Acta Herpetologica 2(2): 69-77, 2007 ISSN 1827-9643 (online) © 2007 Firenze University Press Molecular DNA variation among Triturus vittatus vittatus (Urodela) from different breeding sites at the southern limit of its distribution Oren Pearlson1, 2, 3, Gad Degani1, 2 1 MIGAL–Galilee Technology Center, P. O. Box 831, Kiryat Shmona 11016, Israel. Corresponding author. Gad Degani, E-mail: gad@migal.org.il 2 School of Science and Technology, Tel Hai Academic College, Galilee, Israel 3 Institute of Evolution, Faculty of Sciences and Science Education, University of Haifa, Israel Abstract. Molecular DNA variation among Triturus vittatus vittatus (striped newt) from different breeding sites at the southern limit of the species distribution (where environmental conditions are most extreme) was studied by the random amplification of polymorphic DNA (RAPD) method that has been found to be appropriate for other Triturus species. Altitudes of the localities ranged between 15-740 m a.s.l. Of the 20 primers employed, OPA-16 was the only one suitable for T. vittatus, revealing a differ- ent band pattern for different populations. Genetic similarity was calculated by band sharing, which demonstrated a high similarity among the Israeli populations. Keywords. Genetic polymorphism, xeric habitat, conservation, extreme conditions, Triturus vittatus vittatus. INTRODUCTION The banded newt, Triturus vittatus vitattus, is an endangered species in Israel (Geffen et al., 1987). Other species and subspecies of this genus are endangered in other regions of the world. Triturus v. vitattus is distributed throughout the western Caucasus, Turkey, Lebanon, Syria, Israel, Iraq and perhaps, Jordan (Borkin et al., 2003; Litvinchuk et al., 2005). The biology and life cycle of Triturus vittatus in Europe and the Mediterranean region have been described by Raxworthy (1989) and Olgun et al. (1997). Until recently, Triturus vittatus was thought to have two subspecies: Triturus v. vittatus, along the east- ern edge of the Mediterranean Sea from Turkey to Israel, where it reaches its southern limit, and T. v. ophryticus in the Caucasus, east and south of the Black Sea. Based on its trunk vertebrae count, genome size and allozyme data, Litvinchuk et al. (2005) suggested that the northern taxon, T. v. ophryticus, is subdivided into two geographic fragments, 70 O. Pearlson and G. Degani the “western group”, with populations from western Anatolian Turkey, and the “eastern group”, composed of populations from the remaining region of Pontic Turkey and the western Caucasus. At the southern limit of T. v. vittatus distribution (in Israel), environmental condi- tions are the most extreme. There, limiting factors are likely to be the breeding sites and dryness of the terrestrial habitat. The biology and life cycle of T. v. vittatus in northern Israel and the Upper Galilee have been described by Degani and Mendelssohn (1983) and Degani (1986a, 1996), while a population in central Israel has been studied by Geffen et al. (1987). Triturus v. vittatus inhabit mainly winter pools that typically contain water until the beginning of summer, although occasionally they have water all year-round (Degani and Kaplan, 1999). Like other newts, T. v. vittatus requires water bodies surrounded by an adequate terrestrial habitat to support both life phases. If either habitat is damaged, a population may be unable to survive. Zajc and Arntzen (2000) utilized fragments of mitochondrial DNA to study evolution- ary aspects of Triturus. Their findings support the sister-taxon relationship of T. vulgaris and T. montadoni and the position of T. vittatus within the subgenus, Triturus, as a sister taxon of the clade of the large-bodied newts, T. marmoratus and T. cristatus. According to the allozyme analysis of Litvinchuk et al. (2005), the genus Triturus is not a monophyletic taxon and representatives of the genus Neurergus are closely related to T. karelinii. Based on these results and reports from earlier studies on mitochondrial DNA analyses in newts by other authors, in which it was demonstrated that the genus Triturus is paraphyletic, a taxonomic scheme was proposed wherein the genus Triturus was divided into four mono- phyletic genera, Triturus s.s., Lophinus, Mensotriton and Ommatotriton. Furthermore, the substantial genetic differences that was found between the northern and southern samples of T. vittatus, together with the unique