Wielstra Lab

A male pygmy marbled newt (left) and a male marbled newt. Pictures by Michael Fahrbach.

Although my work is heavily focused on crested newts, there are two marbled newt species in the genus Triturus as well. In a new paper out in Molecular Phylogenetics and Evolution, led by my former MSc student Christos Kazilas, we present a ‘next-generation phylogeography’ of marbled newts. There is remarkable little gene flow between the two marbled newt species compared to crested newts. This finding strongly supports the species status of the two marbled newts – in case anyone was still in doubt. Furthermore, both marbled newt species are composed of two genetically distinct groups. We could expose these intricate patterns thanks to the massive amount of DNA data that is easily generated with the Triturus sequence capture protocol.

Reference: Kazilas, C., Dufresnes, C., France, J., Martínez-Solano, Í., Kalaentzis, K., de Visser, M.C., Arntzen, J.W., Wielstra, B. (2024). Spatial genetic structure in European marbled newts revealed with target enrichment by sequence capture. Molecular Phylogenetics and Evolution 194: 108043.

In an article aimed at high school students for the journal Frontiers for Young Minds my former student Nienke Prins and I explain hybrid zone movement. Obviously we also mention the best example of hybrid zone movement there is: crested newts. Please have a look here.

(You can also see a previous Frontiers for Young Minds piece from our lab on balanced lethal systems here.)

Reference: Prins, N., Wielstra, B. (2024). Moving hybrid zones; when two species meet, mate, and compete. Frontiers for Young Minds 12: 1207354.

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 655487.

In the Dune area of Meijendel, close to Leiden, a population of crested newts occurs. The history of this population is dodgy. While genetic data could not confirm that this population is introduced, the isolated position and apparent rapid expansion speak in favor of such a scenario. Previously we reported on an aberrantly colored newt from this population, with a pattern reminding of a Koi carp. Last spring we caught another odd individual, this time almost completely pale yellow.

Catching two of these weird newts in rapid succession in the same area: surely that cannot be a coincidence? I suspect this high freak-frequency fits with an introduction history for the Meijendel crested newts. The establishment of an introduced population involves similar genetic bottlenecking and inbreeding dynamics as the establishment of island populations and island populations also tend to show a higher incidence of color deviations.

Reference: Elfering, R., Bijlsma, L., Mannix, S., Plomp, S., Theodoropoulos, A., Wielstra, B. (2023). Kamsalamanders met kleurafwijking uit Meijendel. Holland’s Duinen 83: 38-39.

Three banded newt species are currently recognized. However, this was not always the case. Previously, my lab has shown that these morphologically similar species are genetically super distinct. That is why we suggested their treatment as distinct species – despite their morphological similarity. The term ‘cryptic species’ is regularly used in such a case.

But is being genetically distinct enough to qualify as a species? If cryptic species meet in nature, it is possible to perform the ultimate test of species status: hybrid zone analysis. Selection against hybrids allows species to persist. Hybrid zone analysis can tell you if natural selection favors purebreds of either species over their genetically mixed offspring.

In a new paper published in Ecology and Evolution, we show that two of the banded newt species meet at a hybrid zone in northern Türkiye. At this hybrid zone, selection against hybrids is evidently very strong. As a consequence, the hybrid zone is extremely narrow. Our study shows that the two banded newts truly are proper species. We have already shown that, with the benefit of hindsight, it is actually possible to distinguish them based on morphological characteristics.

Reference: Kalaentzis, K., Arntzen, J.W. Avcı, A., van den Berg, V., Beukema, W. France, J., Olgun, K., van Riemsdijk, I., Üzüm, N., de Visser, M.C., Wielstra, B. (2023). Hybrid zone analysis confirms cryptic species of banded newt and does not support competitive displacement since secondary contact. Ecology and Evolution 13(9): e10442.

