Balanced Lethal Systems: An Evolutionary Mystery

When a female crested newt lays her clutch of about three-hundred eggs, half of these will never hatch. What an incredible waste! It turns out that these newts suffer from a deadly hereditary disease called a balanced lethal system. Here is how it works: in a balanced lethal system, there are two distinct versions of a particular chromosome. Newts need both to live. However, which version a fertilized newt egg gets from its father and mother is random. Therefore, there is a 50% chance that it receives the same version twice—and misses the other half of the critical pair. Hence, half of the eggs end up with the wrong chromosome combination and die. The newts are not the only example; balanced lethal systems are found in some plants and insects as well. Why something so disadvantageous as a balanced lethal system would ever evolve is still a big, evolutionary mystery!

Oxidative Stress Parameters in Goitrogen-Exposed Crested Newt Larvae (Triturus spp.): Arrested Metamorphosis

Thiourea is an established disruptor of thyroid hormone synthesis and is frequently used as an inhibitor of metamorphosis. The changes caused by thiourea can affect processes associated with the oxidative status of individuals (metabolic rate, the HPI axis, antioxidant system). We investigated the parameters of oxidative stress in crested newt (Triturus spp.) larvae during normal development in late larval stage 62 and newly metamorphosed individuals, and during thiourea-stimulated metamorphosis arrest in individuals exposed to low (0.05%) and high (0.1%) concentrations of thiourea. Both groups of crested newts exposed to thiourea retained their larval characteristics until the end of the experiment. The low activities of antioxidant enzymes and the high lipid peroxidation level pointed to increased oxidative stress in larvae at the beginning of stage 62 as compared to fully metamorphosed individuals. The activities of catalase (CAT) and glutathione-S-transferase (GST) and the concentration of sulfhydryl (SH) groups were significantly lower in larvae reared in aqueous solutions containing thiourea than in newly metamorphosed individuals. The high thiourea concentration (0.1%) affected the antioxidative parameters to the extent that oxidative damage could not be avoided, contrary to a lower concentration. Our results provide a first insight into the physiological adaptations of crested newts during normal development and simulated metamorphosis arrest.

Ontogenetic Changes of the Aquatic Food Uptake Mode in the Danube Crested Newt (Triturus dobrogicus Kiritzescu 1903)

The study of the feeding mechanisms in vertebrates requires an integrative approach since the feeding event consists of a chain of behaviors. In the present study we investigated the food uptake behavior in different ontogenetic stages in the Danube crested newt (Triturus dobrogicus). We focused on the coordination in the kinematics of the elements of the locomotor and the feeding systems at the transition between the approach of the newt to the prey and the food uptake start. In the feeding strategy of the larvae of T. dobrogicus, the phase of food search is replaced by an initial “food detection phase.” In both larvae and adult specimens, the animals approached the food to a close distance by a precise positioning of the snout besides the food item. The larvae were able to reach food items offered at over 80° relative to the longitudinal midline of the head. When the food was offered at a large distance or laterally, the food uptake was either not successful or the coordination chain at the transition between food approach and food uptake was interrupted. In young larvae we detected an abrupt change in the activity of the locomotor system and the feeding system. The larvae approached the food by tail undulation and after reaching the final position of attack, no further activity of the locomotor apparatus was detectable. The larvae used a pure form of inertial suction to ingest food. In pre-metamorphic larvae and adults we registered an integrated activation of the locomotor apparatus (both limbs and tail) and the feeding apparatus during prey capture in the form of compensatory suction. The drastic change in the feeding mode of the pre-metamorphotic larvae and the adults compared to the younger larvae in T. dobrogicus may indicate the evolutionary development of a defined relation in the activity of the locomotor system and the control of the feeding apparatus. We propose that in newts, the interaction between the control execution in both systems switched from successive (body movement – feeding) into integrated (body movement – body movement and feeding) during the ontogeny. The main trigger for such a switch (at least in T. dobrogicus) is the formation of functional limbs during the late larval development.

How reliable is the habitat suitability index as a predictor of great crested newt presence or absence?

