Abstract
In amphibians, morphological differentiation and disparity at the larval and post-metamorphic ontogenetic stages can diverge, owing to various contrasting environments and different selective pressures. In the monophyletic clade of nine Triturus newt species, five different morphotypes can be recognized, but information on larval morphology is limited. Here we explore divergence of larval morphology in Triturus ivanbureschi, T. macedonicus, and their F1 hybrids. These two genetically and morphologically distinct crested newt species hybridize in nature and form a relatively wide hybrid zone in the central part of the Balkan Peninsula. Using a geometric morphometric approach and multivariate statistics, we evaluated differences of tail size and shape, colouration pattern, and the presence of a tail filament at the mid-larval stage in larvae reared under controlled laboratory conditions. We chose the tail as the main propulsive organ crucial for locomotion, feeding, and escaping predators. We found that Triturus ivanbureschi and T. macedonicus larvae differ in tail shape, but not in tail size. Two groups of F1 hybrid larvae (obtained from reciprocal crossing) were similar to each other, but differed from the parental species in size and shape of the tail, colouration pattern, and the presence of a tail filament. Our results indicate that, like adults, larvae diverge morphologically and hybrid larvae do not exhibit intermediate morphology of the parental species.
Introduction
In amphibians, the free-living aquatic larvae phenotypically differ greatly from the post-metamorphic stages and adults. Larval and metamorphosed stages are adapted to various contrasting environments and are exposed to different selective pressures. Therefore, morphological divergence among species at the larval and adult stages is not necessarily equivalent (Escoriza and Hassine, 2017; Sherratt et al., 2017). Within salamandrid salamanders, large-bodied newts of the genus Triturus have been widely studied. The morphological differentiation of Triturus species is primarily based on the Wolterstorff index or the relation between trunk length and limb length (Wolterstorff, 1923; Arntzen and Wallis, 1994). Trunk lengthening is characterized by increase in the number of vertebrae in the trunk region, which coincides with the annual number of months that adults spend in the water (Arntzen, 2003; Slijepčević et al., 2015). Accordingly, the nine Triturus species (Wielstra et al., 2013; Wielstra and Arntzen, 2016) can be sorted into different ecomorphotypes which form a cline from the predominantly terrestrial T. marmoratus and T. pygmaeus with a short and stout body and 12 thoracic vertebrae to the slender and elongated largely aquatic T. cristatus and T. dobrogicus with 15-17 thoracic vertebrae (Arntzen, 2003; Slijepčević et al. 2015). Recent studies of morphological differentiation between Triturus species explored divergences in adult body form (Vukov et al., 2011; Ivanović and Arntzen, 2014), the axial skeleton (Arntzen et al., 2015; Slijepčević et al., 2015), vertebra size and shape (Ratnikov and Litvinchuk, 2007, 2009; Urošević et al., 2016; Govedarica et al., 2017), the limb skeleton (Ivanović et al., 2008b; Tomašević Kolarov, Ivanović and Kalezić, 2011), the cranial skeleton (Ivanović et al., 2007, 2008a, 2013; Cvijanović et al., 2014), and colouration patterns (e.g. Arntzen and Wallis, 1999; Arntzen, 2003). On the contrary, information on larval morphological variation is scarce. Some differences of body shape among T. dobrogicus, T. cristatus, T. ivanbureshi, and T. macedonicus were found at the mid-larval stage, with T. ivanbureschi (syn. T. arntzeni) and T. macedonicus being more similar to each other than to T. dobrogicus and T. cristatus (Ivanović, Cvijanović and Kalezić, 2011). However, at the time when the latter study was conducted, genetic structure of the population was unknown, and the population used to represent T. ivanbureschi (syn. T. arntzeni) belonged to the T. macedonicus and T. ivanbureschi hybrid zone (Arntzen, Wielstra and Wallis, 2014). Differences of pigmentation pattern in Triturus larvae were also found (Litvinchuk and Borkin, 2009; Cvijanović, Ivanović and Kalezić, 2015).
It is well known that phenotypic plasticity affects larval morphological traits due to different environmental factors, such as the presence of predators or canopy cover (Van Buskirk and Schmidt, 2000; Schmidt and Van Buskirk, 2005; Van Buskirk, 2009, 2011). However, information about changes in Triturus larval morphological traits resulting from hybridization is obscure. Hybridization is an important force in shaping Triturus interspecific morphological variability (Crnobrnja-Isailović et al., 1997; Arntzen et al., 2009; Slijepčević et al., 2015). Thus, hybrid phenotypes can be similar to paternal or maternal species due to dominance or the maternal effect, intermediate between parental species, or they can exceed the values of both parental phenotypes (Spurway, 1953; Vallée, 1959; Pfennig, Chunco and Lackey, 2007; Vinšálková and Gvoždík, 2007; Litvinchuk and Borkin, 2009; Slijepčević et al., 2015).