_{This project has received funding from the European Union’s Horizon 2020 research and innovation programme (under the Marie Skłodowska-Curie grant agreement No. 655487) and the ‘Nederlandse organisatie voor Wetenschappelijk Onderzoek’ (NWO Open Programme 824.14.014)}

Befitting its intimidating scientific name, Ichthyosaura, the alpine newt is an interesting beast. The alpine newt is characterized by hugely divergent mtDNA lineages that may well reflect cryptic species (genetically distinct but morphologically similar species). The distribution of these cryptic species is still incompletely understood. On top of that, alpine newts have been introduced outside their natural range on a massive scale. While the alpine newt has even colonized New Zealand, it is in the Netherlands, the UK and Ireland where the introduced population has truly exploded.

In the context of their BSc research project in my lab, Jody Robbemont and Sam van Veldhuijzen studied an enormous number of samples, from both the native and introduced alpine newt range. Many international collaborators contributed to this dataset. Furthermore, we relied on a large network of volunteers for NGO RAVON to collect skin swabs of alpine newts – a nice example of citizen science.

In a paper out in Amphibia-Reptilia we use the mtDNA barcoding technique to take a closer look at the alpine newt. We manage to delineate the geographical distribution of the cryptic alpine newt species in much more detail then before and home in on the regions where they presumably meet in nature. Our improved alpine newt mtDNA phylogeography also allows us to determine the provenance of many introduced populations. Four out of seven of the highly distinct alpine newt mtDNA lineages are implicated! The alpine newt provides a textbook example of the uncoordinated and simply chaotic nature of species invasion.

Reference: Robbemont, J., van Veldhuijzen, S., Allain, S.J.R., Ambu, J., Boyle, R., Canestrelli, D., Ó Cathasaigh, É., Cathrine, C., Chiocchio, A., Cogalniceanu, D., Cvijanović, M., Dufresnes, C., Ennis, C., Gandola, R., Jablonski, D., Julian, A., Kranželić, D., Lukanov, S., Martínez-Solano, I., Montgomery, R., Naumov, B., O’Neill, M., North, A., Pabijan, M., Pushendorf, R., Salvi, D., Schmidt, B., Sotiropoulos, K., Stanescu, F., Stanković, D., Stapelton, S., Šunje, E., Szabolcs, M., Vacheva, E., Willis, D., Zimić, A., France, J., Meilink, W.R.M., Stark, T., Struijk, R.P.J.H., Theodoropoulos, A., de Visser, M.C., Wielstra, B. (2023). An extended mtDNA phylogeography for the alpine newt illuminates the provenance of introduced populations. Amphibia-Reptilia 44(3): 347-361.

Reference: Wielstra, B., Boer, I. den, France, J., de Visser, M., Struijk, R. (2023). MtDNA barcoding van exotische amfibieën in de duinen. RAVON 89(2): 26-29.

Reference: de Visser, M., Prins, N., France, J., Struijk, R., Wielstra, B. (2023). Exotische amfibieën in de duinen ontmaskerd met mtDNA barcoding. Holland’s Duinen 82(1): 25-29.

Reference: Struijk, R.P.J.H., Prins, N., Koster, S., Putters, N., Jansen, N., Esselaar, J., de Visser, M., France, J., Wielstra, B. (2024). Verspreiding, voortplanting en geografische herkomst van de knoflookpad in Callantsoog. RAVON 26(1): 2-5.

The job of immune genes is to fight off pathogens. However, pathogens don’t take this beating lying down. Pathogens adapt to evade the immune genes, forcing the immune genes to counter-adapt in return. To improve your chances against infection in this evolutionary arms race, would it not be neat if you could borrow immune genes that evolved in another species? Hybridization could facilitate this. Through repeated hybridization, genes can flow between species: a process known as introgression. In a study led by Tomasz Gaczorek and Wiesław Babik, published in Molecular Ecology, we test to what extent the important immune genes of the major histocompatibility complex (MHC) are exchanged between different crested newt species. It turns out that MHC genes introgress more extensively than random genes – exactly what you would expect if natural selection were to favor exotic immune genes.

