Herpetology Notes, volume 13: 1041-1044 (2020) (published online on 14 December 2020)

First record of facultative paedomorphosis in the Turkish smooth newt, Lissotriton schmidtleri (Raxworthy, 1988), from Greece

Konstantinos Sotiropoulos1,*, Konstantinos Moustakas1, and Elisavet-Aspasia Toli1

Paedomorphosis is a life history strategy, where Among the five species of European newts that are in caudate amphibians sexually mature individuals found in Greece (Valakos et al., 2008; Wielstra et al., retain larval characteristics (i.e. gills) and remain 2014; Wielstra et al., 2018), facultative paedomorphosis in the aquatic habitat for their entire (or most) life has been reported for three of them; the alpine newt, span (Whiteman, 1994; Denoël et al., 2005). This Ichthyosaura alpestris (five localities; Denoël et alternative phenotype is considered to be the product al., 2001; Denoël, 2004), the Greek smooth newt, of the combined action of environmental and genetic Lissotriton graecus (three localities; Sotiropoulos et al., factors, and is related to the costs and benefits of 2008; Sotiropoulos et al., 2017) and the Macedonian each environment to the development and survival crested newt, Triturus macedonicus (one locality; of the individual (Denoël et al. 2009). Facultative Sotiropoulos et al., 2017). paedomorphosis has been reported in many European So far, only eight populations of Lissotriton newts, especially in members of genus Lissotriton schmidtleri from Turkey have been reported to exhibit (Kalezić and Džukić, 1985; Litvinchuk et al., 1996; facultative paedomorphosis (Bozkurt et al., 2016 and Çevik et al., 1997; Litvinchuk, 2001; Sidorovska et al., references therein; Kizil et al., 2016) (Figure 1). In 2003; Sotiropoulos et al., 2008; Denoël et al., 2009; this paper we present the first record of facultative Skorinov et al., 2009; Çiçek and Ayaz, 2011; Covaciu- paedomorphosis in L. schmidtleri from Greece. Marcov et al., 2011; Gvoždík et al., 2013; Picolli, 2013; Bozkurt et al., 2015; Bozkurt et al., 2016; Sotiropoulos et al., 2017; Denoël et al., 2019). Within the Lissotriton species complex, the “smooth newt” (Lissotriton vulgaris sensu lato and L. montandoni) currently comprises of six species; L. vulgaris (Linnaeus, 1758), L. montandoni (Boulenger, 1860), L. kosswigi (Freytag, 1955), L. graecus (Wolterstorff, 1905), L. lantzi (Wolterstorff, 1914) and L. schmidtleri (Raxworthy, 1988), and is widely distributed through Europe and adjacent Asia (Wielstra et al., 2018).

Figure 1. Geographic distribution of L. schmidtleri, along 1 Molecular Ecology & Conservation Genetics Lab, Department with the locations of all known populations exhibiting of Biological Applications & Technology, School of Health facultative paedomorphosis. Black dots = literature records Sciences, University of Ioannina, GR 45110 Ioannina, (Bozkurt et al., 2016 and references therein); star = new Greece. record from Aghios Kosmas (Macedonia Prefecture, NE * Corresponding author. E-mail: [email protected] Greece). 1042 Konstantinos Sotiropoulos et al.

