Wielstra Lab

Cryptic species are species that look similar – and therefore are classified as a single species, or have been so up to recently – but that have very different DNA. The alpine newt serves as a case in point. It has long been considered to comprise multiple cryptic species, but previous studies lacked the genetic resolution required to sort out the situation. In a paper led by Stephanie Koster and Anagnostis Theodoropoulos, just out in Molecular Ecology, we crack the alpine newt cryptic species complex

We use the NewtCap protocol to obtain the genomic context required to disentangle the intricate evolutionary history of the alpine newt. We uncover five distinct species that we call the southern alpine newt (Mesotriton veluchiensis, blue on the distribution map below), the Vlasina alpine newt (which needs a formal species description still, working on it, grey below), the Apennine alpine newt (M. apuanus, orange), the Reiser’s alpine newt (M. reiseri, green), and the Northern alpine newt (M. alpestris, red). Hopefully, by being formally recognized as distinct species, these five awesome alpine newt species can be better protected.

Reference: Koster, S., Theodoropoulos, A., Beukema, W., Ambu, J., Babik, W., Canestrelli, D., Chiocchio, A., Cogalniceanu, D., Cvijanović, M., de Visser, M.C., Dufresnes, C., France, J., Hyseni, A., Jablonski, D., Kranželić, D., Lukanov, S., Martínez-Solano, I., Naumov, B., Pabijan, M., Salvi, D., Schmidt, B., Sotiropoulos, K., Stanescu, F., Stanković, D., Šunje, E., Szabolcs, M., Vacheva, E., Vörös, J., Zimić, A., Wielstra, B. (2026). Five hidden species in a widespread European vertebrate: disentangling the alpine newt cryptic species complex through genomic phylogeography. Molecular Ecology 35(5): e70300.

One reason that makes crested newts such a cool study system is that the species involved meet at hybrid zones. Where their ranges come into contact, they crossbreed. However, the low fitness of the hybrid offspring prevents the species from blending into one another. The consequence is a sharp transition between species distribution ranges. My previous work on crested newt hybrid zones mainly focused on Turkey and the Balkan Peninsula. Hybrid zones further north have remained poorly studied in comparison. It is time to change that!

In an Amphibia-Reptilia paper led by MSc student Arilah van Eden, we use lots of DNA data to refine the position, shape and dynamics of the hybrid zone between the Danube and Italian crested newts. The hybrid zone is narrow and runs pretty much from north to south, east of the Alps. At the top we see an elevated amount of DNA derived from the Danube crested newt inside the Italian crested newt. We link this to the Italian newt moving around the Alps when glacial conditions receded after the Last Glacial Maximum. Another crested newt hybrid zone tackled!

Reference: van Eden, A.J., Theodoropoulos, A., Arntzen, J.W., Czurda, J., Kranželić, D., Mačát, Z., Mikulíček, P., Reiter, A., Schmidt, B., Stanković, D., Vek, M., Vörös, J., Wielstra, B. (2026). The position, shape and dynamics of the hybrid zone between the Danube and Italian crested newt based on genome-wide data. Amphibia-Reptilia TBA.

Most animals are diploid, meaning they have two copies of each chromosome. One of these copies they inherited from their mother and the other one from their father. Sometimes the number of chromosome copies differs. In a new paper out in Journal of Heredity we explore the occurrence of triploidy, where individuals have three instead of two copies of a chromosome, in the hybrid zone between crested and marbled newts. In a large set of hybrids, we find quite some newts that appear to have twice as many alleles (gene versions) of one of the parent species. We test two of these individuals with different techniques and independently confirm that they truly are triploids. Thus, triploidy is not that rare in Triturus.

Reference: Arntzen, J.W., McCartney-Melstad, E., Litvinchuk, S., Wielstra, B. (2026). Triploidy in a pair of hybridizing salamanders at the far end of the speciation continuum. Journal of Heredity TBA.

This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (Grant Agreement No. 802759).

How do you determine the ratio of male and female newts in a population? You could try to sex the adults by eye. For younger animals, we now have genetic tools that can accomplish the same thing. But what if I told you, you do not even have to go through the effort of capturing animals? All the newts in a pond shed DNA into the environment. If you collect this environmental DNA, you would obtain a population-level DNA sample from which sex ratios could be inferred.