Reference: Gaczorek, T., Marszałek, M., Dudek, K., Arntzen, J.W., Wielstra, B., Babik, W. (2023) Interspecific introgression of MHC genes in Triturus newts: evidence from multiple contact zones. Molecular Ecology 32(4): 867-880.

The first crested newt I ever saw was in Meijendel, a dune area close to Leiden (where I studied biology at the time). This crested newt population is odd because it is completely isolated from the main distribution range. Could it be that these newts were introduced here?

There are other unusual amphibians in Meijendel too. Around the start of the 21^st century tree frogs suddenly appeared. These have rapidly expanded their range and are now omnipresent. Less known is that a population of midwife toad has also become established here. Surely these guys must have been introduced!

I have always wondered where all these animals came from. When I set up my own lab in Leiden, I finally had the opportunity to take a closer look. Or rather, I had a large team of BSc students do the work!

The papers from their projects have now been published in Amphibia-Reptilia. We use mtDNA barcoding to determine the provenance of outlier populations in the Dutch coastal dunes. Because the species involved show geographical variation in mtDNA across their distribution ranges, we can link odd populations to the part of the range where their mtDNA naturally occurs.

For crested newts and midwife toads we unfortunately cannot say much about their origin, except that they derive from ‘somewhere in western Europe’, rather than from elsewhere in the range. The reason is that there is practically no genetic variation in western Europe, because this part of the range was colonized relatively recently, after the last glacial period subsided.

With the tree frogs it is a different story. These actually belong to three different species, two of which, the Italian and the eastern tree frog, are not even native to the Netherlands! Also spadefoot toads (not introduced in Meijendel but near Callantsoog) are highly distinct from the (threatened) native populations in the Netherlands. They derive from deep in central Europe. These examples showcase the power of mtDNA barcoding!

Reference: 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? Amphibia- Reptilia 44(1): 19-26.

Reference: Vliegenthart, C., van de Vrede, M., den Boer, I., Gilbert, M.J., Lemmers, P., France, J., de Visser, M.C., Struijk, R.P.J.H., Wielstra, B. (2023). The limits of mtDNA analysis for determining the provenance of invasive species: a midwife toad example. Amphibia- Reptilia 44(1): 27-33.

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

Reference: 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. Amphibia- Reptilia 44(1) 11-18.

Adult marbled and crested newts have two versions – a long and a short one – of their largest chromosome: chromosome 1. They randomly transmit either the long or the short version to each of their sex cells, resulting in an equal ratio of sex cells with the long or short version. When egg and sperm cells fuse upon fertilization, by chance half of the resulting embryos will have either two long or two short versions. Such individuals die before they even hatch. A situation in which only individuals with two distinct versions of a chromosome survive – and the ones that have the same version twice perish – is called a balanced lethal system.

In a new paper out in Philosophical Transactions of the Royal Society B: Biological Sciences we provide a new hypothesis on how balanced lethal systems could evolve. Balanced lethal systems pose an evolutionary paradox, because they are extremely wasteful, but must have evolved in the face of natural selection. Emma Berdan did a postdoc in my lab on my ERC Starting Grant project. Together with Alexandre Blanckaert, Emma took the lead in this study (actually part of a special issue that Emma and I co-edited). We tested if a balanced lethal system could be a potential end point of a ‘supergene system’.

Supergenes are large chunks of DNA that contain many genes and come in different versions. Crucially, the different versions of the same supergene cannot exchange DNA anymore because, for example, one of them has been flipped around in the genome. Due to the different orientation of the two supergene versions, they are not recognized as equivalent during the production of sex cells, when typically the DNA of both parents gets reshuffled to produce a new, unique genetic combination, different from each parent (a process known as recombination). As a consequence of suppressed recombination in supergenes, alleles (gene variants) of many genes can co-evolve independently on the different versions and, together, eventually encode widely different phenotypes. This can be a good thing: distinct phenotypes may provide unique benefits. If this were the case, then balancing selection would tend to preserve both supergene versions in the same population.

