Herpetology Notes, volume 10: 255-260 (2017) (published online on 20 May 2017) First record of facultative paedomorphosis in the Macedonian crested newt (Triturus macedonicus) and an additional record for the Greek smooth newt (Lissotriton vulgaris) from Greece: implications on species conservation and preservation of alternative ontogenetic trajectories Konstantinos Sotiropoulos*, Konstantinos Moustakas, Konstantinos Konstantinidis, Vasiliki Mantzana-Oikonomaki, Souzanna Siarabi and Anastasios Bounas Heterochrony, defined as processes altering the rate essential in preservation of the evolutionary potential of or timing of developmental events relative to the same species (Denoël et al., 2005b; Denoël, 2007; Denoël and events in the ancestors, produces phenotypic variation Ficetola, 2015). with significant contribution in micro and macro- Among European newts, facultative paedomorphosis evolutionary processes (Gould, 1977; McNamara, has been frequently reported for the alpine newt, 2012). A case of heterochrony is paedomorphosis, Ichthyosaura alpestris (Laurenti, 1768), and the smooth which is expressed in caudate amphibians as aquatic newt, Lissotriton vulgaris (Linnaeus, 1758) (Kalezić gilled larvae acquire sexual maturity bypassing and Džukić , 1985; Litvinchuk et al., 1996; Denoël metamorphosis. Facultative paedomorphosis is et al., 2001; Sidorovska et al., 2003; Çiçek and Ayaz, frequently observed in newts and salamanders in a large 2011; Covaciu-Markov et al., 2011; Gvozdik et al., diversity of habitats and is an environmentally induced 2013; Litvinchuk, 2013; Pizzuti-Piccoli, 2013; Bozcurt polymorphism that results in the coexistence of both et al., 2015; Kizil et al., 2016). However, facultative phenotypes–the paedomorphs and the metamorphs– in paedomorphosis is rare in the crested newts (Triturus the same reproductive habitat (Semlitsch and Wilbur, species complex). For instance, in the Macedonian 1989; Denoël et al., 2005a). One explanation to the crested newt, Triturus macedonicus (Karaman, 1922), persistence of facultative paedomorphosis in natural only five records from a restricted area in Montenegro populations is that it allows individuals to cope with have been reported so far (Kalezić et al., 1994; Denoël environmental heterogeneity and increase their fitness, et al., 2009; Slijepcevic, 2015). The Montenegrin karst since each phenotype is being adapted to spatially or area is already appreciated as a recognizable centre of temporally available resources (Whiteman, 1994; Ryan facultative paedomorphosis, where local populations and Semlitsch, 1998; Denoël and Ficetola, 2014). This of three different species have found to contain both process, besides its expected contribution towards metamorphic and paedomorphic individuals (Džukić possible speciation events, also increases biodiversity at et al., 1990; Breuil, 1992; Denoël et al., 2001; Ćirović, the intraspecific level. Therefore, preservation of such 2004; Denoël et al., 2009). Additionally, coexistence of alternative ontogenetic pathways can be considered as alternative morphs of two different species (L. vulgaris and T. macedonicus) was found in only five sites, which is also a very rare pattern. Following Denoël (2007), Montenegro appears to be a hotspot for paedomorphosis because of its high density of populations with Molecular Ecology & Conservation Genetics Lab, Department of Biological Applications & Technologies, School of Health paedomorphosis and mainly because all three species Sciences, University of Ioannina, GR 45110 Ioannina, present this alternative life-history trait. Greece Four species of European newts are found in Greece * Corresponding author. E-mail: [email protected] (Valakos et al., 2008). Among them, facultative 256 Konstantinos Sotiropoulos et al. pond situated at the University of Ioannina Campus (39.6141°N; 20.8437°E, 497 m a. s. l). The closest paved road is approximately 50m away and the closest building at approx. 180m. The pond is circular in shape with a mean annual diameter of 18 m and maximum depth of 1.3m in early spring (mean surface area 254 m2, mean water volume 165 m3), while approximately 75% of pond’s bottom is covered with submerged aquatic vegetation (Figure 1A). The surrounding habitat is characterized mostly by abandoned cultivations and extended pastures of herbaceous vegetation, with sparse deciduous trees, either individually or in small stands. The pond has not been used for livestock or cultivation watering for the last 15 years, upon the establishment of the University Campus in the area, while no other use or activity is in place for the same time period. The pond is used as a breeding site for Macedonian crested newts (Triturus macedonicus) and four anuran species (Pelophylax epeiroticus, Bufo