required to get a better understanding
of the number of different geographical stocks
involved and where these occur in syntopy.

Based on our extended sampling across the
natural range, a good number of introduced
alpine newt localities can now be connected
to a native source population. Yet, the genetic
homogeneity in temperate Europe makes it dif-
ficult to pinpoint the provenance of many others
(a well-known limitation of the mtDNA bar-
coding of invasive species, see e.g., Vliegen-
hart et al., 2023). We can confirm that mul-
tiple distinct sources are involved. How did

Downloaded from Brilj.com 04/16/2024 12:28:35PM

This article is licensed under a Creative Commons Attribution 4.0 International license.
occur in syntopy. A similar situation has previ-
ously been observed in e.g., Spain (van Riems-
dijk et al., 2018) and the Netherlands (Kuijt et
al., 2023), where the mtDNA of two recently
recognized species of *Ommatotriton* newts and
*Hyla* treefrogs were found together in intro-
duced populations. Such a situation may stem
from the fact that cryptic species are typically
mixed in the pet trade, particularly before they
have been characterized as such in major taxo-
nomic lists. According to current taxonomy, the
alpine newt is regarded a single species, despite
the presumed presence of cryptic species (Spey-
broeck et al., 2020). We urge for an evalua-
tion of the extent of genetic admixture between
the potential cryptic species within the intro-
duced alpine newt populations based on many
unlinked nuclear DNA markers.

In Ireland and Northern Ireland we find
a single haplotype (EWES_38) that was pre-
viously reported from an introduced popula-
tion in Larzac, southern France (Arntzen et
al., 2016). This haplotype clearly belongs to
the Eastern/Western Europe & Spain mtDNA
clade. However, despite our increased sam-
pling, which spans the previously hypothe-
sized source in western Switzerland (Arntzen
et al., 2016), we could not locate this haplo-
type in the natural distribution range. Given
the apparently restricted geographical distribu-
tion of this haplotype in the wild, it is likely
that the ancestors of these geographically dis-
tinct introduced populations derive from the
same source. Note that Arntzen et al. (2016)
identified a haplotype in two individuals from
Larzac, France (GenBank Accession numbers
KR107546 and KR107549) that is quite dis-
tinct from any of the haplotypes known through-
out the natural range – also when considering
the enhanced sampling of the current study –
and appeared ‘intermediate’ between Spanish
and Eastern/Western European haplotypes. We
noticed that this haplotype is identical to mul-
tiple haplotypes found throughout Eastern and
Western Europe for our 241 internal primer
stretch, while carrying three fixed differences.

compared to Spanish haplotypes. Yet, in the
remainder of the sequence, the opposite pattern
is occasionally found. Therefore, we considered
it likely that this haplotype represents a chimeric
sequence and therefore opted to exclude it from
our dataset.

Wales, Scotland and two out of three local-
ities from England harbour a different haplo-
type (EWES_1) of the Eastern/Western Europe
& Spain mtDNA clade; a haplotype which is
widely distributed across the natural range of
this mtDNA clade and is also the most com-
mon haplotype observed in the introduced local-
ities in the Netherlands. A genome-scale dataset
would be required to try and provide further
insight on the provenance of populations bear-
ing this naturally widespread haplotype.

In one of those two England localities where
the Eastern/Western Europe & Spain mtDNA
clade is present, we also find the same Italian
mtDNA clade haplotype (IT_7) that has also
been introduced in Oudeland in the Netherlands
(see above) as well as in New Zealand (Arntzen
et al., 2016). Again this raises a question about
the extent of genetic admixture between two
potential cryptic alpine newt species. The same
Italian mtDNA clade haplotype is the sole hap-
lotype present in the third locality from Eng-
land we studied. Many more introduced alpine
newt localities, in large part revealed by cit-
izen science records, are known from Eng-
land (Allain and Lynn, 2021) and Scotland
(C. Cathrine, unpublished data); additional sam-
pling is required to get a better understanding
of the number of different geographical stocks
involved and where these occur in syntopy.

Based on our extended sampling across the
natural range, a good number of introduced
alpine newt localities can now be connected
to a native source population. Yet, the genetic
homogeneity in temperate Europe makes it dif-
ficult to pinpoint the provenance of many others
(a well-known limitation of the mtDNA bar-
coding of invasive species, see e.g., Vliegen-
hart et al., 2023). We can confirm that mul-
tiple distinct sources are involved. How did
... downloaded from Brüll.com 04/16/2024 12:28:35PM
... terms
of the CC BY 4.0 license.
https://creativecommons.org/licenses/by/4.0/
these alpine newts get introduced? In most cases this is unknown. Alpine newts are common in the pet trade and release by owners seems the most plausible route (Arntzen et al., 2016), with an additional possible transmission route being 'hitchhiking' (perhaps as eggs) in aquatic vegetation (Schmeller et al., 2020). Ironically, alpine newts in Ratho, Scotland were introduced because they were mistakenly thought to be the native great crested newt (*T. cristatus*); the population is now expanding (C. Cathrine, unpubl. data).

A particular risk when closely related species or distinct populations of a single species are brought into contact with native counterparts is 'genetic pollution', where native gene copies are driven to extinction, because they are replaced by invasive ones (Rhymer and Simberloff, 1996; Huxel, 1999; Meilink et al., 2015; Todesco et al., 2016; Dufresnes et al., 2017). In this context, the introduction of highly distinct alpine newt mtDNA clades, presumably representing cryptic species, close to native populations, is of particular concern. Given the limitations associated with the use of a single DNA marker such as mtDNA, genome-scale data would be required to screen for potential genetic pollution (Wielstra et al., 2016).

### Swabbing by citizen scientists

This project benefited from citizen science. Involving citizens in scientific projects helps to reduce the gap between science and society (Devictor et al., 2010; Bonney et al., 2016). Most of our samples from the Netherlands were collected by volunteers of NGO RAVON (Reptile, Amphibian and Fish Conservation Netherlands) to whom we sent swabs with detailed instructions on how to use these. Evidently, this was an efficient way to amass geographically dense sampling (fig. 1, supplementary table S1).

We show that the success rate of skin swabbing, in terms of obtaining an mtDNA barcode, is the same as for buccal swabbing. Skin swabs have a wider application than simple mtDNA barcoding. Both skin and buccal swabs have

been used to obtain nuclear DNA data, including microsatellite (Broquet et al., 2007; Prunier et al., 2012) and KASP genotyping (Fahrbach et al., 2021; de Brouwer et al., 2023). Buccal swabs are also being applied in amphibians to obtain genome-scale datasets via RAD-sequencing (Ambu and Dufresnes, 2023) and we would recommend testing skin swabs for this purpose as well (while DNA concentrations of skin swabs are lower than those of buccal swabs, many should yield sufficient DNA for RAD-sequencing).

To the best of our knowledge, the stress associated with buccal and skin swabbing on amphibians has never been quantified. However, it is reasonable to assume that skin swabbing is relatively less stressful than buccal swabbing (Ringler, 2018). Our study illustrates the potential of the technique, particularly in projects on aquatic amphibians where conventional molecular techniques are applied and citizen scientists are involved.