During a field survey in northeast Greece on April (Pabijan et al., 2015; Corduk et al., 2017; Wielstra et al., 22, 2019, gilled smooth newts were observed in 2018) and were assigned to L. schmidtleri. However, a cement reservoir situated in a recreational area all captured paedomorphic males express a “graecus” approximately 1 Km to the east of the village of external morphology (i.e. smooth dorsal crest) and not Aghios Kosmas (Macedonia Prefecture, Greece) the typical “schmidtleri” morphology (i.e. denticulated (41.0837°N, 24.6708°E, 447 m a. s. l.) (Figure 1). The dorsal crest) (Figure 2). reservoir is rectangular in shape (22X15m, depth ~3m) Since the specific population is located at the and approximately 30% of its bottom is covered with westernmost edge of L. schmidtleri distribution submerged aquatic vegetation. Newts were visible (Figure 1), and at the “graecus-schmidtleri” transition throughout the reservoir. The surrounding habitat is characterised mostly by old cultivations and extended pastures of herbaceous vegetation, with stands of deciduous and evergreen trees and shrubs. The reservoir is used for livestock or cultivation watering, and is also used as a breeding site for Balkan crested newts (Triturus ivanbureschi) and at least two anuran species (Pelophylax ridibundus, Bufo bufo). A total of nine gilled smooth newts were captured (6 males and 3 females) by dipnets, anaesthetised in a 0.2 % w/v aquatic solution of Ethyl 3-aminobenzoate methanesulfonate (MS222), sexed and measured to the nearest 0.1 mm with digital callipers. After recovery they were photographed in a portable aquarium and then released to the point of capture. Verification of paedomorphs was based on the presence of external gills, the overall adult morphology (size and colouration pattern), while the morphology of the cloaca was further considered in assessing sexual maturity, since it offers a rapid and highly reliable trait to discriminate paedomorphs from over-wintering larvae, as occasionally overwintering larvae of newts may reach relatively large body sizes (Covaciu-Marcov and Cicort-Lucaciu, 2009; Denoël, 2017) (Figure 2). The mean snout-vent length, SVL (i.e. the distance from the tip of the snout to the posterior edge of the cloacal opening), along with ± SD and minimum-maximum measures, was 34.9 ± 2.2 mm (32.5-36.8 mm, n=3) for females and 36.5 ± 2.3 mm (33.8-40.1 mm, n=6) for males. Since no metamorphic individuals were observed or captured during this session, comparison of the SVL of the gilled specimens with published measurements of metamorphic and sexually mature individuals of a nearby population (Zarkadia, 41.0187°N, 24.6473°E, 124 m a. s. l.), showed that body dimensions of the Figure 2. Lateral (A) and ventrolateral (B) view of paedomorphs were within the range of metamorphs male paedomorphic L. schmidtleri from Aghios Kosmas (SVL males: 34.1-40.3 mm, n=8; SVL females: 32.6- (Macedonia Prefecture, NE Greece). Notice the well- 36.5 mm, n=10) (Sotiropoulos, 2004; Sotiropoulos et developed external gills as well as the adult morphology al., 2008). and colouration patterns; (C) Lateral view of a mature male Metamorphic smooth newts from Aghios Kosmas metamorphic L. schmidtleri from Evros delta (NE Greece). have been included in previous phylogenetic analyses Photos by Konstantinos Sotiropoulos. First record of facultative paedomorphosis in the Turkish smooth newt 1043 area, we proceeded in assessing the taxonomic status et al., 2019; Toli et al., 2020), future conservation- of the paedomorphs. For this purpose, buccal swab oriented work should also focus on phenotypically samples were obtained from four individuals and total diverse but spatially localised variation. Within this genomic DNA was extracted using the NucleoSpin context, the importance of small elements of the Tissue kit (Macherey-Nagel) following the landscape should also be considered in conservation manufacturer’s protocol. A fragment of approximately practices as potential reserves of biodiversity (Denoël 900 base-pairs (bp), covering a large part of the and Ficetola, 2015). mitochondrial ND4 gene with the associated tRNAs, was amplified following the procedures described Acknowledgments. We thank Mathieu Denoël and an in Corduk et al. (2017). The amplified fragments anonymous reviewer for helpful comments and suggestions on an earlier version of the manuscript. We are grateful to Proff. Jacek were sequenced by CeMIA (Cellular & Molecular Szymura for his joyful assistance during this fieldwork. The work Immunological Applications, Larisa, Greece). was implemented under a special permit (ΩΚΖΤ4653Π8-422) Sequences were edited and aligned in MEGA X issued by the Hellenic Ministry of the Environment. (Kumar et al., 2018), and the final alignment included 834bp. All four sequences were identical to each References other and to the sequence with Acc. No. KJ852618 Arevalo, E., Davis, S.K., Sites, J.W. Jr (1994): Mitochondrial in GenBank, which corresponds to haplotype E15 of DNA sequence divergence and phylogenetic relationships the current smooth newt phylogeny (Corduk et al., among eight chromosome races of the Sceloporus grammicus 2017). The specific haplotype belongs to “Clade E” complex (Phrynosomatidae) in Central Mexico. Systematic which is distributed in western Turkey, the northeastern Biology 43: 387–418. part of Greece and eastern Bulgaria, and comprises L. Babik, W., Branicki, W., Crnobrnja-Isailovic, J., Cogălniceanu, D., Sas, I., Olgun, K., Poyarkov, N.A., Garcia-Paris, M., schmidtleri (Babik et al., 2005; Corduk et al., 2017; Arntzen, J.W. (2005): Phylogeography of two European newt Pabijan et al., 2015). species – discordance between mtDNA and morphology. The fact that all sequenced paedomorphs bear Molecular Ecology 14: 2475–2491. schmidtleri’s haplotype, while male paedomorphs Bozkurt, E., Olgun K., Wielstra B. (2015): First record of express a ‘graecus-like’ crest morphology (Figure 2) facultative paedomorphism in the Kosswig’s newt Lissotriton (vulgaris kosswigi (Freytag, 1955) (Urodela; Salamandridae), might be due to the limited development of secondary ) sexual characters, such as the dorsal crest, a situation endemic to northwestern Turkey. Turkish Journal of Zoology 39: 976–980. that has been observed also in other paedomorphic Bozkurt, E., Tural, M., Ulutaş, G., Üzüm, N., Olgun, K. (2016): newts (Çevik et al., 1997; Denoël et al., 2001; Winandy Two new paedomorphic population records of the smooth and Denoël, 2015; Kizil et al., 2016). newt, Lissotriton vulgaris schmidtleri (Raxworthy, 1988) This is the first case of facultative paedomorphosis (Urodela, Salamandridae), from Western Turkey. Russian in a Greek population of L. schmidtleri. Facultative Journal of Herpetology 23: 158–162. paedomorphosis has now been reported for four out Çevik, E., Atatür, M.K., Arikan, H., Akyurtlakli, N. (1997): Occurrence of neotenic Triturus vulgaris (Urodela: of the five newt species occurring in Greece, thus Salamandridae) larvae in western Anatolia. Israel Journal of classifying Greece among other important areas for the Zoology 43: 301–304. preservation of intra-specific adaptive diversity. Çiçek, K., Ayaz, D. 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