Two genetically distinct species, Triturus ivanbureschi and T. macedonicus, belong to well-separated lineages within the monophyletic clade of large-bodied newts (Wielstra and Arntzen, 2011). They hybridize in nature and form a relatively wide hybrid zone in the central and eastern part of Serbia (Arntzen, Wielstra and Wallis, 2014). Triturus macedonicus has widened its range and transected the T. ivanbureschi range, forming an introgression zone (ca. 54,000 km2). Within the introgression zone, hybrid individuals have T. macedonicus nuclear DNA, but T. ivanbureschi mtDNA, which is considered as a T. ivanbureschi footprint of its previous range (Wielstra and Arntzen, 2012). A confirmed natural T. macedonicus-T. ivanbureschi hybrid population is found in eastern Serbia (Wielstra and Arntzen, 2014; Wielstra et al., 2014).
We here explore the morphological variation and examine the differences between T. macedonicus and T. ivanbureschi and their F1 hybrids [obtained from reciprocal crossing T. ivanbureschi (♀) × T. macedonicus (♂) and T. macedonicus (♀) × T. ivanbureschi (♂)] at the mid-larval stage. Our aims were to determine if there are differences of tail morphology between the two species and ascertain the position of F1 hybrid phenotypes relative to the parental phenotypes. We chose to analyse tail morphology because the tail is the main propulsive organ at the larval stage allowing locomotion while foraging or escaping predators.
Materials and methods
Experimental setting
Adults of Triturus ivanbureschi were collected from Zli Do, Serbia (42°25′N; 22°27′E). Triturus macedonicus adults were collected from Ceklin, Montenegro (42°21′N; 18°59′E). Both populations are south of the hybrid zone and have known genetic characteristics (Wielstra et al., 2013). Triturus ivabureschi larvae were obtained from six gravid females collected in the spring of 2014 and transferred to the laboratory of the Institute for Biological Research “Siniša Stanković”. Other larvae were obtained from experimental crossings performed after hibernation in the laboratory. To prevent insemination before experimental crossing, females and males were kept separately. Experimental mating of 1) T. macedonicus ♀ × ♂ (T. macedonicus larvae), 2) T. ivanbureschi ♀ × T. macedonicus ♂ (T. ivanbureschi-mothered F1 hybrids), and 3) T. macedonicus ♀ × T. ivanbureschi ♂ (T. macedonicus-mothered F1 hybrids) were carried out in March of 2017. After insemination, four to eight females per crossing were transferred to separate aquaria to lay eggs. Plastic strips were provided for oviposition and eggs were collected daily. Eggs and embryos were kept submerged in dechlorinated tap water in plastic Petri dishes until hatching.
In order to minimize environmental induction of morphological variation, we raised larvae under the same, controlled laboratory conditions. Hatchlings were transferred to separate plastic containers half filled with dechlorinated tap water (a single individual per container). The temperature was kept stable between 18 and 19°C. Water was changed every other day. Larvae were fed with Artemia sp. at early stages and Tubifex sp. at later stages every second day.
Data collection
The analysed larvae (N = 150) had fully developed limbs and tail (at the beginning of stage 62 according to Glücksohn, 1932). The following sample sizes were analysed: T. ivanbureshi, N = 32; T. macedonicus, N = 36; T. ivanbureschi-mothered F1 hybrids, N = 35; and T. macedonicus-mothered F1 hybrids, N = 47. Larvae were photographed in Petri dishes positioned in the centre of the optical field, sideways with their left body side uppermost using a Moticam 2000 camera attached to a Nikon SMZ800 stereo zoom microscope and a Sony DSC-F828 digital camera. The distance to the stereo microscope or camera lens was consistent to minimize and equalize the distortion and image aberration because of parallax (Mullin and Taylor, 2002).
To capture tail shape and size, 17 landmarks (fig. 1a) were digitized in the tpsDig2 program (Rohlf, 2006). Landmarks 1 and 2 were positioned at the base of the tail at the level of the outer edge of the cloaca and insertion of the hind limbs. Landmark 9 was the tip of the tail. Other landmarks were equally spaced along the outer edge of the tail fin and tail muscle. To obtain equal angular spacing between landmarks, we used the MakeFan6 program from the Integrated Morphometrics Program (IMP) series (Sheets, 2000).
