We have tested an existing phylogeny of Triturus against independent molecular data. Two PCR-amplified fragments of the mtDNA molecule with relatively low (12S rRNA) and high rates of evolution (ATPase) were employed to test some of the earlier and more recent speciation events of the Triturus radiation, respectively. The choosen fragments showed evolutionary rates that were anticipated and that were appropriate for addressing phylogenetic questions at this taxonomic level. Faster evolving protein-coding ATPase genes can be reliable tracers of evolutionary history among close relatives (as within the T. vulgaris species group), where silent, third codon position substitutions account for most variation (Zardoya and Meyer, 1996). On the other hand, if the saturation of transitions is suspected, it is imperative to reduce the emphasis on, or even eliminate, this class of substitutions from phylogenetic analysis (Disotell et al., 1992; Knight and Mindell, 1993). Although intuitively appealing, differential weighting may downweight informative transitions in conserved regions and upweight transversions primarily present in more variable regions of the molecule, thereby obscuring phylogenetic relationships (Titus and Larson, 1995; Mindell and Tacker, 1996). Given the observed transition bias, saturation was not prevalent among the compared sequences. The observation that with weighting the bootstrap support for congruent sections of competing phylogenetic solutions dropped and the phylogenetic resolution decreased, supports this view.
Our analysis presents a hypothesis that is concordant with well-supported areas in the established Triturus phylogeny. As to the specific questions we raised, some appear to be solved while others are not. The new data do not help to elucidate the relationship between T. boscai and T. italicus. Their monophyly was suggested by allozyme data and, ambiguously, by behavioral data (Arntzen and Sparreboom, 1989). The observation of ‘flamenco’ behavior in the sexual repertoire of T. helveticus (M. Faria, pers. comm.) and T. marmoratus pygmaeus (T. Halliday, pers. comm., M. Sparreboom, pers. comm.) further erodes the support for T. boscai - T. italicus monophyly. Similarly, the current study does not convincingly clarify the position of T. alpestris. The phylogenetic position of this species could also not be satisfactorily resolved from behavioral data (Arntzen and Sparreboom, 1989). Bolkay (1928) placed it in a third subgenus with intermediate characteristics, in between Triturus and Paleaotriton.
The 12S rRNA sequence data support the placement of T. vittatus as the sister taxon to the T. marmoratus species group in the subgenus Triturus, as was inferred from its breeding behavior (Arntzen and Sparreboom, 1989). This challenges the inferences from the phenetic analyses of osteological (Rafinski and Pecio, 1989), karyological (Bucci-Inocenti et al., 1983), morphological (Giacoma and Balletto, 1988) and immunological data (Busack et al., 1988). According to these studies, T. vittatus is more similar to T. alpestris or the small-bodied newts of the subgenus Palaeotriton. Immunological data (Busack et al., 1988) suggested a sister taxon relationship for T. v. vulgaris and T. helveticus. The ATPase sequences provide strong support for the monophyly of T. vulgaris and T. montandoni, in line with allozyme data (Rafinski and Arntzen, 1987).
The rate of 12S rRNA molecular evolution in some Palearctic salamandrids was calibrated as approximately 3 mA of lineage independence per percent sequence divergence and 7 mA per percent sequence divergence when only transversions are considered (Caccone et al., 1994). Applying these rates provides an estimate of 12-19 mA for the age of the T. vittatus lineage (versus the T. marmoratus species group) in the subgenus Triturus and an estimate of 14-19 mA for the T. boscai lineage (versus the T. vulgaris species group) within the subgenus Palaeotriton. This is in line with estimates from fossil, biochemical, molecular and biogeographical data that converge to 14-15 mA for the T. vittatus lineage and to 13-15 mA for the T. boscai lineage (reviewed in Oosterbroek and Arntzen, 1992).
The monophyly of the genus Triturus, although widely accepted, is defined on the basis of somewhat vague character state descriptions (‘a suite of behavioral character states’ and ‘a high level of sexual dimorphism’ - Halliday, 1977), that are not explicit synapomorphies. Moreover, a feature such as the potential for interbreeding (Wolterstorff and Herre, 1935) is explicitely a plesiomorphy.The monophyly of the genus Triturus has recently been put into question in a molecular phylogenetic study of the family Salamandridae (Titus and Larson, 1995) and in a molecular biogeographical study of Euproctus (Caccone et al., 1994). In both of them only two Triturus species were involved. With just the representatives of the genera Cynops, Neurergus and Paramesotriton as out-groups, a denser in-group taxon sampling does not challenge the hypothesis of Triturus monophyly. However, 12S and 16S mtDNA sequence data indicate that the genera Cynops, Neurergus and Paramesotriton themselves may be in-group taxa relative to Triturus (Fig. 4B). In the light of these results, their a priori choice as out-groups to the genus Triturus may have been unfortunate.