**Acknowledgements.** Many citizen scientists, all part of RAVON's network of volunteers that conduct monitoring, took skin swabs for us (supplementary table S5). Permits for fieldwork are listed in supplementary table S6. Dutch land owner organizations that provided field permits are Het Flevo-landschap, Geldersch Landschap & Kasteelen, Landgoed de Utrecht, Natuurmonumenten (Society for Preservation of Nature Monuments in the Netherlands) and Staatsbosbeheer (State Forestry). Land owners in Wales (UK) provided permission for pond surveys and collection of tissue samples. Thanks also go to the National Parks and Wildlife Service (NPWS) Ireland and Northern Ireland Environment Agency for permissions. Apenheul Primate Park, Caledonian Conservation, Hortus Botanicus Leiden, Societas herpetologica Slovenica, Wiesław Babik, Sergé Bogaerts, David O'Brien, Ivona Burić, David Eastwood, Michael Fahrbach, Alberto Gosá, Ana Ivanović, Robert Jehle, Fairlie Kirkpatrick, Đurdica Majetić, Saša Marić, Helena Martínez, Alfredo G. Nicieza, Radostina Pravcheva, Gregorio Sánchez Montes, Krisztián Szabó, Judit Vörös and Piotr Zieliński helped to obtain samples. Belgium DNA extractions were provided by the Research Institute for Nature and Forest (INBO). DS was partially supported by the Slovenian Research Agency (P1-0255). MS was funded by the National Research, Development and Innovation Office of Hungary (NKFIH-OTKA K134391). MON & AT were supported by an Erasmus+ internship.

Downloaded from britl.com 04/16/2024 12:28:35PM

distributed under the terms

of the CC BY 4.0 license.
these alpine newts get introduced? In most cases this is unknown. Alpine newts are common in the pet trade and release by owners seems the most plausible route (Arntzen et al., 2016), with an additional possible transmission route being 'hitchhiking' (perhaps as eggs) in aquatic vegetation (Schmeller et al., 2020). Ironically, alpine newts in Ratho, Scotland were introduced because they were mistakenly thought to be the native great crested newt (*T. cristatus*); the population is now expanding (C. Cathrine, unpubl. data).

A particular risk when closely related species or distinct populations of a single species are brought into contact with native counterparts is 'genetic pollution', where native gene copies are driven to extinction, because they are replaced by invasive ones (Rhymer and Simberloff, 1996; Huxel, 1999; Meilink et al., 2015; Todesco et al., 2016; Dufresnes et al., 2017). In this context, the introduction of highly distinct alpine newt mtDNA clades, presumably representing cryptic species, close to native populations, is of particular concern. Given the limitations associated with the use of a single DNA marker such as mtDNA, genome-scale data would be required to screen for potential genetic pollution (Wielstra et al., 2016).

### Swabbing by citizen scientists

This project benefited from citizen science. Involving citizens in scientific projects helps to reduce the gap between science and society (Devictor et al., 2010; Bonney et al., 2016). Most of our samples from the Netherlands were collected by volunteers of NGO RAVON (Rep tile, Amphibian and Fish Conservation Nether lands) to whom we sent swabs with detailed instructions on how to use these. Evidently, this was an efficient way to amass geographically dense sampling (fig. 1, supplementary table S1).

We show that the success rate of skin swabbing, in terms of obtaining an mtDNA barcode, is the same as for buccal swabbing. Skin swabs have a wider application than simple mtDNA barcoding. Both skin and buccal swabs have

been used to obtain nuclear DNA data, including microsatellite (Broquet et al., 2007; Prunier et al., 2012) and KASP genotyping (Fahrbach et al., 2021; de Brouwer et al., 2023). Buc cal swabs are also being applied in amphibians to obtain genome-scale datasets via RAD- sequencing (Ambu and Dufresnes, 2023) and we would recommend testing skin swabs for this purpose as well (while DNA concentrations of skin swabs are lower than those of buccal swabs, many should yield sufficient DNA for RAD-sequencing).

To the best of our knowledge, the stress associated with buccal and skin swabbing on amphibians has never been quantified. However, it is reasonable to assume that skin swabbing is relatively less stressful than buccal swabbing (Ringler, 2018). Our study illustrates the poten tial of the technique, particularly in projects on aquatic amphibians where conventional molec ular techniques are applied and citizen scientists are involved.

**Acknowledgements.** Many citizen scientists, all part of RAVON's network of volunteers that conduct monitoring, took skin swabs for us (supplementary table S5). Permits for fieldwork are listed in supplementary table S6. Dutch land owner organizations that provided field permits are Het Flevo-landschap, Geldersch Landschap & Kasteelen, Land goed de Utrecht, Natuurmonumenten (Society for Preserva tion of Nature Monuments in the Netherlands) and Staatsbosbeheer (State Forestry). Land owners in Wales (UK) pro vided permission for pond surveys and collection of tissue samples. Thanks also go to the National Parks and Wildlife Service (NPWS) Ireland and Northern Ireland Environment Agency for permissions. Apenheul Primate Park, Cale donian Conservation, Hortus Botanicus Leiden, Societas herpetologica Slovenica, Wiesław Babik, Sergé Bogaerts, David O'Brien, Ivona Burić, David Eastwood, Michael Fahrbach, Alberto Gosá, Ana Ivanović, Robert Jehle, Fairlie Kirkpatrick, Đurdica Majetić, Saša Marić, Helena Martínez, Alfredo G. Nicieza, Radostina Pravcheva, Gregorio Sánchez Montes, Krisztián Szabó, Judit Vörös and Piotr Zieliński helped to obtain samples. Belgium DNA extractions were provided by the Research Institute for Nature and For est (INBO). DS was partially supported by the Slove nian Research Agency (P1-0255). MS was funded by the National Research, Development and Innovation Office of Hungary (NKFIH-OTKA K134391). MON & AT, were sup

Downloaded from britl.com 04/16/2024 12:28:35PM

ported by an Erasmus+ internship

distributed under the terms
**Supplementary material.** Supplementary material is available online at:
https://doi.org/10.6084/m9.fishshare.23308559

## References

Allain, S.J.R., Lynn, V.J. (2021): Distribution of the alpine newt *Ichthyosaura alpestris* in Great Britain updated using social media. Herpetol. Bull. **158**: 28-31.

Allendorf, F.W., Hohenlohe, P.A., Luikart, G. (2010): Genomics and the future of conservation genetics. Nat. Rev. Genet. **11**: 697-709.

Ambu, J., Dufresnes, C. (2023): Buccal swabs for amphibian genomics. Amphib.-Reptil. **44**: 249-255.

Ambu, J., Martínez-Solano, I., Suchan, T., Hernandez, A., Wielstra, B., Crochet, P.-A., Dufresnes, C. (submitted): Genomic phylogeography illuminates deep cyto-nuclear discordances in midwife toads (*Alytes*). Mol. Phylogenet. Evol. **183**: 107783.

