HERPETOLOGICA ROMANICA Vol. 6, 2012, pp.51-67 ISSN: 1842-9203 Article No. 121103

Molecular DNA variation in felixarabica from various breeding sites in northern Israel

Gad DEGANI1,2,*, Reut NAGAR2 and Svetlana YOM-DIN1

1. MIGAL–Galilee Technology Center, P. O. Box 831, Kiryat Shmona 11016, Israel. 2. School of Science and Technology, Tel Hai Academic College, Upper Galilee 12210, Israel * Corresponding author, G. Degani, E-mail: [email protected]

Abstract. A genetic study was carried out on tree (Hyla felixarabica) larvae from of different locations and altitudes in northern Israel. Cytochrome b and 12S were amplified for sequencing and used for the assessment of genetic variation by random amplification of polymorphic DNA (RAPD PCR). The cytochrome b fragment varied at the nucleotide positions, 64, 124, 143 and 145 among H. felixarabica populations of various breeding sites. Among the populations, there was a high genetic identity, 98.4-100%, as revealed by nine sequences from specimens of populations at each location that were analyzed by Arlequin software. According to this analysis, the Dir-Hanna Pond population had a lower identity than the other populations, and Matityahu Pond was a close second. All other populations had a highly similar mitochondrial cytochrome b gene. The variation among the populations was higher compared to the variation within the populations according to the cytochrome b gene. The 12S gene sequence varied among breeding site populations in Israel, with respect to 14 nucleotide positions. The phylogenetic trees that were constructed from the 12S sequences demonstrated that Fara Pond, the most northwestern breeding site,differed the most from the other populations, with Leshem Pond, Jauda Spring and Elrom Pond following, respectively. The variation among the populations according to 12S was 89.64% and within the populations was 10.36%. Among the populations studied, there were 5 - 14 identical bands, according to the OPA-16 primer, and 7 - 14 identical bands according to the OPA-18 primer. When comparisons were made between paired populations using the OPA-16 primer, there were 0 - 7 common bands, and when using the OPA-18 primer, there were 0 – 8. A few populations had a very low similarity, compared to other populations, e.g., populations from Leshem Pond and Jauda Spring (with the OPA- 16 primer).

Key words: Mitochondrial DNA, Genetic Variation Hyla felixarabica, Hyla savignyi, Northern Israel.

©Romanian Herpetological Society,Cluj-Napoca / Oradea, Romania, 2012 Herpetol. Rom, 6, http://herpetofauna.uv.ro/herprom.html 2012, Romania 52 Degani, G. et al.

INTRODUCTION

The genus Hyla comprises 31 species from Holarctic regions (North America, Asia, Europe and the northern part of Africa), and is clustered into four species groups: arborea, cinerea, versicolor and eximia (Faivovich et al. 2004, Faivovich et al. 2005). Stock et al. (2008) carried out a study using mitochondrial and nuclear markers of the Hyla species and subspecies from all currently recognized circum- Mediterranean areas. Based on their study in the Middle East, Hyla savignyi exists in Cyprus, inhabiting well-illuminated, broad-leafed and mixed forests, bush and shrub lands, meadows, gardens, vineyards, orchards, parks, lakeshores and low riparian vegetation. They live mostly in arboreal habitats, on bushes, shrubs and trees. In Israel, the of three varies from H. arborea to H. savignyi, based on mitochondrial DNA (mtDNA) and nuclear DNA fragments (Stock et al. 2008). Andersen et al. (2004), who studied the , H. arborea, in Denmark in suitable habitats and population sizes, examined its population struc- ture, based on the genetic variation of 12 polymorphic DNA microsatellites. Salducci et al. (2005) investigated the taxonomic diversity and phylogenetic diver- sity in French Guiana of two genera, Hyla and Scinax, using sequences of two mito- chondrial genes (16S rDNA and 12S rDNA.) and two nuclear genes (tyrosinase and 18S rRNA). Grach et al. (2007) described the endemic population of another species in Is- rael, H. heinzsteinitzi, based on morphological characterizations, coloration and call structure. H. heinzsteinitzi is located in a small area among three Judean Hills, at three sites within a 6x13 km range, at altitudes of 730 - 895 m above sea level (ASL). In northern Israel, the tree frog is one of the most important . Its spawning occurs in stagnant waters, such as ponds, swamps and reservoirs (Degani 1986, Degani et al. 1999, Degani 1982). Nevo and Yang (1979) studied the genetic variation of eight populations of H. savignyi (H. felixarabica) from Israel along two transects of increasing aridity from north to south, using allozymic variation in proteins encoded by 27 loci. The re- sults of this study showed that polymorphisms were associated with climal pat- terns of increasing aridity southwards and eastwards, and that central populations harbor more genetic variations than marginal ones. Nevo and Yang (1979) sug- gested that the genetic variation in H. savignyi, according to protein polymor- phisms, is based on climatic selection, rather than on stochastic processes or neu- trality. He concluded that the environmental variation model seems to be the best predictor of genetic variation in this species.

