High Incidence of Dorsal Dark Spots in North-Western Greek Populations of Alpine Newts Ichthyosaura Alpestris (Salamandridae, Caudata)
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Herpetology Notes, volume 8: 589-598 (2015) (published online on 06 December 2015) High incidence of dorsal dark spots in north-western Greek populations of alpine newts Ichthyosaura alpestris (Salamandridae, Caudata) Haritakis Papaioannou1, Anna Batistatou2, Maria-Christina Mavrogiorgou3, Constantinos Konidaris4, Stathis Frillingos5, Evangelos Briasoulis3, Katerina Vareli3,6 and Ioannis Sainis3,* Abstract. Large dorsal dark spots were observed in a number of alpine newts Ichthyosaura alpestris inhabiting Lake Gistova, the highest altitude alpine lake in the Balkans ever studied. The trait is mainly exhibited by female paedomorphs. Histopathological examination excluded any pathological cause of pigmented skin and revealed that even in paedomorphic skin sections, the altered pigmentation pattern was located only in regions with metamorphic characteristics. Phylogenetic analysis of the previously unstudied Gistova’s newt population based on mtDNA sequences revealed that it belongs to the D2 subclade, comprising only two other newt populations of the wider geographic region that interestingly, also exhibit the trait. The putative genetic or environmental basis for dark spot appearance in these populations is discussed. Keywords: alpine lakes, Balkan Peninsula, black spots, 16S mitochondrial rRNA, mitochondrial cytb gene. Introduction small animal with a total length of about 80 to 100 mm for males and up to 120mm for females (Sotiropoulos, The Balkan Peninsula is considered to be a major et al., 2008; Vukov et al., 2011). Phylogenetic analysis hotspot for European biodiversity (Griffiths et al., revealed the existence of a putative ancestor relict 2004; Strong et al., 2008). Balkan low-altitude lakes have been intensively studied in terms of biodiversity, as many of them are among the most ancient European lakes and thus old enough to feature endemic faunas and floras (Frogley et al., 2001; Frogley and Preece, 2007; 1 Department of Environmental and Natural Resources Albrecht and Wilke, 2008; Vareli et al., 2009; Wilke Management, University of Patras, 30100 Agrinio, Greece et al., 2010; Albrecht et al., 2012). In contrast, Balkan 2 Laboratory of Pathological Anatomy, Department of alpine lakes are less well studied, due to their tiny size Medicine, School of Health Sciences, University of Ioannina, and difficult access. Originated in the end of the last 45110 Ioannina, Greece 3 glacial period, only a few thousand years ago, their Interscience Molecular Oncology Laboratory (iMol), Cancer Biobank Center (UICBC), University of Ioannina, 45110 species assemblages probably derive from a process Ioannina, Greece of recent colonization from neighbouring, geologically 4 Laboratory of Biochemistry, Department of Biological older habitats (Shehu et al., 2009). Greek alpine lakes Applications and Technology, University of Ioannina, 45110, are small, often fishless and located at high-altitude Ioannina Greece areas above 1900m, near the summit of mountains. 5 Laboratory of Biological Chemistry, Department of Medicine, They are called “dragon-lakes” (Drakolimnes in Greek) School of Health Sciences, University of Ioannina, 45110 because their main vertebrate inhabitant, the alpine newt Ioannina, Greece 6 Laboratory of Biology, Department of Biological Applications (Ichthyosaura alpestris), is reminiscent of the shape and and Technology, University of Ioannina, 45110, Ioannina appearance of a small dragon. Greece Ichthyosaura alpestris (initially referred to as Triturus * Corresponding author e-mail: alpestris and more recently as Mesotriton alpestris) is a [email protected], [email protected] 590 Haritakis Papaioannou et al. lineage (Clade A) originating from south-eastern 1988) newt populations from lakes of the northern Serbia and two other lineages designated Western Pindus area were characterized as potentially endemic and Eastern further divided in Clades B, C and D, E and it has been reported that newts from lakes Tymphi respectively (Sotiropoulos et al., 2007). Clades D and and Smolikas exhibit colour variants (e.g. specimens E representing southern and central-northern Balkan with large black spots). However, no effort was made populations were further subdivided, suggesting greater in this study, either to examine histopathologically isolation into multiple refugia (Sotiropoulos et al., these colour variants or to reveal any correlation to 2007). More recently, Recuero and colleagues, by using population’s demographics. a combined analysis of mtDNA, nuclear (nDNA) and Thermoregulation, UV protection and avoiding allozyme data tried to resolve the evolutionary history detection from predators have been proposed as putative of the species I.alpestris (Recuero et al., 2014). The adaptive functions of colour patterns in amphibians phylogenetic hypotheses based on mtDNA from this (Rudh and Qvarnström, 2013). In common frogs the study recovered the same lineages and phylogenetic degree of melanism increases as a function of latitude structure as suggested earlier (Sotiropoulos et al., and altitude (Alho et al., 2010; Vences et al., 2002). 