Arévalo, E., Davis, S.K., Sites, J.W., Jr. (1994): Mitochondrial DNA sequence divergence and phylogenetic relationships among eight chromosomous races of *Sceloporus grammicus* complex (Phrynosomatidae) in central Mexico. Syst. Biol. **43**: 387-418.

Arntzen, J.W., Smithson, A., Oldham, R.S. (1999): Marking and tissue sampling effects on body condition and survival in the newt *Triturus cristatus*. J. Herpetol **33**: 567-576.

Arntzen, J.W., King, T.M., Denoël, M., Martínez-Solano, I., Wallis, G.P. (2016): Provenance of *Ichthyosaura alpestris* (Caudata: Salamandridae) introductions to France and New Zealand assessed by mitochondrial DNA analysis. Herpetol. J. **26**: 49-56.

Avise, J.C. (2000): Phylogeography: the History and Formation of Species. Harvard University Press, Cambridge, Massachusetts.

Babik, W., Branicki, W., Crnobrnja-Isaïlovic, J., Cogalniceanu, D., Sas, I., Olgun, K., Poyarkov, N.A., Garcia-Paris, M., Arntzen, J.W. (2005): Phylogeography of two European newt species – discordance between mtDNA and morphology. Mol. Ecol. **14**: 2475-2491.

Bellard, C., Cassey, P., Blackburn, T.M. (2016): Alien species as a driver of recent extinctions. Biol. Lett. **12**: 20150623.

Bonney, R., Phillips, T.B., Ballard, H.L., Enck, J.W. (2016): Can citizen science enhance public understanding of science? Public Underst. Sci. **25**: 2-16.

Broquet, T., Berset-Braendli, L., Emaresi, G., Fumagalli, L. (2007): Buccal swabs allow efficient and reliable microsatellite genotyping in amphibians. Conserv. Genet. **8**: 509-511.

Chiocchio, A., Bisconti, R., Zampiglia, M., Nascetti, G., Canestrelli, D. (2017): Quaternary history, population genetic structure and diversity of the cold-adapted alpine newt *Ichthyosaura alpestris* in Peninsular Italy. Sci. Rep. **7**: 2955.

Creemers, R., van Delft, J.J.C.W. (2009): De amfibieën en reptielen van Nederland. In: Nederlandse Fauna 9. Nationaal Natuurhistorisch Museum Naturalis & European Invertebrate Survey, Leiden.

de Brouwer, J., Helder, B., France, J., de Visser, M.C., Struijk, R.P.J.H., Wielstra, B. (2023): An isolated crested newt population in Dutch coastal dunes: distribution relict or introduction? Amphib.-Reptil. **44**: 19-26.

Devictor, V., Whittaker, R.J., Beltrame, C. (2010): Beyond scarcity: citizen science programmes as useful tools for conservation biogeography. Divers. Distrib. **16**: 354-362.

Diagne, C., Leroy, B., Vaissière, A.-C., Gozlan, R.E., Roiz, D., Jarić, I., Salles, J.-M., Bradshaw, C.J.A., Courchamp, F. (2021): High and rising economic costs of biological invasions worldwide. Nature **592**: 571-576.

Dufresnes, C., Di Santo, L., Leuenberger, J., Schuerch, J., Mazepa, G., Grandjean, N., Canestrelli, D., Perrin, N., Dubey, S. (2017): Cryptic invasion of Italian pool frogs (*Pelophylax bergeri*) across western Europe unraveled by multilocus phylogeography. Biol. Invasions **19**: 1407-1420.

Dufresnes, C., Berroneau, M., Dubey, S., Litvinchuk, S.N., Perrin, N. (2020): The effect of phylogeographic history on species boundaries: a comparative framework in *Hyla* tree frogs. Sci. Rep. **10**: 5502.

Dufresnes, C., Jablonski, D. (2022): A genomics revolution in amphibian taxonomy. Science **377**: 1272.

Džukić, G., Cvijanović, M., Urošević, A., Vukov, T.D., Tomašević Kolarov, N., Slijepčević, M., Ivanović, A., Kalezić, M.L. (2015): The batrachological collections of the institute for biological research "Siniša Stanković", University of Belgrade. Bulletin of the Natural History Museum **8**: 118-167.

Fahrbach, M., de Visser, M.C., Wielstra, B. (2021): The hybrid zone between the Italian and northern crested newts (*Triturus carnifex* and *T. cristatus*) reaches Germany. Salamandra **57**: 428-434.

Fibla, M., Ubach, A., Oromí, N., Montero-Mendieta, S., Camarasa Arbós, S., Pascual, M., Martinez Silvestre, A., Montori, A. (2015): Población introducida de tritón alpino (*Mesotriton alpestris*) en el Prepirineo catalán. Boletín de la Asociación Herpetologica Espanola **26**: ms749.

Fijarczyk, A., Nadachowska, K., Hofman, S., Litvinchuk, S.N., Babik, W., Stuglik, M., Gollmann, G., Choleva, L., Cogălniceanu, D.A.N., Vukov, T., Džukić, G., Szymura, J.M. (2011): Nuclear and mitochondrial phylogeography of the European fire-bellied toads *Bombina bombina* and *Bombina variegata* supports their independent histories. Mol. Ecol. **20**: 3381-3398.

Frost, D.R. (2021): Amphibian Species of the World: an Online Reference. Version 6.1 (23 March 2021). Electronic Database accessible at http://research.amnh.org/herpetology/amphibia/index.html. American Museum of Natural History, New York, USA.

Funk, W.C., Donnelly, M.A., Lips, K.R. (2005): Alternative views of amphibian toe-clipping. Nature **433**: 193.

Gerlach, U., Fahrbach, M. (2016): Morphologie folgt Genetik: Ein Merkmal zur Unterscheidung zweier Kammmolcharten aus der Türkei. Terraria/elaphe **59**: 76.

Hebert, P.D.N., Cywinska, A., Ball, S.L., deWaard, J.R. (2003): Biological identifications through DNA barcodes. PLoS Soc. Lond. **B** **270**: 313-321.

via Open Access. This article is distributed under the terms of the CC BY 4.0 license.
**Supplementary material.** Supplementary material is available online at:
https://doi.org/10.6084/m9.fishshare.23308559

## References

Allain, S.J.R., Lynn, V.J. (2021): Distribution of the alpine newt *Ichthyosaura alpestris* in Great Britain updated using social media. Herpetol. Bull. **158**: 28-31.

Allendorf, F.W., Hohenlohe, P.A., Luikart, G. (2010): Genomics and the future of conservation genetics. Nat. Rev. Genet. **11**: 697-709.

Ambu, J., Dufresnes, C. (2023): Buccal swabs for amphibian genomics. Amphib.-Reptil. **44**: 249-255.

Ambu, J., Martínez-Solano, I., Suchan, T., Hernandez, A., Wielstra, B., Crochet, P.-A., Dufresnes, C. (submitted): Genomic phylogeography illuminates deep cyto-nuclear discordances in midwife toads (*Alytes*). Mol. Phylogenet. Evol. **183**: 107783.