PhD student Emilie Didaskalou demonstrates the potential of this approach in a paper in Molecular Ecology Resources. At the crested newt colony in Belgrade, Serbia, she put newts of known sex together in tanks. Emilie then took water samples and confirmed it was possible to detect if there were more or less males present compared to females from environmental DNA. Certainly this is a research avenue worthy to explore further!

Reference: Didaskalou, E.A., France, J., Cvijanović, M., Trimbos, K.B., Vučić, T., Ajduković, M., Ivanović, A., Wielstra, B., van Bodegom, P.M., Stewart, K.A. (2026). Unlocking  demography: An eDNA-based toolkit to measure sex ratios from populations. Molecular Ecology Resources 26(1): e70089.

Over a decade ago, Christine Grossen and colleagues raised an interesting hypothesis that the two versions of chromosome 1 in Triturus newts, 1A and 1B – responsible for the balanced lethal system – used to be distinct versions of a former Y chromosome. The exact scenario is too complicated to reproduce here, but suffice to say it persuaded us to test if 1A and 1B correspond to what is the Y chromosome in Lissotriton newts (the sister lineage of Triturus). In a paper out in Genome Biology and Evolution, my PhD student James France determined which chromosomes in Lissotriton newts correspond to the Y chromosome and which correspond to Triturus’ 1A and 1B. These are clearly not the same: a finding that does not bode well for Christine’s elegant hypothesis. Interestingly, the actual Y chromosomes of Lissotriton and Triturus are also not the same. This means that, on an evolutionary timescale, newts must have switched between Y chromosomes. How often has this happened? That is an exiting question to be addressed in future research.

Oh yeah, we can now also genetically determine the sex of Triturus newts, using the same approach as James previously perfected in Lissotriton newts.

Reference: France, J., Babik, W., Cvijanović, M., Dudek, K., Ivanović, A., Vučić, T., Wielstra, B. (2025). Identification of Y-chromosome turnover in newts fails to support a sex chromosome origin for the Triturus balanced lethal system. Genome Biology and Evolution 17(9): evaf155.

‘Target enrichment by sequence capture’ is a nice technique to get plenty of DNA data, for a specific, predetermined part of the genome. You just extract DNA from a sample, cut it up, label it, and then pull out your genes of interest with complementary probes. Such a genome-reduction step is particularly helpful if you study species with large and complicated genomes, such as newts. In a paper out in Ecology and Evolution, my PhD student Manon de Visser pushes the limits of the Triturus sequence capture protocol, to see how broadly it could be applied within the family of newts and true salamanders, a.k.a. the Salamandridae. Conveniently, the protocol – which we refer to as NewtCap – works across the entire family. With NewtCap, standardized data, suitable to address a wide range of questions, can be collected for any newt or true salamander.

Reference: de Visser, M.C., France, J., McCartney-Melstad, E., Bucciarelli, G., Theodoropoulos, A., Shaffer, H.B., Wielstra, B. (2025). NewtCap: an efficient target capture approach to boost genomic studies in Salamandridae (true salamanders and newts). Ecology and Evolution 15(8): e71835.

It is quite easy to tell apart males and females of the smooth newt species complex (Lissotriton vulgaris and related species) when observing adults in ponds during the breeding season. They clearly differ by their secondary sexual characteristics: males are splendidly colored and sport a crest, while females are more drab. But what about animals outside the breeding season – or juveniles, larvae and embryos? To sex these you would need to resort to genetic methods. However, this is not straightforward: while newts have huge genomes, their sex-chromosomes are hardly diverged.

In the first paper resulting from his PhD thesis, out in Molecular Ecology Resources, my student James France undertook a massive study to design sex markers for the smooth newts. He compared two distinct ways of design: 1) looking at consistent differences in random DNA obtained from a sizable sample of males and females; and 2) looking at the distribution of DNA differences that are only present in the offspring of, in this case, the father (because in smooth newts the males have an X and a Y version of the sex chromosome). To cut a long story short: the conclusion of James’ study is that the first method works best. Based on the data collected, he designed a genetic tool that allows any member of the smooth newt species complex to be sexed. James’ approach to genetically sex salamanders should be broadly applicable.