2007), but nDNA and allozyme analyses revealed more Dark markings in montane common frogs were found to complex phylogenetic relationships. Nevertheless it is be valuable to attain faster heating rates of higher body of interest that both nDNA and allozyme data support temperatures, and thereby performance benefits (Vences also that southern Balkan populations (clade D) is a et al., 2002). In larvae of newts and salamanders, UV discrete phylogenetic lineage (Recuero et al., 2014). induces skin darkening implying that melanization may Paedomorphosis is common in a vast number of newt have a protective role against UV radiation. However, populations living in the south margin of the species direct experimental evidence for such a role is missing geographical range, mainly in the Italian and Balkan (Rudh and Qvarnström, 2013). Peninsulas (Denoël et al., 2001). It is a polymorphism On the other hand, in the spotted salamander widespread in newts and salamanders and by which Ambystoma maculatum, UV induces DNA damage larval characters, such as gills and gill slits, are despite the increased melanin production in this species retained in adults (paedomorphs) (Whiteman, 1994). (Lesser et al., 2001). Furthermore, dark spots in some The adults characterized by absence of gill slits are Urodele amphibian species (e.g. Calotriton arnoldi, called metamorphs. In the five Greek paedomorphic Triturus cristatus) were found to be of cancerous origin populations examined so far, the percentage of (Koussoulakos, et al., 1994; Martínez-Silvestre et al., paedomorphs in the total population ranged between 2011). 23% and 77% (Denoël et al., 2001). Paedomorphosis is Based on these facts, we set the following specific under genetic control in several species of salamanders objectives for our study: (a) to investigate the histological (Voss and Shaffer, 1997). A genetic basis favouring nature of the dark spots, (b) to study the distribution of paedomorphosis in I. alpestris is also suspected (Denoël the trait by sex and life history type, (c) to investigate et al., 2001). Moreover, resource partitioning between the presence/absence of the trait in other geographically morphs is in favour of the maintenance of polymorphism neighbouring populations and (d) to resolve the in this species (Denoël and Joly, 2001a; Denoël and phylogenetic relationship of Gistova’s unstudied newt Joly, 2001b). population to other newt populations exhibiting the The Greek alpine newt was classified as a subspecies same colour trait. named Ichthyosaura alpestris veluchiensis (Breuil and Parent, 1988; Denoël, 2004). Although I. alpestris Materials and Methods specimens in northwestern Greece are found in various Study Area water bodies above 1190m such as ponds, drinking troughs and watering basins (Denoël, 2004), large Our study area comprises three alpine lakes (Figure. populations inhabit only the alpine lakes of the region 1). Lake Gistova, located on Mt. Grammos (40o21’N, (Denoël and Schabetsberger, 2003). 20o47’E), is the highest lake in Greece (2360m a.s.l) In our survey conducted in October 2012, large dorsal (Figure 1). It is a fishless elliptical lake (150m x 70m), dark spots were observed in a number of newts from with a maximum depth of about 4m, characterized by Lake Gistova, the highest altitude lake of Greece (2360m a muddy bottom, rocky shores, surrounded by alpine a.s.l) and one of the highest in the Balkans (Sehu et al., pasture. Aquatic vegetation is absent along the shoreline 2009). Interestingly, in a 1988 study (Breuil and Parent, and in the lake. High incidence of dorsal dark spots in Ichthyosaura alpestris, north-western Greece 591 Figure 1. A) Distribution of genetically, already studied I. alpestris populations in SW Balkans. B) A satellite view of the study area in the mountains of NW Greece. The studied populations are indicated with square dots. Known, but genetically unstudied populations are indicated with red dots. (a: Drakolimni Gistova, b: Drakolimni Smolika, c: Drakolimni Tymphis). (Satellite photos by Google-earth) [1. Jankovac (Papuk Mt. Croatia), 2. Kutarevo (Lika , Croatia), 3. Zegar (Usljerbka, Croatia), 4. Joseva (Vajero Mt. Serbia), 5. Prokosko Lake (Vranica Mt. Bosnia Herzegovina), 6. Ljubina bara (Juzni Kucaj Mt. Serbia), 7. Seljani (Rogatica, Bosnia Herzegovina), 8. Zminicko Lake (Sinjavina Mt. Montenegro), 9. Kapetanovo Lake (Lukavica Mt. Montenegro), 10. Bukumirsko Lake (Zijovo Mt. Montenegro), 11. Vlasina