Arévalo, E., Davis, S.K., Sites, J.W., Jr. (1994): Mitochondrial DNA sequence divergence and phylogenetic relationships among eight chromosomous races of *Sceloporus grammicus* complex (Phrynosomatidae) in central Mexico. Syst. Biol. **43**: 387-418.

Arntzen, J.W., Smithson, A., Oldham, R.S. (1999): Marking and tissue sampling effects on body condition and survival in the newt *Triturus cristatus*. J. Herpetol **33**: 567-576.

Arntzen, J.W., King, T.M., Denoël, M., Martínez-Solano, I., Wallis, G.P. (2016): Provenance of *Ichthyosaura alpestris* (Caudata: Salamandridae) introductions to France and New Zealand assessed by mitochondrial DNA analysis. Herpetol. J. **26**: 49-56.

Avise, J.C. (2000): Phylogeography: the History and Formation of Species. Harvard University Press, Cambridge, Massachusetts.

Babik, W., Branicki, W., Crnobrnja-Isaïlovic, J., Cogalniceanu, D., Sas, I., Olgun, K., Poyarkov, N.A., Garcia-Paris, M., Arntzen, J.W. (2005): Phylogeography of two European newt species – discordance between mtDNA and morphology. Mol. Ecol. **14**: 2475-2491.

Bellard, C., Cassey, P., Blackburn, T.M. (2016): Alien species as a driver of recent extinctions. Biol. Lett. **12**: 20150623.

Bonney, R., Phillips, T.B., Ballard, H.L., Enck, J.W. (2016): Can citizen science enhance public understanding of science? Public Underst. Sci. **25**: 2-16.

Broquet, T., Berset-Braendli, L., Emaresi, G., Fumagalli, L. (2007): Buccal swabs allow efficient and reliable microsatellite genotyping in amphibians. Conserv. Genet. **8**: 509-511.

Chiocchio, A., Bisconti, R., Zampiglia, M., Nascetti, G., Canestrelli, D. (2017): Quaternary history, population genetic structure and diversity of the cold-adapted alpine newt *Ichthyosaura alpestris* in Peninsular Italy. Sci. Rep. **7**: 2955.

Creemers, R., van Delft, J.J.C.W. (2009): De amfibieën en reptielen van Nederland. In: Nederlandse Fauna 9. Nationaal Natuurhistorisch Museum Naturalis & European Invertebrate Survey, Leiden.

de Brouwer, J., Helder, B., France, J., de Visser, M.C., Struijk, R.P.J.H., Wielstra, B. (2023): An isolated crested newt population in Dutch coastal dunes: distribution relict or introduction? Amphib.-Reptil. **44**: 19-26.

Devictor, V., Whittaker, R.J., Beltrame, C. (2010): Beyond scarcity: citizen science programmes as useful tools for conservation biogeography. Divers. Distrib. **16**: 354-362.

Diagne, C., Leroy, B., Vaissière, A.-C., Gozlan, R.E., Roiz, D., Jarić, I., Salles, J.-M., Bradshaw, C.J.A., Courchamp, F. (2021): High and rising economic costs of biological invasions worldwide. Nature **592**: 571-576.

Dufresnes, C., Di Santo, L., Leuenberger, J., Schuerch, J., Mazepa, G., Grandjean, N., Canestrelli, D., Perrin, N., Dubey, S. (2017): Cryptic invasion of Italian pool frogs (*Pelophylax bergeri*) across western Europe unraveled by multilocus phylogeography. Biol. Invasions **19**: 1407-1420.

Dufresnes, C., Berroneau, M., Dubey, S., Litvinchuk, S.N., Perrin, N. (2020): The effect of phylogeographic history on species boundaries: a comparative framework in *Hyla* tree frogs. Sci. Rep. **10**: 5502.

Dufresnes, C., Jablonski, D. (2022): A genomics revolution in amphibian taxonomy. Science **377**: 1272.

Džukić, G., Cvijanović, M., Urošević, A., Vukov, T.D., Tomašević Kolarov, N., Slijepčević, M., Ivanović, A., Kalezić, M.L. (2015): The batrachological collections of the institute for biological research "Siniša Stanković", University of Belgrade. Bulletin of the Natural History Museum **8**: 118-167.

Fahrbach, M., de Visser, M.C., Wielstra, B. (2021): The hybrid zone between the Italian and northern crested newts (*Triturus carnifex* and *T. cristatus*) reaches Germany. Salamandra **57**: 428-434.

Fibla, M., Ubach, A., Oromí, N., Montero-Mendieta, S., Camarasa Arbós, S., Pascual, M., Martinez Silvestre, A., Montori, A. (2015): Población introducida de tritón alpino (*Mesotriton alpestris*) en el Prepirineo catalán. Boletín de la Asociación Herpetologica Espanola **26**: ms749.

Fijarczyk, A., Nadachowska, K., Hofman, S., Litvinchuk, S.N., Babik, W., Stuglik, M., Gollmann, G., Choleva, L., Cogălniceanu, D.A.N., Vukov, T., Džukić, G., Szymura, J.M. (2011): Nuclear and mitochondrial phylogeography of the European fire-bellied toads *Bombina bombina* and *Bombina variegata* supports their independent histories. Mol. Ecol. **20**: 3381-3398.

Frost, D.R. (2021): Amphibian Species of the World: an Online Reference. Version 6.1 (23 March 2021). Electronic Database accessible at http://research.amnh.org/herpetology/amphibia/index.html. American Museum of Natural History, New York, USA.

Funk, W.C., Donnelly, M.A., Lips, K.R. (2005): Alternative views of amphibian toe-clipping. Nature **433**: 193.

Gerlach, U., Fahrbach, M. (2016): Morphologie folgt Genetik: Ein Merkmal zur Unterscheidung zweier Kammmolcharten aus der Türkei. Terraria/elaphe **59**: 76.

Hebert, P.D.N., Cywinska, A., Ball, S.L., deWaard, J.R. (2003): Biological identifications through DNA barcodes. PLoS Soc. Lond. **B** **270**: e13-21.

https://creativecommons.org/licenses/by/4.0/
Huxel, G.R. (1999): Rapid displacement of native species by invasive species: effects of hybridization. *Biol. Conserv.* **89**: 143-152.

Jakóbik, J., Janowski, P., Błażuk, J., Narczyński, T., Pabijan, M. (2019): An alpine newt (*Ichthyosaura alpestris*) population on the Baltic coast of Poland. *Herpetol. Notes* **12**: 923-930.

Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K.F., von Hae-seler, A., Jermiin, L.S. (2017): ModelFinder: fast model selection for accurate phylogenetic estimates. *Nat. Methods* **14**: 587-589.

Koster, S., Prins, N., Dufresnes, C., France, J., de Visser, M.C., Struijk, R.P.J.H., Wielstra, B. (2023): The conservation paradox of an introduced population of a threatened species: spadefoot toads in the coastal dunes of the Netherlands. *Amphib-Reptil.* **44**: 11-18.

Kuijt, M., Oskam, L., den Boer, I., Dufresnes, C., France, J., Gilbert, M., de Visser, M., Struijk, R.P.J.H., Wielstra, B. (2023): The introduction of three cryptic tree frog species in the Dutch coastal dunes challenges conservation paradigms. *Amphib.-Reptil.* **44**: 1-10.

