Herpetology Notes, volume 10: 255-260 (2017) (published online on 20 May 2017)

First record of facultative paedomorphosis in the Macedonian crested ( macedonicus) and an additional record for the Greek ( 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 , facultative paedomorphosis evolutionary processes (Gould, 1977; McNamara, has been frequently reported for the , 2012). A case of heterochrony is paedomorphosis, alpestris (Laurenti, 1768), and the smooth which is expressed in caudate as aquatic newt, Lissotriton vulgaris (Linnaeus, 1758) (Kalezić gilled larvae acquire sexual maturity bypassing and Džukić , 1985; Litvinchuk et al., 1996; Denoël . Facultative paedomorphosis is et al., 2001; Sidorovska et al., 2003; Çiçek and Ayaz, frequently observed in newts and 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 For Ioannina each body (NW Greece).trait the mean L: 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 7.0 coloration pattern of typical metamorphic individuals, (StatSoft, Inc., 2004). No significant difference in SVL further excluding the possibility to be an overwintering were found between sexes (U = 2186; Z = 0.812; p = that has attained a large body size. 0.417). On a second short visit at the pond on 16 February 2017, by the same four persons, among 10 captured

1

Figure 2. Lateral (A) and ventral (B) view of the female paedomorphic T. macedonicus from Ioannina basin (NW Greece). Notice the well-developed external gills as well as the adult morphology and coloration patterns; (C) Ventral view of the paedomorphic female T. macedonicus (bottom) and a conspecific mature metamorphic male (top) caught at the specific sampling site. Arrows point at the external gills. Inset photo presents a close-up view of the cloaca; (D) Ventral view of the second paedomorphic female T. macedonicus. Arrows point at the external gills. Photos by Konstantinos Sotiropoulos. 258 Konstantinos Sotiropoulos et al.

Macedonian crested newts (seven males, three food availability (Moustakas and Sotiropoulos, 2015), females), another paedomorphic female was identified are factors that could offer habitat stability and favour the (Figure 2D). Both belly pattern and body proportions expression of paedomorphosis. However, additional and showed that it was not the female captured five days detailed research is needed in order to better understand before (Figure 2C, D). This second female weighted at and identify the factors responsible for the occurrence 10.3 g and measured L = 125.4 mm and SVL = 68.3 of paedomorphosis in the specific site. During older mm. Comparison of both specimens with conspecific surveys in Epirus (Denoël, 2004; Sotiropoulos et al., metamorphic and sexually mature females of the same 2013) and although biodiversity in the entire populations, on the basis of L and SVL, showed that area of Ioannina basin is monitored regularly during the body dimensions of the paedomorphic female were last two years, no other incidence of paedomorphosis within the range of metamorphics (Table 1). in smooth and Macedonian crested newts has been Concluding, all measured external morphological recorded so far. Definitely, a more detailed survey of traits prove that the two individuals were fully mature ponds and other water bodies in the area is required, to paedomorphic females and constitute the first record of fully realise the exact distribution of alternative morphs. facultative paedomorphosis in Triturus macedonicus at However, this record should not be overlooked, since it the southern edge of its distribution (Figure 3). outlines the importance of the area in sustaining a rich The expression of facultative paedomorphosis in both biodiversity. Ioannina basin is a sensitive ecosystem species at the same breeding site, although unequal that includes, entirely or partially, five sites of the between species, highlights the importance of the European Network of Protected Areas, NATURA 2000 environment in the appearance of paedomorphosis (GR2130005, GR2130008, GR2130011, GR2130012, (Breuil, 1992; Denoël et al., 2005b). The specific site, GR2130013). Apart of the increased species richness, a permanent and undisturbed aquatic habitat, seems to the area seems important for the preservation of intra- provide a stable environment for newts. Lack of severe specific diversity as well. water level fluctuations, lack of disturbance by activities The incidence of facultative paedomorphosis in T. or any kind of exploitation or use, along with increased macedonicus, as well as coexistence with paedomorphs of L. vulgaris, has been reported from only two areas so far (Montenegro and Epirus prefecture, NW Greece) (Figure 3). Both areas have been served as refugia for the species during the Last Glacial Maximum (WIelstra et al., 2013), and are among areas of increased biodiversity in the Balkans (Griffiths et al., 2004), as well. Additionally, both areas also harbour paedomorphic populations of a third newt species in the Balkans, the alpine newt (Ichthyosaura alpestris) (Denoël et al., 2001; Denoël, 2004). All these unique properties enhance their role as hot spots for polyphenism in newts and should be designated as areas of high conservation priority for the protection of adaptive intraspecific variation (Denoël, 2007). Fish introductions in Montenegro had a documented negative impact on paedomorphic populations, leading to extirpation of the 47%, 25%, and 20% of the historical presence of paedomorphosis in I. alpestris, L. vulgaris, and T. macedonicus, respectively (Denoël et al., 2009). In Ioannina basin, fish introductions in small permanent Figure 3. Geographic distribution of T. macedonicus, along ponds –mostly for recreational fishing by the locals- with the locations of all known populations exhibiting have been recorded during the last decade. This practice facultative paedomorphosis. Black dots = literature records could lead to local extinctions, further compromising (Denoël et a., 2009); star = new record from Ioannina basin the conservation status and the evolutionary potential of (Epirus Prefecture, NW Greece). In all sites, paedomorphic T. the species. Therefore, the design of protection actions macedonicus coexist with paedomorphic L. vulgaris. for habitats and species in the area of Ioannina should First record of facultative paedomorphosis in the Macedonian crested newt 259 include effective management practices, at local (e.g. at Biological Reviews 80: 663-671. pond scale) and regional scale, such as prevention of Denoël, M., Dzukic, G., Kalezic, M.L. (2005b). Effect of fish introductions in small ponds, or even fish removal widespread fish introductions on paedomorphic newts in Europe. Conservation Biology 19: 162–170. from such ponds (Denoël and Winandy, 2015). Future Denoël, M. (2007): Priority areas of intraspecific diversity: Larzac, conservation-oriented work should focus, not only on a global hotspot for facultative paedomorphosis in amphibians. species distributions, but also on phenotypically diverse Conservation 10: 110-116. but spatially localized variation. The importance of Denoël, M., Ficetola, G.F., Ćirović, R., Radović, D., Džukić, G., small elements of the landscape as potential reserves of Kalezić, M.L., Vukov, T.D. (2009): A multi-scale approach to biodiversity should also be considered in conservation facultative paedomorphosis of european newts () practices. Given that T. macedonicus (as a member of the in the Montenegrin karst: Distribution pattern, environmental variables, and conservation. Biological Conservation 142: 509- T. cristatus species complex), is an Annnexe II species 517. of the E.U. Directive No 92/43, such considerations Denoël, M., Ficetola, G.F. 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Accepted by Wouter Beukema