Wielstra Lab

Hybrid zones are the regions where different species meet, mate and produce offspring. In a study just published in the Biological Journal of the Linnean Society we explore nine Triturus hybrid zones (all but one existing in nature), using a large amount of genetic markers (forty allozymes and a mitochondrial gene) and a highly informative morphological character (the number of rib-bearing vertabrae), for a huge number of newts (well over 700). Hybrid zones are narrow and individuals show less hybridity than would be expected under random mating, which shows that the different species are genetically pretty isolated. The degree of genetic isolation increases with genetic divergence. We do find mitochondrial DNA in the ‘wrong’ species, which illustrates that individual genes can be exchanged between species that for the rest manage to remain distinct. The number of ribs is a good indicator of species identity. Although there is some variation within species, most of this occurs close to hybrid zones, suggesting hybridization is to blame here. This synthesis marks the end of an era in a way: a lot of the data was already collected decades ago. It is also a strong base for future research on the Triturus hybrid zones using the new Ion Torrent protocol.

Reference: Arntzen, J.W., Wielstra, B., Wallis, G.P. (2014). The modality of nine Triturus newt hybrid zones, assessed with nuclear, mitochondrial and morphological data. Biological Journal of the Linnaean Society 113(2): 604-622.

I conducted this work as a Newton International Fellow.

In a paper published in Annales Zoologici Fennici we tested the influence of shared ancestry and climate on the variation in life history traits in Triturus newts. Some variation can be explained by climate, some by shared ancestry and some by both.

Reference: Vukov, T.D., Cvijanović, M., Wielstra, B., Kalezić M.L. (2014). The roles of phylogeny and climate in shaping the variation in life-history traits observed in the newt genus Triturus (Caudata, Salamandridae). Annales Zoologici Fennici 51(5): 445-456.

My application for a Newton International Fellowship has been succesful! I will conduct this two-year postdoctoral fellowship, using the Ion Torrent protocol I designed during my postdoc at Naturalis to study hybridzones in Triturus, in the lab of Professor Terry Burke at the University of Sheffield.

The crested newt traditionally referred to as ‘Triturus karelinii’ comprises three distinct mitochondrial DNA clades. These clades are found in the east, the centre and the west of the range. The difference between mitochondrial DNA clades is comparable to the difference between the mitochondrial DNA of recognized crested newt species. We wanted to see if the three mitochondrial DNA clades might in fact represent different species.

This plot shows individuals (thin bars) within populations (thick bars) roughly ordered from west to east. Based on their genotype, individuals are allocated (0-100%) to three geographical genetic groups (represented by different colors).

In a paper published in Molecular Phylogenetics and Evolution we show that, based on three nuclear genes, there are three discrete geographical groups, in line with the three species hypothesis. We suggest that these three groups should be considered distinct species, but as it is as yet unclear if they can be distinguished based on morphology, we refer to them as ‘cryptic species’ for now.

Here you see the three cryptic species that make up the T. karelinii-group of crested newts. An intriguing finding is that asymmetric DNA introgression from the western group into the central group (the red-green hatched area). We suggest that this pattern can be explained by the central group having expanded its range at the expense of the western group, while the two hybridized in the process. An interesting hypothesis to test in a future study!

Reference: Wielstra, B., Baird, A.B., Arntzen, J.W. (2013). A multimarker phylogeography of crested newts (Triturus cristatus superspecies) reveals cryptic species. Molecular Phylogenetics and Evolution 67(1): 167-175.

Hybridizing species sometimes exchange genes in nature (a phenomenon called introgression). Introgression has particularly been documented for mitochondrial DNA. This might mean mitochondrial DNA is more susceptible to introgression, but it is also the case that researchers have simply studied mitochondrial DNA more. Usually introgression is restricted to close to the hybrid zone. There are however more pronounced examples. It does not get more extreme than in smooth newts, where the original mitochondrial DNA of Lissotriton montandoni has been completely replaced by that of L. vulgaris.

A male Lissotriton montandoni on the left and a male L. vulgaris on the right. Pictures by team Babik.

In a paper published in Molecular Ecology we extensively sample both mitochondrial and nuclear DNA for L. montandoni and surrounding L. vulgaris populations. We also use species distribution modelling to determine range dynamics of L. montandoni since the Last Glacial Maximum. We show that mitochondrial DNA introgression occurred several times, and at different moments, in different parts of the range of L. montandoni. This is probably related to the inferred range fragmentation under glacial conditions, and  independent range expansion from these range fragments into L. vulgaris territory when the climate ameliorated. In contrast, there is little evidence of recent nuclear gene flow between the species.

A visualization of complete mitochondrial DNA capture in response to species displacement and range reduction. We are dealing with two species here, let’s call them green and red. These species possess distinct mitochondrial DNA. Panels are ordered chronologically. In the panels, dots reflect localities, while boxes represent rough outlines of the ranges. Background shading (green or red) reflects species identity, while localities are colored according to the mitochondrial DNA type present (so dots are green or red).