distribution, external morphology and coloration differences of the latter, led them to suggest that the northern populations of T. vittatus be designated a distinct species, T. ophryticus. Mikulicek and Pialek (2003) used RAPD PCR analysis to elucidate the difference between the crested newt species, T. cristatus, T. dobrogicus, and T. carnifex, allowing identification of genetic relatedness among popula- tions of different breeding sites, as an indication of the degree of isolation between the sites, an important aspect for developing management strategies to protect the species. Weisrock et al. (2006), in their study, resolved a non-monophyletic history for Triturus. They divided Triturus species into four main parts: a clade of the T. cristatus species, the T. vulgaris and T. boscai clade, the T. alpestris clade and the T. vittatus clade, whose sister taxon is Neurergus. Their findings strongly support previous studies, in which the Medi- terranean island Euproctus species, E. montanus (Corsica) and E. platycephalus (Sardinia), form a strongly supported clade, as well. Beebee et al. (1999) used molecular techniques in order to identify three putative newt hybrids of the two parent species, T. vulgaris and T. helveticus. RAPD analysis provided straightforward and reliable proof, producing consist- ent results with specimens from widely separated parts of their geographic ranges. Hybrids were identified by amplification of both the mitochondrial cytochrome b and ATPase genes, including partial sequencing of the latter. One hybrid had a T. vulgaris mother, while the other two had T. helveticus mothers. Much research has focused on the genetic variables of Urodela in different geographi- cal regions (e.g. Gibbs et al., 1998; Steinfartz et al., 2000; Lecis and Norris, 2004; Riberon 71Molecular DNA variation among Triturus vittatus vittatus et al., 2004). However, genetic variation among populations in natural habitats around breeding sites located relatively near each other has received little attention (Lecis and Norris, 2004). Various methods have been employed to study genetic variation in amphib- ians, including albumins in serum (Degani, 1986b), isozymes (Veith et al., 1992), RAPD PCR (Degani et al., 1999; Mikulicek and Pialek, 2003) and mitochondrial DNA sequenc- ing (Weisrock et al., 2001). The present study, by means of RAPD PCR, examines genetic differences, among T. v. vittatus populations in five different breeding sites in the southern-most region of its dis- tribution, where environmental conditions are most extreme. MATERIAL AND METHODS Study sites Nahalit Ponds (Figs. 1B, 2b): these two ponds are located side by side in cattle grazing land in the Upper Galilee (longitude 243657, latitude 776401) 665 m a.s.l. The ponds fill up with water Fig. 1. The five ponds colonized by newts examined in the study: (A) Dovev Pond, (B) Nahalit Pond, (C) Matityahu Q. Pond, (D) Amiad water holes, (E) Afeka. 72 O. Pearlson and G. Degani that seeps through chicken farms and cattle pens. The smaller pond holds water from January to May, has an area of ca 50 m2 and a depth in the center of ca 2 m. The other one has an area of ca 1000 m2, a depth in the center of ca 0.8 m and contains water from January to June. Both have water plants, such as Ranunculus aquatilis, Scirpus tuberosus and Scirpus tabernae-montanus. Adult Triturus inhabit the ponds from mid-January until the beginning of April, and larvae were found between mid-March and June, when the pond dries up. Dovev Pond (Figs. 1A, 2a): the pond is a nature reserve site in the Upper Galilee (longitude 239158, latitude 772801) at an altitude of 740 m a.s.l. The pond is located among apple orchards with water from December to July. It has an area of ca 300 m2, a depth in the center of ca 1.5 m and water plants, such as Ranunculus aquatilis, Scirpus tuberosus and Scirpus tabernae-montanus. Adult Triturus can be observed from mid-December until the beginning of April, while larvae can be dis- cerned from mid-March until July, when the pond dries up. Amiad water hole (Figs. 1D, 2d): The water hole is man made and is located in the south- ern part of the Upper Galilee (longitude 251721, latitude 757994, altitude: 212 m a.s.l.). The pond is located in cattle grazing land and holds water from December to July. The water hole has a radius of 0.7 m, an area of ca 3 m2, a depth in the center of ca 1.5 m and water plants, such as