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.

In this study, we used genetic approaches to assess the conservation status of a protected amphibian species, the Macedonian crested newt, Triturus macedonicus, in Northern Pindos National Park (Epirus, Greece). Mitochondrial DNA sequences and multilocus genotypes of individuals from 38 breeding sites were used to infer their phylogenetic position and to detect and measure genetic variation patterns, population genetic structure, and levels of gene flow. The examined individuals fell within two major clades of the Macedonian crested newt phylogeny, being geographically separated by the Aoos River valley and Vikos Gorge. Both groups constitute separate gene pools, bearing private haplotypes and alleles, and the groups were found to be highly differentiated in both their mitochondrial and microsatellite markers. Thus, they meet all of the criteria needed to be characterized as evolutionary significant units (ESUs) that deserve a separate conservation status. Within each ESU, the particularly high inter-population differentiation and low migration rates imply a lack of connectivity between breeding sites and local isolation in favorable habitats. Our results can inform future programs and actions towards the conservation and management of the Macedonian crested newt in the area.

Salamanders have large and complex genomes, hampering whole genome sequencing. However, reduced representation sequencing provides a feasible alternative to obtain genome-wide data. We present NewtCap: a sequence capture bait set that targets c.7k coding regions across the genomes of all true salamanders and newts (the family Salamandridae, also known as ‘salamandrids’). We test the efficacy of NewtCap, originally designed for the Eurasian Triturus newts, in 30 species, belonging to 17 different genera, that cover all main Salamandridae lineages. We also test NewtCap in two other salamander families. We discover that NewtCap performs well across all Salamandridae lineages (but not in the salamander families Ambystomatidae and Hynobiidae). As expected, the amount of genetic divergence from the genus Triturus correlates negatively to capture efficacy and mapping success. However, this does not impede our downstream analyses. We showcase the potential of NewtCap in the contexts of; 1) phylogenomics, by reconstructing the phylogeny of Salamandridae, 2) phylogeography, by sequencing the four closely related species comprising the genus Taricha , 3) hybrid zone analysis, by genotyping two Lissotriton species and different classes of interspecific hybrids, and 4) conservation genetics, by comparing Triturus ivanbureschi samples from several wild populations and one captive-bred population. Overall, NewtCap has the potential to boost straightforward, reproducible, and affordable genomic studies, tackling both fundamental and applied research questions across salamandrids.

High throughput sequencing technologies have become essential in the fields of evolutionary biology and genomics. When dealing with non-model organisms or genomic gigantism, sequencing whole genomes is still relatively costly and therefore reduced-genome representations are frequently obtained, for instance by ‘target capture’ approaches. While computational tools exist that can handle target capture data and identify small-scale variants such as single nucleotide polymorphisms and micro-indels, options to identify large scale structural variants are limited. To meet this need, we introduce PAV-spotter: a tool that can identify presence/absence variation (PAV) in target capture data. PAV-spotter conducts a signal cross-correlation calculation, in which the distribution of read counts per target between samples of different a priori defined classes – e.g. male versus female, or diseased versus healthy – are compared. We apply and test our methodology by studying Triturus newts: salamanders with gigantic genomes that currently lack an annotated reference genome. Triturus newts suffer from a hereditary disease that kills half their offspring during embryogenesis. We compare the target capture data of two different types of diseased embryos, characterized by unique deletions, with those of healthy embryos. Our findings show that PAV-spotter helps to expose such structural variants, even in the face of medium to low sequencing coverage levels, low sample sizes, and background noise due to mis-mapped reads. PAV-spotter can be used to study the structural variation underlying supergene systems in the absence of whole genome assemblies. The code, including further explanation, is available through the PAV-spotter GitHub repository: https://github.com/Wielstra-Lab/PAVspotter .

The marsh frog ( Pelophylax ridibundus complex) has been dubbed the number one amphibian invader in Western Europe. In The Netherlands, marsh frogs are native to the north, centre and west of the country, whereas pool frogs ( P. lessonae ) are mainly found in the south and east; their hybridogenetic hybrid, the edible frog ( P . klepton esculentus ), is distributed throughout The Netherlands. Over the past decades, the southeastern province of Limburg has been overrun by marsh frogs. To expose the invasion history of marsh frogs in The Netherlands, we mtDNA barcoded hundreds of water frogs from across the country. Throughout The Netherlands, we primarily detect pool frog mtDNA: the only mtDNA type that is expected to occur here in any native water frog, regardless of taxon, due to asymmetric introgression. By contrast, we identify four distinct marsh frog mtDNA haplotypes in Limburg, originating from four regions of the native marsh frog range: (1) Central, Eastern and Southeastern Europe, (2) western Balkan Peninsula, (3) western and northern Turkey, Iran and eastern Ukraine into Russia, up to the Ural Mountains and (4) central southern Turkey. Additionally, two presumably introduced Central, Eastern and Southeastern European marsh frog haplotypes are detected locally elsewhere in The Netherlands, within the native marsh frog range. Our results align with the broader pattern that invasive marsh frogs constitute a ‘cocktail’ of genetically diverse lineages. As the invasion front in Limburg approaches native water frog populations, we urge that conservation measures be taken promptly.

We introduce multiplex Kompetitive Allele-Specific PCR (mxKASP): a modification of ‘classical’ KASP genotyping that allows zygosity to be determined in diploid organisms. Rather than targeting a SNP associated with a single marker, mxKASP targets two non-homologous markers. We show proof of concept by applying mxKASP to the balanced lethal system in Triturus newts, in which individuals are known to possess either: (1) zero copies of the 1A version of chromosome 1 and two copies of the 1B version; (2) one copy of 1A and one copy of 1B; or (3) two copies of 1A and zero copies of 1B. mxKASP is successful in amplifying both a 1A and a 1B marker in a single reaction (if present), allowing the zygosity of individuals to be inferred. We independently confirm our mxKASP results with a multiplex PCR approach. We argue that mxKASP can be applied to rapidly and economically determine zygostity in diploid organisms, for a large number of samples at once.