In I) the ranges of the two species are geographically separated. At this stage, green mitochondrial DNA is only found in localities of the green species, and red mitochondrial DNA in localities of the red species. However, the green species is doing well for itself and, over the generations, its population increases. Surplus individuals from the right edge of the range start colonizing new localities further right. In these localities, numbers start increasing again and some offspring colonize new localities even further right, and so on. With time, the green species expands its range to the right, towards that of the red one.

In II) the ranges of the two species have come into contact. Where they meet, the two species start reproducing with one another (hybridization), resulting in offspring that are a mix of both green and red. Initially there are relatively few green individuals, but they keep pouring into the into the hybrid zone, while this is not the case for red individuals. Hybrids already present mate with these green individuals, and their offspring again mate with green individuals, and so on. Over time, individuals in the hybrid zone become ‘greener’. However, because at the initial stage of invasion by the green species most matings will concern individuals that also have red genes, some of these red genes rather than their green counterparts could locally get fixed in (introgress into) the green species by chance (and several processes that we won’t go into now might reinforce introgression). In this case mitochondrial DNA introgresses. Hence, at the right edge of the green species range, you start to see localities where individuals belong to the green species, but possess mitochondrial DNA typical of the red species (red dots in a green range).

In III) the individuals in the initial hybrid zone have become all green, except their mitochondrial DNA. Still the green species keeps expanding its range further to the right. Once again green individuals meet red ones, start hybridizing and gradually take over, and so on. In consequence, the hybrid zone between the two species moves towards the right, as the green species replaces the red species. Yet, the members of the green species at the frontier, as well as the red individuals they hybridize with, only possess red mitochondrial DNA. So the region to the right of the initial hybrid zone becomes ‘greener’, but the overturn between green and red mitochondrial DNA still aligns with that initial hybrid zone. One way to put this is that the red mitochondrial DNA ‘surfs the wave’ of the green species expansion. In effect you end up with red localities on a green background over a considerable area.

In IV) the hybrid zone between the green and the red species has stabilized at a region where the green species does not have an edge over the red species as it did before. At the same time the part of the green species’ range where it still possessed the green mitochondrial DNA type becomes unsuitable and here the green species goes extinct (black background). As a result, there are now only members of the green species left that possess red mitochondrial DNA, while the original green mitochondrial DNA has been lost.

Reference: Zieliński, P., Nadachowska-Brzyska, K., Wielstra, B., Szkotak, R., Covaciu-Marcov, S., Cogălniceanu, D., Babik, W. (2013). No evidence for nuclear introgression despite complete mtDNA replacement in the Carpathian newt (Lissotriton montandoni). Molecular Ecology 22(7): 1884-1903.

The Triturus karelinii-group of crested newts comprises three mitochondrial DNA lineages, but no morphological differences are known. In a paper published in Zoologischer Anzeiger we analyse skull shape to see if there are differences between lineages. While we do not find three discrete geographical groups, the observed differences among lineages are also not smaller than between lineages and another crested newt species, T. macedonicus. We just need to keep looking for morphological differences.

Reference: Ivanović, A., Üzüm, N., Wielstra, B., Olgun, K., Litvinchuk, S.N., Kalezić, M.L., Arntzen, J.W. (2013) Is mitochondrial DNA divergence of Near Eastern crested newts (Triturus karelinii group) reflected by differentiation of skull shape? Zoologischer Anzeiger 252(2): 269-277.

The Quaternary Ice Age heavily influenced the distribution of species. During the colder glacial periods, species went extinct in part of their range, while during warmer interglacial periods, they could recolonize these regions again. This contraction-expansion pattern left its mark on genetic diversity across species’ ranges, with high diversity in regions where species survived continuously, and low diversity in regions where they were periodically wiped out. Past range shifts can also be visualized by projecting species distribution models, based on the environmental conditions currently experienced, on climate reconstructions of the past.

In a study published in Frontiers in Zoology we explore how all the marbled and crested newts species (so the entire genus Triturus) responded to the climate change associated with the Ice Age. We conduct a phylogeographical survey, meaning we sequence a lot of mitochondrial DNA, for many populations throughout each of the species ranges, and look at variation in genetic diversity across species ranges. Additionally, we compare species distribution models projected on current and on past climate layers (the Last Glacial Maximum, about 21,000 years ago).

A visualization of the biogeographical scenario proposed, showing the positions of glacial refugia in dark shades and the regions postglacially colonized in light shades for each individual species. In grey areas we infer that one species displaced another as they shifted their ranges.

By combining the two independent techniques of phylogeography and species distribution modelling, we obtain a more complete understanding of the historical biogeography of the crested and marbled newts than both approaches would have provided on their own.

Reference: Wielstra, B., Crnobrnja-Isailović, J., Litvinchuk, S.N., Reijnen, B., Skidmore, A.K., Sotiropoulos, K., Toxopeus, A.G., Tzankov, N., Vukov, T., Arntzen, J.W. (2013). Tracing glacial refugia of Triturus newts based on mitochondrial DNA phylogeography and species distribution modeling. Frontiers in Zoology 10: 13.