However, the lack of recombination between the supergene versions also comes at a cost: when genes on one supergene version get broken, they cannot be replaced by working copies on the alternative supergene version anymore – and the other way around. The official term for this irreversible accumulation of broken genes is Muller’s Ratchet. Now what if both supergene versions acquire unique broken genes? Then you are only viable if you possess both supergene versions, and a balanced lethal system is born! We wanted to see what it would take for this to happen in nature.

We simulate a situation in which two different versions of a supergene first evolve in separate populations and randomly acquire bad mutations here. The two are subsequently united, because one population donates its supergene version to the population with the alternative supergene version via hybridization. Now each supergene version can correct for the bad mutations on the other one. We explore how the two supergene versions could be maintained together by balancing selection long enough for them to degrade to the point at which each of them cannot function on its own anymore because genes have become broken. The tricky thing is that, as soon as one supergene version starts to perform relatively worse, natural selection tends to remove it from the population. Degradation of the supergene versions needs to be symmetrical! Only when the population is tiny, and the broken genes are fully compensated by their complements on the alternative supergene version, do we sometimes see balanced lethal systems appear in our simulations.

We need to look at Triturus newts and see if we can find evidence of hybridization associated with chromosome 1: could it be that one of the versions of chromosome 1 was donated to the ancestral Triturus population by another newt species? Actually, introgression (genetic exchange between species) abounds in the salamander family that the crested and marbled newts belong to. When we get our hands on Triturus genomes we can also test for the signature left by the tiny population size that we predict is required for the origin of the balanced lethal system. Did the ancestral Triturus population go through such a bottleneck? And when we home in on the broken genes on each version of chromosome 1, we expect that the task of these genes is fully taken over by their counterparts on the alternative version of chromosome 1. Lots of exciting ideas to test!

Reference: Berdan, E.L., Blanckaert, A., Butlin, R.K., Flatt, T., Slotte, T., Wielstra, B. (2022). Mutation accumulation opposes polymorphism: Supergenes and the curious case of balanced lethals. Philosophical Transactions of the Royal Society B 377(1856): 20210199.

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

Phylogeography – the study of the geographical genetic structure within species or groups of closely related species – was until recently typically based on one or a few genes. Nowadays, many genes can be consulted. This allows us to better address questions about taxonomy, species relationships and hybridization. We adapted the Triturus Ion Torrent protocol and took another look at the banded newts, using two orders of magnitude more markers than before.

If you were not convinced already: we can now be very confident that there are three species of banded newt. The Anatolian and Caucasian banded newts appear to be more closely related to each other than to the southern banded newt, but support is not great. These two newt species probably meet in nature and are known to hybridize under artificial conditions. Although the putative hybrid zone remains unsampled, we show that the geographical extent of gene flow between the two must be limited – in sharp contrast to Triturus newts in the region! Another interesting aspect is that there are distinct genetic groups within banded newt species. So, while there we have learned a lot about banded newts the last few years, there are also still some cool outstanding questions (…fortunately!).

Reference: van Riemsdijk, I. Arntzen, J.W., Babik, W., Bogaerts, S., Franzen, M., Kalaentzis, K., Litvinchuk, S.N., Olgun, K. Wijnands, J.W.P.M., Wielstra, B. (2022). Next-generation phylogeography of the banded newts (Ommatotriton): a phylogenetic hypothesis for three ancient species with geographically restricted interspecific gene flow and deep intraspecific genetic structure. Molecular Phylogenetics and Evolution 167: 107361.

_{This project has received funding from the European Union’s Horizon 2020 research and innovation programme (under the Marie Skłodowska-Curie grant agreement No. 655487) and the ‘Nederlandse organisatie voor Wetenschappelijk Onderzoek’ (NWO Open Programme 824.14.014).}