Introgression of immune genes in crested newts

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.

DNA barcoding invasive amphibians in the Dutch dunes

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?

Staring at a crested newt. Picture by Christos Kazilas

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!

Tree frog (left) and midwife toad. Pictures by Ingrid den Boer

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!

Students hard at work. Pictures by me and Manon de Visser

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.

Swabbing a crested newt larva (left) and a juvenile tree frog for DNA. Pictures by Ingrid den Boer

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.

DNA extraction in the lab. Pictures by Ingrid den Boer

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!

Common spadefoot toad adult (left) and larvae. Pictures by Richard Struijk

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.

One way to evolve a balanced lethal system

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’.

Michael Fahrbach provided the picture that graces the cover of our special issue.

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.

Male Triturus newts congregate in ponds and put on an elaborate, ritualized dance to entice the females. After mating, female Triturus newts carefully wrap each of their eggs in a protective layer of vegetation (plastic will also do). A bit silly that, after all this effort, half of the eggs of Triturus newts do not even hatch! Pictures by Michael Fahrbach.

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).

More resolution for the beautiful banded newts

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.

The 35 banded newts studied neatly sort into three species. Pictures: Michael Fahrbach & Sergé Bogaerts.

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).}

Lab tech position available in the Wielstra lab

Please note that the deadline to apply for this position has now passed.

In the context of my ERC Starting Grant project I will hire a lab tech to begin early 2022. Please see the advertisement here.

The distinct marbled and crested newts still exchange genes

A crested newt (left) and a marbled newt (right), with a first generation hybrid between the two species in the middle. Picture credits: hybrid by Paolo Mazzei and parent species by Michael Fahrbach.

In a big chunk of France, the crested newt Triturus cristatus and the marbled newt T. marmoratus live alongside one another. Sometimes things get frisky: about one in 25 adult Triturus newts here is a first generation (F1) hybrid between the two species. The fitness of these F1 hybrids is pretty lousy, but it turns out they are not a complete evolutionary dead end either. In a paper just out in Evolutionary Applications we show that deeper generation hybrids do exist, albeit very rarely. The non-zero fitness of hybrids provides a window of opportunity for gene flow between the two parent species. This gene flow appears to be skewed from marbled toward crested newts (confirming a previous finding that linked this pattern to hybrid zone movement). There are also some weird biases in which F1 hybrids survive (expanding upon a previous finding). If the mother is a crested newt, the majority of offspring is female (meaning more males die). On the other hand, offspring of marbled newt mothers are mostly males (so here more females die). You really need DNA to unveil all this stuff!

Reference: Arntzen, J.W., Jehle, R., Wielstra, B. (2021). Genetic and morphological data demonstrate hybridization and backcrossing in salamanders at the far end of the speciation continuum. Evolutionary Applications 14(12): 2784-2793.

Koi crested newt

This spring we conducted fieldwork in the Dutch dune area Meijendel, close to Leiden. One of our target species was the northern crested newt (Triturus cristatus). There is a crested newt population in Meijendel that is completely isolated from the rest of the species’ range. While dipnetting away in a pond full of crested newts, my student Bas Helder caught his first crested newt ever. And it was a weird one: a partially flavistic male, reminiscent of a Koi carp. We wrote a little note about it (in Dutch) in RAVON’s newsletter Schubben & Slijm.

Picture by Manon de Visser

Reference: Helder, B., de Brouwer, J., Ouwehand, J., de Visser, M., Wielstra, B. (2021). Koi-kamsalamander. Schubben & Slijm 48: 8.

Frontiers for Young Minds piece on balanced lethal systems

In an article aimed at high school students for the journal Frontiers for Young Minds we introduce the evolutionary mystery posed by balanced lethal systems. We use crested newts as an example throughout. Please have a look here.

Reference: Meilink, W.R.M., France, J., de Visser, M.C., Wielstra, B. (2021). Balanced lethal systems: an evolutionary mystery. Frontiers for Young Minds 9: 632945.

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). The PhD position of WRMM is supported by the Nederlandse organisatie voor Wetenschappelijk Onderzoek (NWO Promotiebeurs voor leraren 023.016.006).

NWO funding for the Wielstra lab

I have been awarded an ENW-M-1 grant in NWO’s Open Competition ENW. With this project I will expand my research line on the balanced lethal system in Triturus newts. We know that only those Triturus offspring that possess two different forms of chromosome 1 survive, while those that receive the same form twice from their father and mother, exactly half of the total number, die. We will study developing embryos and their genes simultaneously, to figure out what exactly goes wrong in the unfortunate embryos.

Italian crested newt genes reach Germany

In the 1970s Jozef F. Schmidtler noticed that crested newts in the Berchtesgadener Land, in the extreme southeast of Germany, show morphological characteristics of the Italian crested newt (Triturus carnifex). A particular striking feature was the bright yellow stripe along the back of some juveniles. While this stripe is typical for the Italian crested newt, it is not usually found in the crested newt species that is widely distributed throughout Germany: the northern crested newt (T. cristatus). Schmidtler published his findings in the journal Salamandra.

The yellow stripe that is observed to various extent in juvenile newts (left) is typical of the Italian crested newt. The throat and belly pattern of adult newts is reminiscent of this species as well.

In a new paper also published in Salamandra we employ a genetic toolkit that was originally designed to screen for ‘genetic pollution’ from the Italian into the northern crested newt. For some reason the Italian crested newt has repeatedly been introduced inside the range of the northern crested newt. Hybridization between the two species poses a complicated conservation concern. The genetic toolkit should be able to pick up ‘Italian alleles’ in the natural hybrid zone just as well. Our results are obvious: alleles of the Italian crested newt are indeed present in the Berchtesgadener Land. In other words, the natural hybrid zone between the northern and Italian crested newts just reaches Germany.

Ancestry is the fraction of Italian crested newt alleles and heterozygosity the fraction of genes for which both an Italian and northern crested newt allele are observed. Therefore, only the bottom left corner of the triangle above would represent a pure northern crested newt; any deviation means Italian alleles are present.

Reference: Fahrbach, M., de Visser, M., Wielstra, B. (2021). The hybrid zone between the Italian and Northern crested newts (Triturus carnifex and T. cristatus) reaches Germany. Salamandra 57(1): 428-434.