Leigh, J.W., Bryant, D. (2015): Popart: full-feature software for haplotype network construction. *Methods Ecol. Evol.* **6**: 1110-1116.

McCarthy, M.A., Parris, K.M. (2004): Clarifying the effect of toe clipping on frogs with Bayesian statistics. *J. Appl. Ecol.* **41**: 780-786.

Meehan, S.T. (2013): IWT National Smooth Newt Survey 2013 report.

Meilink, W.R.M., Arntzen, J.W., van Delft, J.J.C.W., Wielstra, B. (2015): Genetic pollution of a threatened native crested newt species through hybridization with an invasive congener in the Netherlands. *Biol. Conserv.* **184**: 145-153.

Mir, R.A., Bhat, K.A., Rashid, G., Ebinezer, L.B., Masi, A., Rakwal, R., Shah, A.A., Zargar, S.M. (2021): DNA barcoding: a way forward to obtain deep insights about the realistic diversity of living organisms. The Nucleus **64**: 157-165.

Müller, A.S., Lenhardt, P.P., Theissinger, K. (2013): Pros and cons of external swabbing of amphibians for genetic analyses. *Eur. J. Wildl. Res.* **59**: 609-612.

Pabijan, M., Babik, W. (2006): Genetic structure in northeastern populations of the alpine newt (*Triturus alpestris*): evidence for post-Pleistocene differentiation. *Mol. Ecol.* **15**: 2397-2407.

Pabijan, M., Rozej, E., Bonk, M. (2009): An isolated locality of the alpine newt (*Mesotriton alpestris*) Laurenti, 1768) in central Poland. *Herpetol. Notes* **2**: 23-26.

Pabijan, M., Zieliński, P., Dudek, K., Stuglik, M., Babik, W. (2017): Isolation and gene flow in a speciation continuum in newts. *Mol. Phylogenet. Evol.* **116**: 1-12.

Palomar, G., Vörös, J., Bosch, J. (2017): Tracking the introduction history of *Ichthyosaura alpestris* in a protected area of central Spain. *Conserv. Genet.* **18**: 867-876.

Pidancier, N., Miquel, C., Miaud, C. (2003): Buccal swabs as a non-destructive tissue sampling method for DNA analysis in amphibians. *Herpetol. J.* **13**: 175-178.

Polich, R.L., Searcy, C.A., Shaffer, H.B. (2013): Effects of tail-clipping on survivorship and growth of larval salamanders. *J. Wildl. Manage.* **77**: 1420-1425.

Poschadel, J.R., Möller, D. (2004): A versatile field method for tissue sampling on small reptiles and amphibians, applied to pond turtles, newts, frogs and toads. *Conserv. Genet.* **5**: 865-867.

Prunier, J., Kaufmann, B., Grolet, O., Picard, D., Pompanon, F., Joly, P. (2012): Skin swabbing as a new efficient DNA sampling technique in amphibians, and 14 new microsatellite markers in the alpine newt (*Ichthyosaura alpestris*). *Mol. Ecol. Resour.* **12**: 524-531.

Pyšek, P., Hulme, P.E., Simberloff, D., Bacher, S., Blackburn, T.M., Carlton, J.T., Dawson, W., Essl, F., Foxcroft, L.C., Genovesi, P., Jeschke, J.M., Kühn, I., Liebhold, A.M., Mandrak, N.E., Meyerson, L.A., Pauchard, A., Pergl, J., Roy, H.E., Seebens, H., van Kleunen, M., Vilà, M., Wingfield, M.J., Richardson, D.M. (2020): Scientists' warning on invasive alien species. *Biol. Rev.* **95**: 1511-1534.

Raffaëlli, J. (2018): Proposal for a new taxonomic arrangement of *Ichthyosaura alpestris* (Laurenti, 1768) (Urodela, Salamandridae), an iconic species with a complex phylogenetic structure. *Alytes* **36**: 178-193.

Rambaut, A., Drummond, A.J., Xie, D., Baele, G., Suchard, M.A. (2018): Posterior summarization in Bayesian phylogenetics using tracer 1.7. *Syst. Biol.* **67**: 901-904.

Rancilhac, L., Irisarri, I., Angelini, C., Arntzen, J.W., Babik, W., Bossuyt, F., Künzel, S., Lüddecke, T., Pasmans, F., Sanchez, E., Weisrock, D., Veith, M., Wielstra, B., Steinfartz, S., Hofreiter, M., Philippe, H., Vences, M. (2021): Phylotranscriptomic evidence for pervasive ancient hybridization among Old World salamanders. *Mol. Phylogenet. Evol.* **155**: 106967.

Recuero, E., Buckley, D., García-París, M., Arntzen, J.W., Cogáñiceanu, D., Martínez-Solano, I. (2014): Evolutionary history of *Ichthyosaura alpestris* (Caudata, Salamandridae) inferred from the combined analysis of nuclear and mitochondrial markers. *Mol. Phylogenet. Evol.* **81**: 207-220.

Rhymer, J.M., Simberloff, D. (1996): Extinction by hybridization and introgression. *Annu. Rev. Ecol. Syst.* **27**: 83-109.

Ringler, E. (2018): Testing skin swabbing for DNA sampling in dendrobatid frogs. *Amphib.-Reptil.* **39**: 245-251.

Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A., Huelsenbeck, J.P. (2012): MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. *Syst. Biol.* **61**: 539-542.

Rozas, J., Ferrer-Mata, A., Sánchez-DelBarrio, J.C., Guirao-Rico, S., Librado, P., Ramos-Onsins, S.E., Sánchez-Gracia, A. (2017): DnaSP 6: DNA sequence polymorphism analysis of large data sets. *Mol. Biol. Evol.* **34**: 3299-3302.

Schmeller, D.S., Utzel, R., Pasmans, F., Martel, A. (2020): *Batrachochytrium salamandrivorans* kills alpine newts (*Ichthyosaura alpestris*) in southernmost Germany. *Salamandra* **56**: 230-232.

Sillero, N., Campos, J., Bonardi, A., Corti, C., Creemers, R., Crochet, P.-A., Crnobrnja Išailović, J., Denoël, M., Ficella, G., Fègolalves, J., Kuzmin, S., Lymerakis, P. (2019): Downloaded from www.biol.berkeley.edu at 04/16/2023 12:28:35PM. This is an unopenable version of the CC BY 4.0 license.
Huxel, G.R. (1999): Rapid displacement of native species by invasive species: effects of hybridization. *Biol. Conserv.* **89**: 143-152.

Jakóbik, J., Janowski, P., Błażuk, J., Narczyński, T., Pabijan, M. (2019): An alpine newt (*Ichthyosaura alpestris*) population on the Baltic coast of Poland. *Herpetol. Notes* **12**: 923-930.

Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K.F., von Haepler, A., Jermiin, L.S. (2017): ModelFinder: fast model selection for accurate phylogenetic estimates. *Nat. Methods* **14**: 587-589.