bufo, Bufo viridis, Hyla arborea). On 28 November 2014, a total of 147 smooth newts (63 males and 84 females) and three Macedonian crested newts (2 males and 1 female) were caught by dip nets, within an hour of sampling by one person. All individuals were anaesthetized in a 0.2 % w/v aquatic solution of Ethyl 3-aminobenzoate methanesulfonate (MS222), weighted, sexed and measured at the nearest 0.1 mm with digital callipers by the same Figure 1. (A) Breeding pond of paedomorphic Lissotriton person, and after recovery released to the point of vulgaris and Triturus macedonicus in Ioannina basin (Epirus capture. Paedomorphic individuals were recognized prefecture, NW Greece); (B) Ventral view of paedomorphic as presenting adult morphology (size and coloration and metamorphic smooth newts (Lissotriton vulgaris) caught pattern) while retaining external gills. The morphology at the specific pond. From bottom to the top: metamorphic of the cloaca was further considered in assessing sexual female, paedomorphic female, paedomorphic male, maturity, since it offers a rapid and highly reliable trait metamorphic male. Arrows point at the external gills. Photos to discriminate paedomorphs from over-wintering by Konstantinos Sotiropoulos. larvae, as occasionally overwintering larvae of newts may reach relatively large body sizes (Covaciu-Marcov and Cicort-Lucaciu, 2009; Denoël, 2017) (Figure 1B). Among the 147 captured smooth newts, 58 males and paedomorphosis has been reported only for the alpine 82 females were paedomorphs while only five males newt (five localities; Denoël et al., 2001; Denoël, 2004) and two females were metamorphic individuals. Sex and for the smooth newt (two localities; Sotiropoulos ratio was 1.4 females/male for paedomorphs and the et al., 2008). morph ratio was approximately 20:1 (paedomorphs/ In this paper we present the first record of facultative metamorph), which means that 95.2% of the captured paedomorphosis in T. macedonicus from Greece, along individuals were paedomorphs. Using snout-vent length with an additional record of paedomorphosis in L. (SVL) as a measure of body size, the mean SVL ( ± SD, vulgaris graecus (Wolterstorff, 1905). along with minimum and maximum measures, was 37.3 During an inventory of amphibians in the Ioannina ± 8.1 mm (29.3-62.1 mm) for paedomorphic females, basin in 2014, we located a breeding site containing 36.2 ± 6.9 mm (25.1-45.9 mm) for paedomorphic males, smooth newts exhibiting facultative paedomorphosis. 41.0 ± 1.0 mm (40.3-41.7 mm) for metamorphic females The habitat regards an artificial (man-made) stone-wall and 39.0 ± 3.9 mm (35.5-41.1 mm) for metamorphic First record of facultative paedomorphosis in the Macedonian crested newt 257 Table 1. Body measurements (in mm) of female metamorphic During a recent sampling session on 11 February and paedomorphic Triturus macedonicus from the breeding 2017, as part of an ongoing project studying resource 1 Table 1. Bodypond measurements in Ioannina (in (NW mm) Greece). of female L: metamorphic total length; and SVL: paedomorphic snout- partitioningTriturus between morphs and species in the site, apart 2 macedonicusvent from length; the breedingN: sample pond size. in ForIoannina each body(NW Greece).trait the meanL: total (± length; SVL: 3 snout-vent length; N: sample size. For each body trait the mean (± SD), as wellfrom as the smooth newts, a total of 23 T. macedonicus were SD), as well as the minimum and the maximum measurements 4 minimum and the maximum measurements are given. captured by dip nets within one hour of sampling by four are given. 5 persons. Among them, one individual (Figure 1A, B) was identified as exhibiting facultative paedomorphosis. The L SVL Metamorphs (N = 7) mean (± SD) 138.7 (13.7) 80.9 (8.3) specimen presented well developed external gills, while min - max 125.8-156.8 70.2-90.8 visual inspection of the cloaca identified it as a mature Paedomorphs (N = 2) mean (± SD) 127.9 (3.5) 70.2 (2.7) female (Figure 2C). The individual was anaesthetized min - max 125.4-130.3 68.3-72.1 in a 0.2% w/v solution of MS222, weighted, measured 6 with digital callipers to the nearest 0.1 mm, and after recovery released to the point of capture. The female weighted at 10.7 g, and measured total body length (L) = 130.3 mm and SVL = 72.1 mm. Additionally, the males. Since the sample size of metamorphs was overall body condition of the specimen refers to a gravid low, we compared body size between sexes only in individual, but this has not been confirmed otherwise paedomorphs. Differences in SVL between sexes in (e.g. inspection of gonads etc). Body coloration also paedomorphs, were tested for statistical significance by highly resembles (practically indistinguishable) the applying a Mann-Whitney U Test in STATISTICA