Analysed morphometric traits. (a) Location of landmarks of larval tail shape. (b) Analysed tail traits and linear measurements: tail length (TL), maximum tail height (MTH), tail muscle height (TMH).
Citation: Amphibia-Reptilia 39, 1 (2018) ; 10.1163/15685381-17000190
We also measured maximum tail height (MTH) and tail muscle height (TMH). Tail length (TL) was measured as the distance between the outer edge of the cloaca and the tail tip (fig. 1b). Snout to vent length (SVL) was determined as the distance from the snout tip to the outer edge of the cloaca. All linear measurements were obtained from photographs using the TMorphGen6 program from the IMP package (Sheets, 2000).
Centroid size (CS) was calculated in the CoordGen6 program from the IMP package (Sheets, 2000). Shape coordinates (Procrustes coordinates) were obtained by Generalized Procrustes Analysis (GPA, Rohlf and Slice, 1990; Dryden and Mardia, 1998) using the MorphoJ software package (Klingenberg, 2011).
Additionally, we examined four tail traits (fig. 1b): 1) the marble colouration pattern (MCP); 2) the amount of dark blotches on the tail edge (DBE); 3) the presence of dark blotches in the tail muscle area (DBM); and 4) the presence of a tail filament (TF).
Statistical analyses
Prior to statistical analyses, linear data were checked for distribution to meet the assumptions of normality.
To take into account effects of larval size on the measured traits, we calculated residual values from the regression of MTH, TMH and TL against SVL. The differences between groups were analysed by ANCOVA with SVL as an independent variable and the residuals of MTH, TMH and TL as dependent variables. The Tukey HSD post-hoc test was used to determine the significance of differences between specific groups. All analyses were made in Statistica 10 software (StatSoft Inc., 2011).
As size-related change in shape (allometry) could account for a significant amount of total variation, multivariate regression of shape variables on log-transformed CS was performed as a preliminary analysis (Monteiro, 1999). For the regression, statistical significance was determined by a permutation test with 1000 iterations (Klingenberg, 2011). Multivariate regression showed that 8.91% of the variation in shape is related to the size. Therefore, residuals from multivariate regression that represent the size-corrected or non-allometric component of shape variation were used in subsequent statistical analyses. Principal Component Analysis (PCA) was done to explore the variation in tail shape within and between species and hybrids. Procrustes distances between mean tail shapes were calculated between each taxon and hybrids. The statistical significance of differences in tail shape was determined using a permutation test with 1000 iterations. The obtained P-values were adjusted with the Bonferroni correction for multiple comparisons. All landmark-based analyses were made using the MorphoJ software package (Klingenberg, 2011).
Correspondence analysis and the G test were applied to evaluate differences in colouration pattern and the presence of a tail tip among species and between species and hybrids using Statistica 10 software (StatSoft Inc., 2011) and Microsoft Excel 2010.
Mean values with standard errors (SE) for the measured traits [snout to vent length (SVL), centroid size (CS), tail length (TL), maximum tail height (MTH), and tail muscle height (TMH)] in the four analysed groups and ANCOVA test results for differences between means with SVL as the covariable and residual values of TL, MTH, and TMH as dependent variables. Species acronyms: T. ivanbureshi-T. iva; T. macedonicus-T. mac.
Results
The ANCOVA test (table 1) showed statistically significant differences between the groups for tail length (TL) and tail muscle height (TMH), but not for maximum tail height (MTH). For TL, the pairwise comparisons (the Tukey HSD test) revealed statistically significant differences only between T. macedonicus and T. macedonicus-mothered F1 hybrids (). For TMH, Triturus ivanbureschi differed from all other groups (). No differences were found between T. macedonicus and hybrid groups ().
Position of larvae in morphospace defined by the first two principal component axes. Confidence ellipses are sized to comprise 75% probability that new sampling would overlap the calculated group’s mean tail shape. The wireframe graph describes shape changes between individuals with maximal scores on the PC1 and PC2 axes. Grey – mean shape; black – shape corresponding to the maximal positive and negative scores. Species acronyms: T. ivanbureshi-T. iva; T. macedonicus-T. mac.
Citation: Amphibia-Reptilia 39, 1 (2018) ; 10.1163/15685381-17000190
The first and second PC axes described 57.31% of total variance (fig. 2). Triturus macedonicus was separated along the first axis from T. ivanbureschi and both hybrid groups. Triturus macedonicus larvae have shorter, but wider tails. The PC2 axis (25.61% of total variance) mostly described within-group variability in the upper or lower direction of the tail tip.