Koster, S., Prins, N., Dufresnes, C., France, J., de Visser, M.C., Struijk, R.P.J.H., Wielstra, B. (2023): The conservation paradox of an introduced population of a threatened species: spadefoot toads in the coastal dunes of the Netherlands. *Amphib-Reptil.* **44**: 11-18.

Kuijt, M., Oskam, L., den Boer, I., Dufresnes, C., France, J., Gilbert, M., de Visser, M., Struijk, R.P.J.H., Wielstra, B. (2023): The introduction of three cryptic tree frog species in the Dutch coastal dunes challenges conservation paradigms. *Amphib.-Reptil.* **44**: 1-10.

Leigh, J.W., Bryant, D. (2015): Popart: full-feature software for haplotype network construction. *Methods Ecol. Evol.* **6**: 1110-1116.

McCarthy, M.A., Parris, K.M. (2004): Clarifying the effect of toe clipping on frogs with Bayesian statistics. *J. Appl. Ecol.* **41**: 780-786.

Meehan, S.T. (2013): IWT National Smooth Newt Survey 2013 report.

Meilink, W.R.M., Arntzen, J.W., van Delft, J.J.C.W., Wielstra, B. (2015): Genetic pollution of a threatened native crested newt species through hybridization with an invasive congener in the Netherlands. *Biol. Conserv.* **184**: 145-153.

Mir, R.A., Bhat, K.A., Rashid, G., Ebinezer, L.B., Masi, A., Rakwal, R., Shah, A.A., Zargar, S.M. (2021): DNA barcoding: a way forward to obtain deep insights about the realistic diversity of living organisms. The Nucleus **64**: 157-165.

Müller, A.S., Lenhardt, P.P., Theissinger, K. (2013): Pros and cons of external swabbing of amphibians for genetic analyses. *Eur. J. Wildl. Res.* **59**: 609-612.

Pabijan, M., Babik, W. (2006): Genetic structure in northeastern populations of the alpine newt (*Triturus alpestris*): evidence for post-Pleistocene differentiation. *Mol. Ecol.* **15**: 2397-2407.

Pabijan, M., Rozej, E., Bonk, M. (2009): An isolated locality of the alpine newt (*Mesotriton alpestris*) Laurenti, 1768) in central Poland. *Herpetol. Notes* **2**: 23-26.

Pabijan, M., Zieliński, P., Dudek, K., Stuglik, M., Babik, W. (2017): Isolation and gene flow in a speciation continuum in newts. *Mol. Phylogenet. Evol.* **116**: 1-12.

Palomar, G., Vörös, J., Bosch, J. (2017): Tracking the introduction history of *Ichthyosaura alpestris* in a protected area of central Spain. *Conserv. Genet.* **18**: 867-876.

Pidancier, N., Miquel, C., Miaud, C. (2003): Buccal swabs as a non-destructive tissue sampling method for DNA analysis in amphibians. *Herpetol. J.* **13**: 175-178.

Polich, R.L., Searcy, C.A., Shaffer, H.B. (2013): Effects of tail-clipping on survivorship and growth of larval salamanders. *J. Wildl. Manage.* **77**: 1420-1425.

Poschadel, J.R., Möller, D. (2004): A versatile field method for tissue sampling on small reptiles and amphibians, applied to pond turtles, newts, frogs and toads. *Conserv. Genet.* **5**: 865-867.

Prunier, J., Kaufmann, B., Grolet, O., Picard, D., Pompanon, F., Joly, P. (2012): Skin swabbing as a new efficient DNA sampling technique in amphibians, and 14 new microsatellite markers in the alpine newt (*Ichthyosaura alpestris*). *Mol. Ecol. Resour.* **12**: 524-531.

Pyšek, P., Hulme, P.E., Simberloff, D., Bacher, S., Blackburn, T.M., Carlton, J.T., Dawson, W., Essl, F., Foxcroft, L.C., Genovesi, P., Jeschke, J.M., Kühn, I., Liebhold, A.M., Mandrak, N.E., Meyerson, L.A., Pauchard, A., Pergl, J., Roy, H.E., Seebens, H., van Kleunen, M., Vilà, M., Wingfield, M.J., Richardson, D.M. (2020): Scientists' warning on invasive alien species. *Biol. Rev.* **95**: 1511-1534.

Raffaëlli, J. (2018): Proposal for a new taxonomic arrangement of *Ichthyosaura alpestris* (Laurenti, 1768) (*Urodela, Salamandridae*), an iconic species with a complex phylogenetic structure. *Alytes* **36**: 178-193.

Rambaut, A., Drummond, A.J., Xie, D., Baele, G., Suchard, M.A. (2018): Posterior summarization in Bayesian phylogenetics using tracer 1.7. *Syst. Biol.* **67**: 901-904.

Rancilhac, L., Irisarri, I., Angelini, C., Arntzen, J.W., Babik, W., Bossuyt, F., Künzel, S., Lüddecke, T., Pasmans, F., Sanchez, E., Weisrock, D., Veith, M., Wielstra, B., Steinfartz, S., Hofreiter, M., Philippe, H., Vences, M. (2021): Phylotranscriptomic evidence for pervasive ancient hybridization among Old World salamanders. *Mol. Phylogenet. Evol.* **155**: 106967.

Recuero, E., Buckley, D., García-París, M., Arntzen, J.W., Cogáñiceanu, D., Martínez-Solano, I. (2014): Evolutionary history of *Ichthyosaura alpestris* (Caudata, Salamandridae) inferred from the combined analysis of nuclear and mitochondrial markers. *Mol. Phylogenet. Evol.* **81**: 207-220.

Rhymer, J.M., Simberloff, D. (1996): Extinction by hybridization and introgression. *Annu. Rev. Ecol. Syst.* **27**: 83-109.

Ringler, E. (2018): Testing skin swabbing for DNA sampling in dendrobatid frogs. *Amphib.-Reptil.* **39**: 245-251.

Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A., Huelsenbeck, J.P. (2012): MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. *Syst. Biol.* **61**: 539-542.

Rozas, J., Ferrer-Mata, A., Sánchez-DelBarrio, J.C., Guirao-Rico, S., Librado, P., Ramos-Onsins, S.E., Sánchez-Gracia, A. (2017): DnaSP 6: DNA sequence polymorphism analysis of large data sets. *Mol. Biol. Evol.* **34**: 3299-3302.

Schmeller, D.S., Utzel, R., Pasmans, F., Martel, A. (2020): *Batrachochytrium salamandrivorans* kills alpine newts (*Ichthyosaura alpestris*) in southernmost Germany. *Salamandra* **56**: 230-232.

Sillero, N., Campos, J., Bonardi, A., Corti, C., Creemers, R., Crochet, P.-A., Crnobrnja Išailović, J., Denoël, M., Ficella, G., Fègolalves, J., Kuzmin, S., Lymerakis, P. (2019). Downloaded from www.cambridge.org by 04/16/2024 at 12:28:35PM terms of the CC BY 4.0 license.
de Pous, P., Rodríguez, A., Sindaco, R., Speybroeck, J., Toxopeus, B., Vieites, D.R., Vences, M. (2014): Updated distribution and biogeography of amphibians and reptiles of Europe. Amphib.-Reptil. **35**: 1-31.

