The phylogeny of crested newts (Triturus cristatus superspecies): nuclear and mitochondrial genetic characters suggest a hard polytomy, in line with the paleogeography of the centre of origin

Newts of the genus Triturus (Amphibia, Caudata, Salamandridae) are distributed across Europe and adjacent Asia. In spite of its prominence as a model system for evolutionary research, the phylogeny of Triturus has remained incompletely solved. Our aim was to rectify this situation, to which we employed nuclear encoded proteins (40 loci) and mitochondrial DNA-sequence data (mtDNA, 642 bp of the ND4 gene). We sampled up to four populations per species covering large parts of their ranges. Allozyme and mtDNA data were analyzed separately with parsimony, distance, likelihood and Bayesian methods of phylogenetic inference. Existing knowledge on taxonomic relationships was confirmed, including the monophyly of the genus and the groups of crested newts (four species) and marbled newts (two species). The genetic coherence of species and subspecies was also confirmed, but not always with high statistical support (depending on taxon, characters under consideration, and method of inference). In spite of our efforts we did not obtain sufficient phylogenetic signal to prefer one out of twelve potential topologies representing crested newts relationships. We hypothesize that the lack of phylogenetic resolution reflects a hard polytomy and represents the (near)-simultaneous origin of crested newt species. Using a calibration point of 24 Ma (million years before present) for the most recent common ancestor of Triturus-species, the crested newt radiation event is dated at 7-6 Ma (scenario 1) or at 11-10 Ma (scenario 2), depending on the application of an allozyme versus mtDNA molecular clock. The first biogeographical scenario involves the spread of crested newts from the central Balkans into four compass directions. This scenario cannot be brought into line with potential vicariance events for south-eastern Europe. The second scenario involves the more or less simultaneous origin of four species of crested newts through large-scale vicariance events and is supported by the paleogeographical reconstruction for the region at the end of the Middle Miocene. The subspecies Triturus carnifex macedonicus carries in one large area the mtDNA that is typical for the neighbouring species T. karelinii, which is attributed to introgression and a recent range shift. It is nevertheless a long distinct evolutionary lineage and we propose to elevate its taxonomic status to that of a species, i.e., from Triturus c. macedonicus (Karaman, 1922) to Triturus macedonicus (Karaman, 1922).