Performance of WI and RBV for species diagnosis
In our previous work concerning the capacity of the Wolterstoff Index to discriminate females of the taxa, we identified several potential problems (Arntzen and Wallis, 1994): 1) statistical representation (the need for a mean value), 2) non-biological variation (preservation and measurement differences), 3) sexual variation (WI is higher for males than for females), allometric variation (WI decreases with size), 4) geographic variation (nearby animals may tend to be more similar within species), 5) hybridisation (hybrids between two species can have intermediate values typical of a third species), and 6) circular reasoning (the need for an independent character set to determine the significance of WI). All of these factors to some extent reduce the efficacy of WI and compromise classification made solely on this basis. The WI purports to capture information useful in taxonomy but in fact confounds the variables limb length, vertebral number, and possibly vertebral length.
Using mtDNA haplotype (Wallis and Arntzen, 1989) we showed that WI makes a good approximation to species classification, but can only be used with confidence for discriminating adult T. dobrogicus from the other species. In contrast, the number of rib-bearing vertebrae (RBV) as assessed by radiography eliminates all but one of these problems. Because RBV is a direct discrete meristic count, stable through the lifetime of the individual, and with limited intraspecific geographic differentiation its use in conjunction with a diagnostic genetic character leaves only the issue of hybridisation to be addressed. Variation caused by hybridisation near regions of parapatry is difficult to disentangle from intraspecific geographic variation on the basis of morphological data alone. Under both scenarios, variation will be most clearly expressed when samples from remote parts of the geographic range are compared. However, the observation that character state changes are consistently in the direction to that of the neighbouring species supports the hypothesis of hybridisation, rather than that of intrinsic geographic variation. For example, the significantly different values in T. carnifex macedonicus (high WI, low RBV compared to T. carnifex carnifex) may well be a result of introgression from T. karelinii, whose mtDNA prevails in some T. carnifex macedonicus populations (Wallis and Arntzen, 1989). Note, however, that the meristic count in hybrids is not necessarily intermediate to that of the parental species, as documented for salmonid fishes (Leary, Allendorf and Knudsen, 1985). If hybridisation between taxa of crested newts is a common phenomenon, it should be possible to find genetic markers for the species covarying with interspecific morphological variation. The observed breakdown of the diagnostic power of RBV in areas where taxa meet may reflect the true nature of characters in a contact zone.
It is possible that RBV is influenced early on in development. Indeed, vertebral count is often highly labile in fish and salamanders (e.g. McDowall, 1970; Jockush, 1997), with cooler conditions generally slowing development and increasing several meristic counts (Barlow, 1961 and references in Jockush, 1997). In Triturus vulgaris the average RBV count increases with the temperature at which the embryos are raised (Orska and Imiolek, 1962), while for other salamander species more complicated environmental effects were found (Lindsey, 1966; Peabody and Brodie, 1975). To address the question to what extent the variation in RBV is genetically determined and to gain insight into the relationship between embryonic development and adult morphology requires experimental work. Triturus dobrogicus might be the best species to work with because it is naturally polymorphic for RBV.