Simberloff, D. (2013): Invasive Species: What Everyone Needs to Know. Oxford University Press.

Sotiropoulos, K., Eleftherakos, K., Dzukic, G., Kaiezic, M.L., Legakis, A., Polymeni, R.M. (2007): Phylogeny and biogeography of the alpine newt *Mesotriton alpestris* (Salamandridae, Caudata), inferred from mtDNA sequences. Mol. Phylogenet. Evol. **45**: 211-226.

Speybroeck, J., Beukema, W., Dufresnes, C., Fritz, U., Jablonski, D., Lymberakis, P., Martínez-Solano, I., Razzetti, E., Vamberger, M., Vences, M., Vörös, J., Crochet, P.-A. (2020): Species list of the European herpetofauna – 2020 update by the taxonomic committee of the societas europaea herpetologica. Amphib.-Reptil. **41**: 139-189.

Šunje, E., Stroil, B.K., Raffaëlli, J., Zimić, A., Marquis, O. (2021): A revised phylogeny of alpine newts unravels the evolutionary distinctiveness of the Bosnian alpine newt – *Ichthyosaura alpestris reiseri* (Werner, 1902). Amphib.-Reptil. **42**: 481-490.

Todesco, M., Pascual, M.A., Owens, G.L., Ostevik, K.L., Moyers, B.T., Hübner, S., Heredia, S.M., Hahn, M.A., Caseys, C., Bock, D.G., Rieseberg, L.H. (2016): Hybridization and extinction. Evol. Appl. **9**: 892-908.

Toews, D.P.L., Brelsford, A. (2012): The biogeography of mitochondrial and nuclear discordance in animals. Mol. Ecol. **21**: 3907-3930.

Trifinopoulos, J., Nguyen, L.-T., von Haeseler, A., Minh, B.Q. (2016): W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. **44**: W232-W235.

Üzüm, N., Avci, A., Olgun, K., Bülbül, U., Fahrbach, M., Litvinchuk, S.N., Wielstra, B. (2019): Cracking cryptic species: external characters to distinguish two recently recognized banded newt species (*Ommatotriton ophyrticus* and *O. nesterovi*). Salamandra **55**: 131-134.

van Riemsdijk, I., Arntzen, J.W., Bogaerts, S., Franzen, M., Litvinchuk, S.N., Olgun, K., Wielstra, B. (2017): The Near East as a cradle of biodiversity: a phylogeography of banded newts (genus *Ommatotriton*) reveals extensive inter-and intraspecific genetic differentiation. Mol. Phylogenet. Evol. **114**: 73-81.

van Riemsdijk, I., van Nieuwenhuize, L., Martínez-Solano, I., Arntzen, J.W., Wielstra, B. (2018): Molecular data reveal the hybrid nature of an introduced population of banded newts (*Ommatotriton*) in Spain. Conserv. Genet. **19**: 249-254.

Vences, M., Susanne Hauswaldt, J., Steinfartz, S., Rupp, O., Goesmann, A., Künzel, S., Orozco-terWengel, P., Vieites, D.R., Nieto-Roman, S., Haas, S., Laugsch, C., Gehara, M., Bruchmann, S., Pabijan, M., Ludewig, A.-K., Rudert, D., Angelini, C., Borkin, L.J., Crochet, P.-A., Crottini, A., Dubois, A., Ficetola, G.F., Galán, P., Geniez, P., Hachtel, M., Jovanovic, O., Litvinchuk, S.N.,

Lymberakis, P., Ohler, A., Smirnov, N.A. (2013): Radi-cally different phylogeographies and patterns of genetic variation in two European brown frogs, genus *Rana*. Mol. Phylogenet. Evol. **68**: 657-670.

Villesen, P. (2007): FaBox: an online toolbox for fasta sequences. Mol. Ecol. Notes **7**: 965-968.

Vliegenthart, C., van de Vrede, M., den Boer, I., Gilbert, M., Lemmers, P., France, J., de Visser, M., Struijk, R.P.J.H., Wielstra, B. (2023): The limits of mtDNA analysis for determining the provenance of invasive species: a mid-wife toad example. Amphib.-Reptil. **44**: 27-33.

Vörös, J., Varga, Z., Martínez-Solano, I., Szabó, K. (2021): Mitochondrial DNA diversity of the alpine newt (*Ichthyosaura alpestris*) in the Carpathian basin: evidence for multiple cryptic lineages associated with Pleistocene refugia. Acta Zool. Acad. Sci. Hung. **67**: 177-197.

Ward, A., Hide, G., Jehle, R. (2019): Skin swabs with FTA® cards as a dry storage source for amphibian DNA. Conserv. Genet. Resour. **11**: 309-311.

Wielstra, B., Beukema, W., Arntzen, J.W., Skidmore, A.K., Toxopeus, A.G., Raes, N. (2012): Corresponding mitochondrial DNA and niche divergence for crested newt candidate species. PLoS ONE **7**: e46671.

Wielstra, B., Crnobrnja-Isailović, J., Litvinchuk, S.N., Reijnen, B.T., Skidmore, A.K., Sotiropoulis, K., Toxopeus, A.G., Tzankov, N., Vukov, T., Arntzen, J.W. (2013): Tracing glacial refugia of *Triturus* newts based on mitochondrial DNA phylogeography and species distribution modeling. Front. Zool. **10**: 13.

Wielstra, B., Arntzen, J.W. (2016): Description of a new species of crested newt, previously subsumed in *Triturus ivanbureschi* (Amphibia: Caudata: Salamandridae). Zootaxa **4109**: 73-80.

Wielstra, B., Burke, T., Butlin, R.K., Schaap, O., Shaf-fer, H.B., Vrieling, K., Arntzen, J.W. (2016): Efficient screening for ‘genetic pollution’ in an anthropogenic crested newt hybrid zone. Conserv. Genet. Resour. **8**: 553-560.

Wielstra, B., Canestrelli, D., Cvikjanović, M., Denoël, M., Fijarczyk, A., Jablonski, D., Liana, M., Naumov, B., Olgun, K., Pabijan, M., Pezzarossa, A., Popgeorgiev, G., Salvi, D., Si, Y., Sillero, N., Sotiropoulos, K., Zieliński, P., Babik, W. (2018): The distributions of the six species constituting the smooth newt species complex (*Lissotriton vulgaris* sensu lato and *L. montandoni*) – an addition to the new atlas of amphibians and reptiles of Europe. Amphib.-Reptil. **39**: 252-259.

Zemanova, M.A. (2019): Poor implementation of non-invasive sampling in wildlife genetics studies. Rethink. Ecol. **4**: 119-132.

Zhang, P., Papenfuss, T.J., Wake, M.H., Qu, L.H., Wake, D.B. (2008): Phylogeny and biogeography of the family Salamandridae (Amphibia: Caudata) inferred from complete mitochondrial genomes. Mol. Phylogenet. Evol. **49**: 586-597.
de Pous, P., Rodríguez, A., Sindaco, R., Speybroeck, J., Toxopeus, B., Vieites, D.R., Vences, M. (2014): Updated distribution and biogeography of amphibians and reptiles of Europe. Amphib.-Reptil. **35**: 1-31.