Statistically significant differences in tail shape were found between all compared pairs, except between the two groups of hybrids. The largest Procrustes distances between mean shapes were between the parental species, Triturus ivanbureschi and T. macedonicus (table 2).
Procrustes distances and P-values from permutation tests (with 1000 permutation rounds and the Bonferroni correction for multiple testing). Species acronyms: T. ivanbureshi-T. iva; T. macedonicus-T. mac.
For the colouration pattern and presence of a tail filament, the following character states were recognized (fig. 3):
- 1. the marble colouration pattern (MCP): close to the tail edge (1), in the middle of the ventral and dorsal parts (2), uniform on both sides (3);
- 2. the amount of dark blotches on the tail edge (DBE): sparse, between 0-5 (1), moderate, between 6-9 (2), numerous, (3);
- 3. the presence of dark blotches in the tail muscle area (DBM): absent (0), present (1); and
- 4. the presence of a tail filament (TF): absent (0), present (1).
Schematic presentation of character states for the chosen tail traits: marble colouration pattern (MCP), amount of the dark blotches on the tail edge (DBE), presence of dark blotches in the tail muscle area (DBM), and presence of a tail filament (TF). See text for detailed information on character states.
Citation: Amphibia-Reptilia 39, 1 (2018) ; 10.1163/15685381-17000190
Correspondence analysis ordination plot and position of the four analysed groups relative to the analysed traits: marble colouration pattern (MCP), amount of dark blotches on the tail edge (DBE), presence of dark blotches in the tail muscle area (DBM), and presence of a tail filament (TF).
Citation: Amphibia-Reptilia 39, 1 (2018) ; 10.1163/15685381-17000190
Correspondence analysis showed that the first two dimensions explain 97.25% of total inertia (fig. 4). Hybrid groups (T. ivanbureschi-mothered F1 hybrids and T. macedonicus-mothered F1 hybrids) clustered together. The characters whose frequencies contribute to differentiation of the hybrid groups from parental species were the presence of a filament at the tail tip (TF1), location of the marble colouration pattern near the edge of the tail (MCP1), and the presence of numerous blotches on the edge of the tail (DBE3). The amount of dark blotches in the tail muscle area (DBM1) distinguished T. ivanbureschi from the other groups. Triturus macedonicus larvae have a uniformly distributed marble colouration pattern on the dorsal and ventral parts of the tail (MCP3) (fig. 4).
Statistically significant differences in frequencies were found for all analysed tail traits among the groups: MCP (G test = 119.49, ), DBE (G test = 14.14, ), DBM (G test = 45.37, ), and TF (G test = 108.97, ). Pairwise comparison (table 3) showed that group pairs differed in the frequency of position of the marble colouration pattern (MCP), except in comparison between hybrid groups. Triturus macedonicus-mothered F1 hybrids differed from T. ivanbureschi-mothered F1 hybrids and T. macedonicus, but not from T. ivanbureschi in frequency of the amount of dark blotches at the edge of the tail (DBE). Triturus ivanbureschi differed from the other groups in frequency of the presence of dark blotches in the middle of the tail (DBM). Hybrid groups diverged from parental species in frequency of the presence of a filament at the tail tip (TF).
Comparison of the frequencies of character states for the following tail traits: MCP – marble colouration pattern; DBE – amount of dark blotches on the tail edge; DBM – presence of dark blotches in the tail muscle area; TF – presence of a tail filament. Species acronyms: T. ivanbureshi-T. iva; T. macedonicus-T. mac.
Discussion
Our analyses of variation in larval tail morphology between two Triturus species (T. ivanbureschi and T. macedonicus) and their F1 hybrids indicated that morphological differentiation is present in their larval stages. Triturus ivanbureschi and T. macedonicus differ in tail shape. Significant differences between the two species were also found for tail muscle height and colouration pattern. The F1 hybrid larvae diverge from parental species in tail size and shape, and have a characteristic pigmented filament at the tail tip. No differences were found between the two groups of F1 hybrids obtained by reciprocal crossing.