The application of a habitat suitability index (HSI) assessment to predict the use of ponds by great crested newts (Triturus cristatus) is commonly used in association with distribution and monitoring projects. Such projects are often used to inform development and planning decision making. However, this type of assessment is frequently misused, and misinterpreted. We used a large, commercially collected environmental DNA (eDNA) survey for great crested newt pond occupancy (489 ponds) to; (1) assess whether it is appropriate to use low HSI scores to rule out occupancy, (2) discuss the use of high HSI scores to identify ponds of high importance for the species and, (3) explore the eDNA detection method. We conclude that there is no evidence to support ruling out pond occupancy based on low HSI scores. However, the conventional view that ponds with HSI scores above 0.7 are of high importance to great crested newts is somewhat supported by the data. Both eDNA and direct observational survey methodologies suffer from sampling error and these need to be acknowledged in the analysis of large data sets.

Establishing Cell Lines from Fresh or Cryopreserved Tissue from the Great Crested Newt (Triturus cristatus): A Preliminary Protocol

This study describes a successful protocol for establishing cell lines from the threatened Triturus cristatus in terms of collection, preparing, establishing, cryopreserving, thawing and quality checking. Different parameters such as media, media change, fresh vs. cryopreserved tissue and seeding density were tested to optimize culture conditions for this species. With fresh tissue, no considerable differences in the use of two different media were found, but with cryopreserved tissue, a combination of ITS (insulin/transferrin/selenite) and 2-mercaptoethanol had a positive effect on growth. Real-time measurements on the cell lines were used, for the first time in amphibian cells, to investigate the effect of different treatments such as media change with or without washing. Media change had a positive impact on the cells, whereas the effect was negative when combined with washing. It is concluded that establishment of cell lines is possible from the great crested newt, especially when using fresh tissue, but much more challenging if the tissue has been cryopreserved. Real-time measurement during cell culture is a useful tool to visualize the sensitivity of amphibian cells during different culture treatments.

Genetic homogeneity in a Pontocaspian crested newt species (Triturus karelinii) suggests recent isolation of its three allopatric range sections

The integration of multilocus datasets and species distribution modelling in phylogeography allows for the reconstruction of more detailed historical biogeographical scenarios than based on mtDNA data alone. We here combine these approaches to investigate the range dynamics of the crested newt Triturus karelinii, an amphibian species endemic to the Pontocaspian region, whose range comprises three allopatric range sections: a Crimean, a Caucasian and a Caspian range section. In a previous mtDNA phylogeographical survey it was suggested that the Caucasian range section was colonized from the Caspian one and that the Crimean range section was subsequently colonized from the Caucasian one. Newly collected nuclear DNA data reveal little genetic differentiation between the three range sections and species distribution modelling suggests that they only recently became isolated. Taken together, our analyses agree with a recent colonization of the Crimean range section, but rather suggest long-term persistence in both the Caspian and Caucasian range sections, with extensive gene flow between the two.

Genetic Divergence Across Glacial Refugia Despite Interglacial Gene Flow in a Crested Newt

MtDNA-based phylogeography has illuminated the impact of the Pleistocene Ice Age on species distribution dynamics and the build-up of genetic divergence. The well-known shortcomings of mtDNA in biogeographical inference can be compensated by integrating multilocus data and species distribution modelling into phylogeography. We re-visit the phylogeography of the Italian crested newt (Triturus carnifex), a species distributed in two of Europe’s main glacial refugia, the Balkan and Italian Peninsulas. While a new 51 nuclear DNA marker dataset supports the existence of three lineages previously suggested by mtDNA (Balkan, northern Italy and southern Italy), the nuclear DNA dataset also provides improved resolution where these lineages have obtained secondary contact. We observe geographically restricted admixture at the contact between the Balkan and northern Italy gene pools and identify a potential mtDNA ghost lineage here. At the contact between the northern and southern Italy gene pools we find admixture over a broader area, as well as asymmetric mtDNA introgression. Our species distribution model is in agreement with a distribution restricted to distinct refugia during Pleistocene glacial cycles and postglacial expansion with secondary contact. Our study supports: (1) the relevance of the north-western Balkan Peninsula as a discrete glacial refugium; (2) the importance of north-eastern Italy and the northern Apennine as suture zones; and (3) the applicability of a refugia-within-refugia scenario within the Italian Peninsula.