Simberloff, D. (2013): Invasive Species: What Everyone Needs to Know. Oxford University Press.

Sotiropoulos, K., Eleftherakos, K., Dzukic, G., Kaiezic, M.L., Legakis, A., Polymeni, R.M. (2007): Phylogeny and biogeography of the alpine newt *Mesotriton alpestris* (Salamandridae, Caudata), inferred from mtDNA sequences. Mol. Phylogenet. Evol. **45**: 211-226.

Speybroeck, J., Beukema, W., Dufresnes, C., Fritz, U., Jablonski, D., Lymberakis, P., Martínez-Solano, I., Razzetti, E., Vamberger, M., Vences, M., Vörös, J., Crochet, P.-A. (2020): Species list of the European herpetofauna – 2020 update by the taxonomic committee of the societas europaea herpetologica. Amphib.-Reptil. **41**: 139-189.

Šunje, E., Stroil, B.K., Raffaëlli, J., Zimić, A., Marquis, O. (2021): A revised phylogeny of alpine newts unravels the evolutionary distinctiveness of the Bosnian alpine newt – *Ichthyosaura alpestris reiseri* (Werner, 1902). Amphib.-Reptil. **42**: 481-490.

Todesco, M., Pascual, M.A., Owens, G.L., Ostevik, K.L., Moyers, B.T., Hübner, S., Heredia, S.M., Hahn, M.A., Caseys, C., Bock, D.G., Rieseberg, L.H. (2016): Hybridization and extinction. Evol. Appl. **9**: 892-908.

Toews, D.P.L., Brelsford, A. (2012): The biogeography of mitochondrial and nuclear discordance in animals. Mol. Ecol. **21**: 3907-3930.

Trifinopoulos, J., Nguyen, L.-T., von Haeseler, A., Minh, B.Q. (2016): W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. **44**: W232-W235.

Üzüm, N., Avci, A., Olgun, K., Bülbül, U., Fahrbach, M., Litvinchuk, S.N., Wielstra, B. (2019): Cracking cryptic species: external characters to distinguish two recently recognized banded newt species (*Ommatotriton ophyrticus* and *O. nesterovi*). Salamandra **55**: 131-134.

van Riemsdijk, I., Arntzen, J.W., Bogaerts, S., Franzen, M., Litvinchuk, S.N., Olgun, K., Wielstra, B. (2017): The Near East as a cradle of biodiversity: a phylogeography of banded newts (genus *Ommatotriton*) reveals extensive inter-and intraspecific genetic differentiation. Mol. Phylogenet. Evol. **114**: 73-81.

van Riemsdijk, I., van Nieuwenhuize, L., Martínez-Solano, I., Arntzen, J.W., Wielstra, B. (2018): Molecular data reveal the hybrid nature of an introduced population of banded newts (*Ommatotriton*) in Spain. Conserv. Genet. **19**: 249-254.

Vences, M., Susanne Hauswaldt, J., Steinfartz, S., Rupp, O., Goesmann, A., Künzel, S., Orozco-terWengel, P., Vieites, D.R., Nieto-Roman, S., Haas, S., Laugsch, C., Gehara, M., Bruchmann, S., Pabijan, M., Ludewig, A.-K., Rudert, D., Angelini, C., Borkin, L.J., Crochet, P.-A., Crottini, A., Dubois, A., Ficetola, G.F., Galán, P., Geniez, P., Hachtel, M., Jovanovic, O., Litvinchuk, S.N.,

Lymberakis, P., Ohler, A., Smirnov, N.A. (2013): Radi-cally different phylogeographies and patterns of genetic variation in two European brown frogs, genus *Rana*. Mol. Phylogenet. Evol. **68**: 657-670.

Villesen, P. (2007): FaBox: an online toolbox for fasta sequences. Mol. Ecol. Notes **7**: 965-968.

Vliegenthart, C., van de Vrede, M., den Boer, I., Gilbert, M., Lemmers, P., France, J., de Visser, M., Struijk, R.P.J.H., Wielstra, B. (2023): The limits of mtDNA analysis for determining the provenance of invasive species: a mid-wife toad example. Amphib.-Reptil. **44**: 27-33.

Vörös, J., Varga, Z., Martínez-Solano, I., Szabó, K. (2021): Mitochondrial DNA diversity of the alpine newt (*Ichthyosaura alpestris*) in the Carpathian basin: evidence for multiple cryptic lineages associated with Pleistocene refugia. Acta Zool. Acad. Sci. Hung. **67**: 177-197.

Ward, A., Hide, G., Jehle, R. (2019): Skin swabs with FTA® cards as a dry storage source for amphibian DNA. Conserv. Genet. Resour. **11**: 309-311.

Wielstra, B., Beukema, W., Arntzen, J.W., Skidmore, A.K., Toxopeus, A.G., Raes, N. (2012): Corresponding mitochondrial DNA and niche divergence for crested newt candidate species. PLoS ONE **7**: e46671.

Wielstra, B., Crnobrnja-Isailović, J., Litvinchuk, S.N., Reijnen, B.T., Skidmore, A.K., Sotiropoulis, K., Toxopeus, A.G., Tzankov, N., Vukov, T., Arntzen, J.W. (2013): Tracing glacial refugia of *Triturus* newts based on mitochondrial DNA phylogeography and species distribution modeling. Front. Zool. **10**: 13.

Wielstra, B., Arntzen, J.W. (2016): Description of a new species of crested newt, previously subsumed in *Triturus ivanbureschi* (Amphibia: Caudata: Salamandridae). Zootaxa **4109**: 73-80.

Wielstra, B., Burke, T., Butlin, R.K., Schaap, O., Shaffer, H.B., Vrieling, K., Arntzen, J.W. (2016): Efficient screening for 'genetic pollution' in an anthropogenic crested newt hybrid zone. Conserv. Genet. Resour. **8**: 553-560.

Wielstra, B., Canestrelli, D., Cvikjanović, M., Denoël, M., Fijarczyk, A., Jablonski, D., Liana, M., Naumov, B., Olgun, K., Pabijan, M., Pezzarossa, A., Popgeorgiev, G., Salvi, D., Si, Y., Sillero, N., Sotiropoulos, K., Zieliński, P., Babik, W. (2018): The distributions of the six species constituting the smooth newt species complex (*Lissotriton vulgaris* sensu lato and *L. montandoni*) – an addition to the new atlas of amphibians and reptiles of Europe. Amphib.-Reptil. **39**: 252-259.

Zemanova, M.A. (2019): Poor implementation of non-invasive sampling in wildlife genetics studies. Rethink. Ecol. **4**: 119-132.

Zhang, P., Papenfuss, T.J., Wake, M.H., Qu, L.H., Wake, D.B. (2008): Phylogeny and biogeography of the family Salamandridae (Amphibia: Caudata) inferred from complete mitochondrial genomes. Mol. Phylogenet. Evol. **49**: 586-597.