Divergence between the parental species
The compared species belong to two different lineages with the most recent common ancestor estimated to have lived ca. 9.4 million years ago (Ivanović and Arntzen, 2014). Differing characteristics of adult phenotypes of the two species in question are already known (see Arntzen, 2003; Litvinchuk and Borkin, 2009; Vukov et al., 2011). We found that at the mid-larval stage, Triturus ivanbureschi larvae have a more muscular tail with shallower fins in comparison with T. macedonicus larvae. As for general tail shape, T. ivanbureschi larvae have a relatively narrower tail compared to T. macedonicus larvae (fig. 2). Also, these two species differ in the position of marble colouration patterns and the presence of dark blotches in the middle of the tail. Triturus macedonicus larvae have a uniformly distributed marble colouration pattern without dark blotches in the middle of the tail, while T. ivanbureschi larvae have a centrally placed marble pattern below and above the lateral line with dark blotches in the middle of the tail (figs 3 and 4).
Divergence between parental species and their hybrids
Previous studies on Triturus newts indicated that hybrids have intermediate morphological traits. This is the case with crosses between Triturus cristatus and T. marmoratus (e.g. Francillon-Vieillot, Arntzen and Géraudie, 1990; Arntzen and Wallis, 1994; Slijepčević et al., 2015), T. cristatus and T. carnifex (Brede et al., 2000), T. carnifex and T. dobrogicus (Vinšálková and Gvoždík, 2007), T. carnifex and T. karelinii (Litvinchuk and Borkin, 2009), and T. dobrogicus and T. karelinii (Litvinchuk and Borkin, 2009). Our study showed that at the larval stage hybrids have intermediate values of tail muscle height relative to the parental species, while T. macedonicus-mothered F1 hybrids have a relatively longer tail than in the maternal species.
The two hybrid groups obtained from crossing T. ivanbureschi (♀) with T. macedonicus (♂) and T. macedonicus (♀) with T. ivanbureschi (♂) were very similar to each other with no differences of tail size or shape. Hybrid larvae also had similar frequencies of external morphological traits, including marble colouration patterns close to the dorsal and ventral edges without dark blotches in the middle part of the tail. However, they diverged from both parental species by having a long filament at the tip of the tail. There has been evidence suggesting that an elongated tail with a tail filament can distract predators from the head and body, thereby increasing the survival rate (Van Buskirk et al., 2003). However, a future survey of the presence of larval tail filament in natural populations outside and within the hybrid zone would provide additional information about the distribution and specificity of this trait in hybrid populations.
Tail morphology and possible functional differences
The functional consequences of tail size and shape variation have been well studied (Webb, 1984; Wassersug and Hoff, 1985; Wassersug, 1989; Weihs, 1989; Liu, Wassersug and Kawachi, 1996). With respect to amphibians, investigations of tail morphological variation have mostly been concerned with swimming performance and response to predators: size-specific swimming performance was significantly related to tail length (see Ackerly and Ward, 2015), but not to tail depth (Van Buskirk and McCollum, 2000). Tail depth (maximum tail height) is important for effectively escaping predators (Van Buskirk, McCollum and Werner, 1997; Van Buskirk and Schmidt, 2000). We detected no difference of tail size, but significant differences of tail shape between T. ivanbureschi and T. macedonicus larvae. Within- and among-population variation of plastic responses in amphibian larvae is well-known (e.g. Laurila, Karttunen and Merilä, 2002) and has been observed in the case of newt larval morphology (Van Buskirk and Schmidt, 2000). T. ivanbureschi and T. macedonicus reproduce in similar aquatic habitats and are exposed to extremely variable environmental conditions within the habitat (Džukić, Vukov and Kalezić, 2016). As we compared larvae raised in the same controlled laboratory conditions, the observed divergence of tail shape most likely is a result of the relatively long independent evolution of these two species.
Many animals, including amphibians, have a homoplastic larval body shape when compared to adults (Smith, Littlewood and Wray, 1995; Wiens, Bonett and Chippindale, 2005). Our results, together with previous findings on morphological divergence of larval body shape in Triturus newts (Ivanović, Cvijanović and Kalezić, 2011) indicate that morphological disparity characterizes premetamorphic stages in this group. However, further studies are needed in order to explore possible ontogenetic changes in morphological disparity during ontogeny. With well resolved phylogenetic relationships (Arntzen et al., 2015; Wielstra and Arntzen, 2016), the Triturus newt group represents a valuable model system for studies of the evolution of ontogenetic trajectories, morphological disparity, and phenotypic plasticity in organisms with a complex biphasic life cycle.
Acknowledgements
We would like to thank two anonymous reviewers for their useful comments and suggestions that improved the interpretation of our data. We also thank undergraduate students of the Faculty of Biology, University of Belgrade for their technical assistance. This work was supported by the Serbian Ministry of Education, Science, and Technological Development (project No. 173043). The experiments were approved by the Ethical Committee of the Institute for Biological Research “Siniša Stanković” (decisions nos. 01-05/14 and 03-03/16). All animals were collected under permits provided by the Ministry of Energy, Development, and Environmental Protection of the Republic of Serbia (permit no. 353-01-75/2014-08) and the Environmental Protection Agency of Montenegro (permit no. UPI-328/4).
