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An Alpine Newt (Ichthyosaura Alpestris) Population on the Baltic Coast of Poland

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An Alpine Newt (Ichthyosaura Alpestris) Population on the Baltic Coast of Poland Herpetology Notes, volume 12: 923-930 (2019) (published online on 23 September 2019) An Alpine newt (Ichthyosaura alpestris) population on the Baltic coast of Poland Joanna Jakóbik1, Paweł Janowski2, Jacek Błażuk3, Tomasz Narczyński4, and Maciej Pabijan1,* Abstract. This study describes a new locality of the Alpine newt located on the Baltic coast of northern Poland, establishing the most northeastern population of this species in Europe. The population inhabits the Tricity Landscape Park (TLP, Trójmiejski Park Krajobrazowy) and is isolated from the main part of the range of this species by about 350 km. By sequencing a fragment of the mitochondrial ND4 gene and incorporating data from recent phylogenetic analyses, we restricted the potential origin of newts from TLP to populations carrying a specific variant of the C3 lineage present in northwestern and central Europe. More precise inference of the source population is hindered by low genetic differentiation within the C3 lineage. We suggest that a human-mediated XXth century introduction (purposeful or accidental) could have established the Alpine newt in TLP, although historical information of the release is currently unavailable. Alternatively, the TLP population may be a relict of a regressing range border due late Holocene climatic shifts and deforestation in the southeastern Baltic region. Keywords. urodele, patchy distribution, introduction, mtDNA Introduction Sura, 2018). Small and isolated localities occur in the highlands of south-central Poland (Pabijan et al., 2009; The Alpine newt, Ichthyosaura alpestris (Laurenti, Kurek and Święciak, 2010) and scattered lowland sites 1768), has a broad distribution encompassing most close to the continuous range in southwestern Poland of western and central Europe, the Balkan Peninsula (Rychła et al., 2002; Najbar et al., 2005). and parts of Italy and Spain (Sillero et al., 2014). Three major mitochondrial DNA (mtDNA) lineages The nominate subspecies is present in western and occur across the range of the Alpine newt (Fig.1; central parts of the continent, while genetically and Sotiroupoulos et al., 2007; Recuero et al., 2014), two morphologically distinct subspecies and lineages occur of these (western and eastern lineages) show further in the southern parts of the distribution (Roček et al., subdivisions in the southern European peninsulas. 2003; Sotiropoulos et al., 2007; Recuero et al., 2014). Western and central Europe, including Poland, are Introduced populations have been documented in Great populated by newts carrying the widespread C3 mtDNA Britain, Holland, central Spain, southern France and lineage (Sotiroupoulos et al., 2007; Recuero et al., 2014). New Zealand (Bell and Bell, 1995; Sillero et al., 2014; Despite limited mtDNA variation within C3, there is Arntzen et al., 2016; Palomar et al., 2017). In Poland, clear geographic substructure with two haplotypes this species is mostly limited to the Carpathians, Sudetes differing by two substitutions in the ND4 gene (Pabijan and Holy Cross mountain ranges and their foothills, and Babik, 2006). The western variant, termed C3-Sud in the southern part of the country (Głowaciński and hereafter, occurs in the Sudetes mountains of Poland and the Czech Republic and westwards into Germany, France and Austria, while the eastern haplotype, hereafter termed C3-Carp, is restricted to the Carpathian mountains of Poland, Ukraine and northern Romania (Fig. 1). 1 Department of Comparative Anatomy, Institute of Zoology Additional haplotypes with limited distributions, e.g. and Biomedical Research, Jagiellonian University, ul. from Hungary and France, occur locally across central Gronostajowa 9, 30-387 Kraków. and western Europe (Recuero et al., 2014; Arntzen et 2 Towarzystwo Herpetologiczne NATRIX. 3 ul. Opolska 10, 80-395 Gdańsk-Oliwa. al., 2016). The Moravian Gate, a depression situated 4 Polskie Towarzystwo Ochrony Przyrody „Salamandra”. between the Carpathian and Sudetes Mountains, divides * Corresponding author: [email protected] the distributions of haplotypes C3-Sud and C3-Carp and 924 Joanna Jakóbik et al. Figure 1. Maximum likelihood tree (lower left) of ND4 sequences of Alpine newts depicting major mtDNA lineages (asterisks on branches denote bootstrap support >70%). Haplotype network (upper left) showing nucleotide variation within the C3 mitochondrial lineage. Map shows the range of the Alpine newt with colors depicting the major mtDNA lineages. Haplotypes C3-Sud and C3-Carp are denoted by filled circles and triangles, respectively. Grey squares indicate introduced populations of unknown provenance; question marks denote areas in which major mtDNA lineage is in need of confirmation. Map based on Sotiropoulos et al. (2007), Recuero et al. (2014), Sillero et al. (2014) and Arntzen et al. (2016). probably constitutes a natural barrier to dispersal for this Material and Methods species since it is devoid of Alpine newt populations. Study area.