Ranunculus aquatilis. Adult Triturus can be found in the water from mid-December until the beginning of April, while larvae can be detected only after the water has dried up in July. Matityahu Quarry Pond (Figs. 1C, 2c): The pond in the Matityahu Quarry is located among apple orchards, in the Upper Galilee (longitude 242783, latitude 774855, altitude: 670 m a.s.l.). The pond contains water from January to June. It is ca 200 m2 and ca 2 m deep in the center. The pond has been dug several times in the last few summers, fills up at the beginning of January and contains water until mid-June. No water plants populate the pond. Adult Triturus were observed from Janu- ary until the beginning of April, and larvae were found from mid-March until June, when the pond dries up. Afeka Pond (Figs. 1E, 2e): the pond is located in the central coastal plain, (longitude 182364, latitude 670453) with a mean altitude of 15 m a.s.l. The pond is situated alongside a section of a Fig. 2. The five breeding sites of Triturus v. vittatus examined in this study: (a) Dovev Pond, (b) Nahalit Pond, (c) Matityahu Q. Pond, (d) Amiad water holes, (e) Afeka. 73Molecular DNA variation among Triturus vittatus vittatus drainage canal. Rainwater, draining from adjacent open fields and from the road, usually fills the pool, beginning in December and lasting until mid-March. It is ca 30 m2 in area and ca 0.5 m deep in the center. The vegetation in the pond consists mostly of Eleocharis palustris and Scirpus marifi- mus. Adult Triturus were found in the water from December until the end of February, and larvae were observed from mid-March until mid-April, when the water dries up. DNA analyses Tissue samples were obtained from Triturus larvae and adults. As previously described by Degani and Mendelssohn (1983), adults migrate to these ponds during the mating season (at the beginning of winter) to breed. There is a large adult population at all five locations (Degani and Kaplan, 1999). Specimens (four individuals per each site) were sampled randomly from the entire area of the water body by hand net. Total genomic DNA was extracted from tail-clipped tissues of adults or from whole tails of larvae. Extraction was carried out by the QIAamp DNA Mini Kit (Qiagen, Germany), after protei- nase K digestion. RAPD PCR amplifications were carried out with 10 mer oligonucleotide primers (Mikulicek and Pialek, 2003) in a 50 µl solution, containing 10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl2, 1 mM of each dNTP, 0.5 µM of primer, 10-500 ng gemonic DNA and 2.5 units of Taq DNA Polymerase (Promega, USA). RAPD fragments were amplified by 45 cycles, including dena- turation for 30 s at 94 °C (initial denaturation for 2 min at 94 °C), primer annealing for 1 min at 35 °C and extension for 2 min at 72 °C. The RAPD amplification ended with an extension at 72 °C for 7 min. To assess the similarity between individuals, band sharing (BS) of the RAPD PCR products was calculated as: BS = 2 × (Nab) / (Na + Nb), where BS = level of band shar- ing between individuals a and b, Nab = number of bands shared by individuals, a and b, Na = total number of bands of individual a and Nb = total number of bands for individual b (Jeffreys and Morton, 1987; Wetton et al., 1987). The PCR patterns were compared only between samples that had been run on a single gel. Differences in BS were examined by the d-test for differences between proportions (Parker, 1976). RESULTS Only one of the 20 primers used in the RAPD PCR was revealed to be suitable for the study of DNA variation among T. v. vittatus (OPA-16; 5’-AGCCAGCGAA, Miku- licek and Pialek, 2003). Other primers either failed to amplify any fragment or only amplified a few fragments, making them inappropriate for a study on variations. In PCRs with the primer OPA-16, the number of bands differed among populations from different sites. There were 12-13 identical bands within populations from Afeka Pond and the Amiad waterhole, 13-14 within the populations of Dovev Pond and Matityahu Quarry and 16-17 in the population at Nahalit Pond. Twelve bands were common to all of the newts. The newts in Nahalit, Amiad and Dovev had one band each that was not observed in any other population. Only three bands, in Nahalit, Amiad and Dovev populations, were not detected in newts from the other ponds. High genetic similarity, calculated by band sharing, was discovered among newts in the ponds at the high alti- tudes, which were found to have less similarity with the newts from the ponds at lower altitudes (Table 1, Figs 3, 4). 