References
Ackerly K.L., Ward A.B. (2015): Linking vertebral number to performance of aquatic escape responses in the axolotl (Ambystoma mexicanum). Zoology. 118: 394-402.
Arntzen J.W. (2003): Triturus cristatus Superspecies – Kammolch-Artenkreis (Triturus cristatus (Laurenti, 1768) – Nördlicher Kammolch, Triturus carnifex (Laurenti, 1768) – Italienischer Kammolch, Triturus dobrogicus (Kiritzescu, 1903) – Donau-Kammolch, Triturus karelinii (Strauch, 1870) – Südlicher Kammolch). In: Handbuch der Reptilien und Amphibien Europas. Schwanzlurche IIA, p. 421-514. Böhme W., Ed., Aula-Verlag, Wiebelsheim.
Arntzen J.W., Wallis G.P. (1994): The ‘wolterstorff index’ and its value to the taxonomy of the crested newt superspecies. Abh. Ber. Naturk. 17: 57-66.
Arntzen J.W., Wallis G.P. (1999): Geographic variation and taxonomy of crested newts (Triturus cristatus superspecies): morphological and mitochondrial data. Contribut. Zool. 68: 181-203.
Arntzen J.W., Jehle R., Bardakci F., Burke T., Wallis G.P. (2009): Asymmetric viability of reciprocal-cross hybrids between crested and marbled newts (Triturus cristatus and T. marmoratus). Evolution. 63: 1191-1202.
Arntzen J.W., Wielstra B., Wallis G.P. (2014): The modality of nine Triturus newt hybrid zones assessed with nuclear, mitochondrial and morphological data. Biol. J. Linn. Soc. 113: 604-622.
Arntzen J.W., Beukema W., Galis F., Ivanović A. (2015): Vertebral number is highly evolvable in salamanders and newts (family Salamandridae) and variably associated with climatic parameters. Contribut. Zool. 84: 85-113.
Brede E.G., Thorpe R.S., Arntzen J.W., Langton T.E.S. (2000): A morphometric study of a hybrid newt population (Triturus cristatus/T. carnifex): Beam Brook Nurseries, Surrey, UK. Biol. J. Linn. Soc. 70: 685-695.
Crnobrnja-Isailović J., Džukić G., Krstić N., Kalezić M.L. (1997): Evolutionary and paleogeographical effects on the distribution of the Triturus cristatus superspecies in the central Balkans. Amphibia-Reptilia. 18: 321-332.
Cvijanović M., Ivanović A., Kalezić M.L., Zelditch M.L. (2014): The ontogenetic origins of skull shape disparity in the Triturus cristatus group. Evol. Dev. 16: 306-317.
Cvijanović M., Ivanović A., Kalezić M.L. (2015): Larval pigmentation patterns of closely related newt species (Triturus cristatus and T. dobrogicus) in laboratory conditions. North-West. J. Zool. 11: 357-359.
Dryden I.L., Mardia K.V. (1998): Statistical Analysis of Shape. Wiley.
Džukić G., Vukov T.D., Kalezić M.L. (2016): The Tailed Amphibians of Serbia. Belgrade. Serbian Academy of Science and Arts.
Escoriza D., Hassine J.B. (2017): Comparative larval morphology in three species of Pleurodeles (Urodela: Salamandridae). Zootaxa. 4237: 587-592.
Francillon-Vieillot H., Arntzen J.W., Géraudie J. (1990): Age, growth and longevity of sympatric Trituruscristatus, T. marmoratus and their hybrids (Amphibia, Urodela): a skeletochronological comparison. J. Herpetol. 24: 13-22.
Glücksohn S. (1932): Äußere Entwicklung der Extremitäten und Stadieneinteilung der Larvenperiode von Triton taeniatus Leyd. und von Triton cristatus Laur. Wilhelm Roux’ Archiv f. Entwicklungsmechanik d. Organismen. 125: 341-405.
Govedarica P., Cvijanović M., Slijepčević M., Ivanović A. (2017): Trunk elongation and ontogenetic changes in the axial skeleton of Triturus newts. J. Morphol. DOI:10.1002/jmor.20733.
Ivanović A., Arntzen J.W. (2014): Evolution of skull and body shape in Triturus newts reconstructed from three-dimensional morphometric data and phylogeny. Biol. J. Linn. Soc. 113: 243-255.