Reference: France, J., Babik, W., Dudek, K., Marszałek, M., Wielstra, B. (2025). Linkage mapping vs Association: A comparison of two RADseq-based approaches to identify markers for homomorphic sex chromosomes in large genomes. Molecular Ecology Resources 25(7): e70019.

In the balanced lethal system in Triturus newts, individuals that inherit either the 1A or the 1B version of chromosome 1 twice will experience developmental arrest and die halfway embryonic development. Before that time, it has been impossible to predict which embryos are viable and which are doomed to die – until now. My PhD students Willem Meilink and Manon de Visser lead a paper, out in Ecology and Evolution, in which they use our genomic insights into the balanced lethal system (stay tuned!) to devise two distinct approaches to determine if an individual only possesses 1A (diseased), only possesses 1B (also diseased), or posses both 1A and 1B (healthy). This is very helpful in the balanced lethal system research program!

Reference: Meilink, W.R.M., de Visser, M.C., Theodoropoulos, A., Fahrbach, M., Wielstra, B. (2025). Determining zygosity with multiplex Kompetitive Allele-Specific PCR (mxKASP) genotyping. Ecology and Evolution 15(6): e71642.

The Near Eastern newts of the genus Neurergus are spectacular salamanders that seem to lack a proper common name. Because many of the species have at one time or another been referred to as ‘something something mountain newt’, let’s go with mountain newts! The taxonomy and systematics of Neurergus newts are a bit of a mess. In a paper led by my former MSc student Stephanie Koster out in Molecular Phylogenetics and Evolution we sort out the situation. We make the case that several species that are sometimes treated as distinct species do not deserve this special status. On the other hand, N. kaiseri should definitely be split into two species. We also notice that the position of N. crocatus in the evolutionary tree differs, depending on which analysis is used. We propose that this is a consequence of gene flow between tree branches. Once again, the Triturus sequence capture protocol proves extremely useful in salamander systematics.

Reference: Koster, S., Polanen, R., Avcı, A., Bogaerts, S., Bozkurt, E., Goudarzi, F., Hemami, M.-R., Olgun, K., Pasmans, F., Steinfartz, S., Üzüm, N., de Visser, M.C., France, J., Theodoropoulos, A., Wielstra, B. (2025). Discordance between phylogenomic methods in Near Eastern mountain newts (Neurergus, Salamandridae). Molecular Phylogenetics and Evolution 211: 108386.

The balanced lethal system in Triturus newts is well-suited to teach biology students basic principles about natural selection and genetic linkage. This was the motivation behind a paper led by my PhD student Willem Meilink published in Ecology and Evolution. We leverage hybrids between two different crested newt species because, due to the genetic divergence between them, we can distinguish the gene versions inherited from the father and from the mother. We trace the inheritance of 30 genetic markers from throughout the genome, including multiple genes positioned on chromosomes 1A and 1B, across three generations. Our experiment perfectly illustrates that we are dealing with two tightly linked subsets of genes, one on chromosome 1A and another on chromosome 1B, that are inherited within an otherwise regularly recombining genome.

We provide evidence to support four hypotheses: 1) upon fertilization, four different genotypes are present, two healthy (1A1B = 1B1A) and two diseased (1A1A and 1B1B) ones; 2) diseased embryos that experience arrested development possess two copies of either chromosome 1A or 1B (1A1A and 1B1B); 3) healthy embryos that survive the balanced lethal system possess a single copy of chromosome 1A and a single copy of chromosome 1B (1A1B = 1B1A); and 4) genes positioned on chromosome 1A or on chromosome 1B are always inherited together (crossing over does not break up these two subsets of genes). We have been running a dedicated lab practical on this ‘Mendelian inheritance with a deadly twist’ in the second year Bachelor course Evolutionary Biology 2 at Leiden University since 2023.

Reference: Meilink, W.R.M., Cvijanović, M., de Visser, M.C., France, J., Ivanović, A., Theodoropoulos, A., Vučić, T., Wielstra, B. (2025). Exposing selection and genetic linkage in the evolutionary enigmatic balanced lethal system in Triturus newts. Ecology and Evolution 15(6): e71591.