74 O. Pearlson and G. Degani Table 1. Band sharing of newts from studied sites (see methods for details). Band Sharing (%) Nahalit Pond Amiad Waterhole Dovev Pond Afeka Pond Matityahu Quarry 90 96 92 88 Nahalit Pond — 80 83 80 Amiad Waterhole — — 88 92 Dovev Pond — — — 88 Fig. 4. Band sharing variations among populations (mean + SE). Fig. 3. RAPD PCR results with the OPA-16 primer. 75Molecular DNA variation among Triturus vittatus vittatus DISCUSSION A low genetic variability was discovered among the different Triturus v. vittatus popu- lations from various altitudes and located at short geographical distances, one from the other, in Israel. Moreover, when these Israeli newt populations, which belong to the sub- species Triturus v. vittatus, were compared by sequence analysis to the other subspecies T. v. ophryticus (found in European Turkey), high genetic variations were observed (Veith et al., 2004). Mikulicek and Pialek (2003) used twenty 10-mer oligonucleotide primers on three crested newt species (Triturus cristatus superspecies) and found 19 of them to yield 2 to 22 bands per primer. In the present study, a very high similarity was detected and only one primer out of 20 (Mikulicek and Pialek, 2003) yielded a reasonable number of bands to show a clear variation among the populations. We believe that the explanation for this result is the very short geographical distances among the populations, together with the fact that all of the habitats were of the unpredictable type, causing the newts to adapt to the wide ecological variations. It has been previously shown that the ecological and bio- logical conditions, suitable for T. v. vitattus, vary, and in unpredictable habitats, such as the rain pools in the southern limit of its distribution, conditions generally correlate with altitude (Degani and Mendelssohn, 1983; Degani, 1986a, 1996; Geffen et al., 1987; Degani and Kaplan, 1999). The effects of habitat and geographic location on genetic variation have been studied in other urodela (Steinfartz et al., 2000; Lecis and Norris, 2004; Riberon et al., 2004). Much research has been conducted on a range of habitats in widespread loca- tions. However, very few studies have examined genetic variation among different popula- tions at breeding sites, covering a relatively small area, as in the present study. Triturus vittatus vittatus is a urodelan, which can be found in different freshwater bodies, such as unpredictable rain pools and springs. Water parameters, such as temper- ature and duration of availability in the winter ponds studied, exert a greater influence on the larvae than does the type of water body. In such ponds, the altitude of the pond influenced the species existing there (Degani and Kaplan, 1999). The results of the present study are very similar to those of Degani et al. (1999), who studied the genetic variation of salamanders from different habitats, using RAPD PCR. They discovered a very low genetic variation between two populations from semi-arid habitats (band sharing was 94%), and in contrast, a high genetic variation between populations from semi-arid and humid habi- tats (85-86% band sharing). Their results agree with the present study, in which ecological conditions affected genetic variation. The foremost methods for studying genetic variation are putative proteins (Degani, 1986b) and enzymes (Veith et al., 1992), differences in DNA fingerprints (RAPD PCR; Degani et al., 1999; Mikulicek and Pialek, 2003) and sequences of conserved and varia- ble genes (Kocher et al., 1989; Macgregor et al., 1990; Caccone et al., 1997). The methods used in this study support previous findings, demonstrating that RAPD PCR is a suitable method for examining small genetic variations among populations, whereas the utiliza- tion of cytochrome b sequencing may be applied when there is a greater taxonomic vari- ation, such as between species (Steinfartz et al., 2000; Lecis and Norris, 2004; Riberon et al., 2004). Although the genetic variation among the Triturus vittatus vittatus populations is relatively low, one of the populations, that of Nahlit Pond, which is the most polluted pond, as mentioned previously, varied more from the other populations. In addition, the 76 O. Pearlson and G. 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