Ivanović A., Vukov T.D., Džukić G., Tomašević N., Kalezić M.L. (2007): Ontogeny of skull size and shape changes within a framework of biphasic lifestyle: a case study in six Triturus species (Amphibia, Salamandridae). Zoomorphology. 126: 173-183.
Ivanović A., Sotiropoulos K., Vukov T.D., Eleftherakos K., Džukić G., Polymeni R.M., Kalezić M.L. (2008a): Cranial shape variation and molecular phylogenetic structure of crested newts (Trituruscristatus superspecies: Caudata, Salamandridae) in the Balkans. Biol. J. Linn. Soc. 95: 348-360.
Ivanović A., Tomašević N., Džukić G., Kalezić M.L. (2008b): Evolutionary diversification of the limb skeleton in crested newts (Trituruscristatus superspecies, Caudata, Salamandridae). Ann. Zool. Fenn. 45: 527-535.
Ivanović A., Cvijanović M., Kalezić M.L. (2011): Ontogeny of body form and metamorphosis: insights from the crested newts. J. Zool. 283: 153-161.
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 (Trituruskarelinii group) reflected by differentiation of skull shape?. Zool. Anz. 252: 269-277.
Klingenberg C.P. (2011): MorphoJ: an integrated software package for geometric morphometrics. Mol. Eco. Resour. 11: 353-357.
Laurila A., Karttunen S., Merilä J. (2002): Adaptive phenotypic plasticity and genetics of larval life histories in two Rana temporaria populations. Evolution. 56: 617-627.
Litvinchuk S.N., Borkin L.J. (2009): Evolution, Systematics and Distribution of Crested Newts (Triturus Cristatus Complex) in the Territory of Russia and Adjacent Countries. Evropeyskiy dom, St. Petersburg.
Liu H., Wassersug R., Kawachi K. (1996): A computational fluid dynamics study of tadpole swimming. J. Exp. Biol. 199: 1245-1260.
Monteiro L.R. (1999): Multivariate regression models and geometric morphometrics: the search for causal factors in the analysis of shape. Syst. Biol. 48: 192-199.
Mullin S.K., Taylor P.J. (2002): The effects of parallax on geometric morphometric data. Comput. Biol. Med. 32: 455-464.
Pfennig K.S., Chunco A.J., Lackey A.C. (2007): Ecological selection and hybrid fitness: hybrids succeed on parental resources. Evol. Ecol. Res. 9: 341-354.
Ratnikov V.Y., Litvinchuk S.N. (2007): Comparative morphology of trunk and sacral vertebrae of tailed amphibians of Russia and adjacent countries. Russ. J. Herpetol. 14: 177-190.
Ratnikov V.Y., Litvinchuk S.N. (2009): Atlantal vertebra of tailed amphibians of Russia and adjacent countries. Russ. J. Herpetol. 16: 57-68.
Rohlf F.J. (2006): tpsDig, version 2.26. State Univ. of New York, Stony Brook, New York. Available at: http://life.bio.sunysb.edu/morph/.
Rohlf F.J., Slice D. (1990): Extensions of the Procrustes method for the optimal superimposition of landmarks. Syst. Biol. 39: 40-59.
Schmidt B.R., Van Buskirk J. (2005): A comparative analysis of predator-induced plasticity in larval Triturus newts. J. Evolution. Biol. 18: 415-425.
Sheets H.D. (2000): Integrated morphometrics package (IMP). Available at: http://www2.canisius.edu/~sheets.
Sherratt E., Vidal-García M., Anstis M., Keogh J.S. (2017): Adult frogs and tadpoles have different macroevolutionary patterns across the Australian continent. Nat. Ecol. Evol. 1: e1385.
Slijepčević M., Galis F., Arntzen J.W., Ivanović A. (2015): Homeotic transformations and number changes in the vertebral column of Triturus newts. PeerJ. 3: e1397.
Smith A.B., Littlewood D.T.J., Wray G.A. (1995): Comparing patterns of evolution: larval and adult life history stages and ribosomal RNA of post-Palaeozoic echinoids. Philos. T. Roy. Soc. B. 349: 11-18.
Spurway H. (1953): Genetics of specific and subspecific differences in European newts. In: Symposia of the Society for Experimental Biology, Number VII. Evolution. Cambridge University Press.
Tomašević Kolarov N., Ivanović A., Kalezić M.L. (2011): Morphological integration and ontogenetic niche shift: a study of crested newt limbs. J. Exp. Zool. Part B. 316: 296-305.