—The Tricity Landscape Park is a mostly This mtDNA phylogeographic break is mirrored by the forested region in the Cassubian lakeland of north- existence of discreet evolutionary units of this species central Poland, close to the Baltic Sea. The young inhabiting the Carpathians and Sudetes as revealed by landscape consists of undulating hills with numerous nuclear DNA variation (Pabijan and Babik, 2006; Babik lakes and small water bodies created by the Weichselian et al., 2008) as well as allozyme (Pabijan et al., 2005) and glaciation. The cool, north-facing slopes and deep skin graft rejection data (Rafinski, 1974). These studies ravines are drained by numerous coldwater streams. also show that the isolated population in the Holy Cross Beech, oak and Scots pine predominate in the forests Mountains (southcentral Poland) is genetically distinct of this region. but most closely related to the Carpathian population. In 2016 several adult Alpine newts were unexpectedly Field methods.—Field surveys of 111 potential found in the Tricity Landscape Park (TLP; amphibian breeding sites were conducted in the spring Polish: Trójmiejski Park Krajobrazowy, German: and early summer of 2015-2016 with the aim of Landschaftsschutzpark Dreistadt), a protected area in obtaining an inventory of the batrachofauna of the TLP northern Poland close to the Baltic coast, adjacent to the (P. Janowski, unpublished). Most of the sites inhabited cities of Gdańsk, Gdynia and Sopot. This Alpine newt by Alpine newts were revisited in 2017 and 2018 to population is geographically distant from other localities confirm the presence of breeding adults and/or larvae of (about 350 km straight line distance to the nearest site). this species. We noted the characteristics of Alpine newt The aim of this study was to determine the provenance breeding habitat (confirmed by the presence of larvae) of the Alpine newts in TLP based on mtDNA variation. by recording variables describing the size, stability, We also mapped the local range of the Alpine newt and connectivity and vegetation of ponds during the time of described its breeding habitat characteristics in this surveys (April-August). area. Molecular methods.—Genomic DNA was extracted from 22 Alpine newts (Table 1), five of which originated An Alpine newt Ichthyosaura alpestris population on the Baltic coast of Poland 925 from TLP. One of the individuals from TLP was an adult using primers ND4 and Leu (Pabijan and Babik, 2006). female (MPFC2190) found dead and partly decomposed After purification, the PCR products were sequenced near site 4 (see Fig. 1). Four larvae (MPFC3220, using Big Dye chemistry (Applied Biosystems) and MPFC3230, MPFC3232, MPFC3233) from different resolved on an ABI 3130 machine (Applied Biosystems). sites (sites 4, 5 and 8, Fig. 1) were sampled by taking Newly obtained sequences were deposited in GenBank tailfin clips in 2018. All other Alpine newt samples (accession numbers MN211320-MN211341). were collected from different locations in central DNA sequence analysis.—Chromatograms were Europe over the past 15 years and stored in alcohol at edited in CodonCode Aligner (v. 2.0.6, Codon Code -20ºC in the amphibian tissue collection maintained at Corporation). The new sequences were aligned with the Department of Comparative Anatomy, Jagiellonian published sequences representing the range-wide University. DNA extraction, PCR and amplicon mtDNA diversity of this species. The final alignment sequencing were conducted according to Pabijan and included the following sequences from Pabijan and Babik (2006). DNA in the decomposed adult female Babik (2006), Recuero et al., (2014) and Arntzen et (MPFC2190) had deteriorated and necessitated the al., (2016): Ambleteuse A3 2,3; Charnohora A2; 12 application of an extraction kit dedicated to retrieving Krempna B1; 40 Beskid Wyspowy; 94, 96 Dulowa B2; trace amounts of DNA from degraded samples (Sherlock 120 Alwernia C1; 154, 177 Prudnik A1; 181, 182 Bielnik; AX, A&A Biotechnology). DNA from all other samples 184 Suchedniów; 215, 218, 249, 250 Muszkowice A1; was extracted using a phenol/chloroform protocol. We 282, 283, 285 Kąty; 315 Złoty Stok A1; 330 Lubawka amplified a fragment of NADH dehydrogenase subunit A1; 303, 312 Riu Marului; 313 Colonia Tomeasa; 268, 4 (ND4), tRNA-His, tRNA-Ser and tRNA-Leu genes 269 Baiu Mts C2; MAB301, MAB302, MAB305 - 1 Table 1. Origin of new mitochondrial ND4 sequences of Alpine newt individuals obtained in this study. Table 1. Origin of newly obtained mitochondrial ND4 sequences of Alpine newt individuals obtained. GenBank accession
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