Urošević A., Slijepčević M.D., Arntzen J.W., Ivanović A. (2016): Vertebral shape and body elongation in Triturus newts. Zoology. 119: 439-446.
Vallée L. (1959): Recherches sur Triturus blasii de l’Isle, hybride naturel de Triturus cristatus Laur. x Triturus marmoratus Latr. B. Soc. Zool. Fr. 31: 1-95.
Van Buskirk J. (2009): Natural variation in morphology of larval amphibians: phenotypic plasticity in nature?. Ecol. Monogr. 79: 681-705.
Van Buskirk J. (2011): Amphibian phenotypic variation along a gradient in canopy cover: species differences and plasticity. Oikos. 120: 906-914.
Van Buskirk J., McCollum S.A. (2000): Functional mechanisms of an inducible defense in tadpoles: morphology and behavior influence mortality risk from predation. J. Evolution. Biol. 13: 336-347.
Van Buskirk J., Schmidt B.R. (2000): Predator-induced phenotypic plasticity in larval newts: trade-offs, selection, and variation in nature. Ecology. 81: 3009-3028.
Van Buskirk J., McCollum S.A., Werner E.E. (1997): Natural selection for environmentally induced phenotypes in tadpoles. Evolution. 51: 1983-1992.
Van Buskirk J., Anderwald P., Lüpold S., Reinhardt L., Schuler H. (2003): The lure effect, tadpole tail shape, and the target of dragonfly strikes. J. Herpetol. 37: 420-424.
Vinšálková T., Gvoždík L. (2007): Mismatch between temperature preferences and morphology in F1 hybrid newts (Triturus carnifex × T. dobrogicus). J. Therm. Biol. 32: 433-439.
Vukov T.D., Sotiropoulos K., Wielstra B., Džukić G., Kalezić M.L. (2011): The evolution of the adult body form of the crested newt (Triturus cristatus superspecies, Caudata, Salamandridae). J. Zool. Syst. Evol. Res. 49: 324-334.
Wassersug R.J. (1989): Locomotion in amphibian larvae (or “Why aren’t tadpoles built like fishes?”). Am. Zool. 29: 65-84.
Wassersug R.J., Hoff K.V.S. (1985): The kinematics of swimming in anuran larvae. J. Exp. Biol. 119: 1-30.
Webb P.W. (1984): Body form, locomotion and foraging in aquatic vertebrates. Am. Zool. 24: 107-120.
Weihs D. (1989): Design features and mechanics of axial locomotion in fish. Am. Zool. 29: 151-160.
Wielstra B., Arntzen J.W. (2011): Unraveling the rapid radiation of crested newts (Triturus cristatus superspecies) using complete mitogenomic sequences. BMC Evol. Biol. 11: 162.
Wielstra B., Arntzen J.W. (2012): Postglacial species displacement in Triturus newts deduced from asymmetrically introgressed mitochondrial DNA and ecological niche models. BMC Evol. Biol. 12: 161.
Wielstra B., Arntzen J.W. (2014): Kicking Triturusarntzeni when it’s down: large-scale nuclear genetic data confirm that newts from the type locality are genetically admixed. Zootaxa. 3802: 381-388.
Wielstra B., Arntzen J.W. (2016): Description of a new species of crested newt, previously subsumed in Triturus ivanbureschi (Amphibia: Caudata: Salamandridae). Zootaxa. 4109: 73-80.
Wielstra B., Crnobrnja-Isailović J., Litvinchuk S.N., Reijnen B.T., Skidmore A.K., Sotiropoulos K., Toxopeus A.N., Tzankov N., Vukov T., Arntzen J.W. (2013): Tracing glacial refugia of Triturus newts based on mitochondrial DNA phylogeography and species distribution modeling. Front. Zool. 10: 13.
Wielstra B., Duijm E., Lagler P., Lammers Y., Meilink W.R.M., Ziermann J.M., Arntzen J.W. (2014): Parallel tagged amplicon sequencing of transcriptome-based genetic markers for Triturus newts with the ion torrent next-generation sequencing platform. Mol. Ecol. Resour. 14: 1080-1089.
Wiens J.J., Bonett R.M., Chippindale P.T. (2005): Ontogeny discombobulates phylogeny: paedomorphosis and higher-level salamander relationships. Syst. Biol. 54: 91-110.
Wolterstorff W. (1923): Übersicht den Unterarten und Formen des Triton cristatus Laur. Blätter für Aquarien- und Terrarien-kunde. 34: 120-126.
Footnotes
Associate Editor: Judit Vörös.