Herpetol. Rom, 6, 2012 Molecular DNA variation in Hyla felixarabica 53

Gvoždik et al. (2010) studied the evolutionary relationships of tree frogs ( family) from the Middle East. The demographic histories of their populations were studied using a combination of mitochondrial and nuclear genes. Southern populations from Yemen, Jordan, southern Syria and extreme northeastern Israel are described the H. felixarabica. The Arabian Peninsula and southern Levant and to the importance of the Dead Sea Rift as a historical barrier geographically separating the new species from H. savignyi. (Gvoždik et al. 2010). The present study investigates the genetic differences of H. felixarabica in the various breeding sites in northern Israel by means of random amplification of po- lymorphic DNA (RAPD PCR) and by molecular mitochondrial DNA (mtDNA) associations.

MATERIALS AND METHODS

Specimens

In order to analyze and characterize H. felixarabica populations in various areas in northern Israel, ten H. felixarabica specimens were collected from each population. As previously de- scribed by Degani (1986), sampling of specimens was carried out randomly from ten breed- ing sites of H. savignyi in northern east Israel, by hand net from the entire area of the water body or in the area surrounding the breeding sites (Fig. 1 and Table 1). The tissues were studied by mitochondrial sequence analysis.

DNA extraction, amplification and sequencing Genomic DNA was extracted from ethanol-preserved tissue samples (clipped or whole tail of larvae) with the QIAamp DNA Mini Kit that employs proteinase K lysis of the tissue and specific DNA binding to the QIAamp silica-gel membrane through which contaminants pass. DNA samples were visualized after electrophoresis on a 0.8% agarose gel that con- tained ethidium bromide. The DNA concentration was measured using NanoDrop100 (Thermo Fisher Scientific, Wilmington, DE, USA). DNA of Hyla cytochrome b and 12S was amplified by PCR and analyzed by RAPD PCR. Primers for DNA amplification of 12S genes were based on those of Veith et al. (1992), and those for cytochrome b were derived from se- quences determined in our lab (Table 2). PCR amplification was performed in a 50 μl solu- tion containing 10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl2, 0.5 mM of each dNTP, 2 μM of primer, 10-500 ng gemonic DNA and 2.5 units of Taq DNA polymerase (Promega, USA) in a PTC-150 MiniCycler (MJ Research, USA) with the following parameters: 3 min denaturation at 94 °C, followed by 36 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 52 °C and elongation at 72 °C for 1 min. An additional 5 min elongation period at 72 °C fol- lowed the last cycle. After amplification, the PCR products were separated by electrophore- sis on a 1.5 % agarose gel that was stained with ethidium bromide. PCR products were puri-

Herpetol. Rom, 6, 2012 54 Degani, G. et al. fied using the HiYield Gel/PCR DNA Fragment Extraction Kit (RBC Bioscience, Taiwan) and were sequenced at the Hy Laboratories (Rehovot, Israel).

Figure 1. The ten breeding sites that were colonized by Hyla savignyi and examined in this study. A – Elrom Pond, B – Fara Pond, C – Matityahu Pond, D – Navoraya Spring, E – Jauda Spring, F – Sasa Pond, G – Leshem Pond, H – Dir-Hanna Pond, I – Balad Spring, J - Gahar Stream.

Table 1. The breeding sites of Hyla savignyi that were examined in this study.

Altitude Longitude Latitude (m ASL) 1060 286429 222677 A – Elrom Pond 446 157022 236119 B – Fara Pond 665 192873 274808 C – Matityahu Pond 663 197970 267307 D – Navoraya Spring 110 205500 259700 E – Jauda Spring 810 186972 270855 F – Sasa Pond 300 174280 249160 G- Leshem Pond 195 184200 252800 H – Dir-Hanna Pond 446 157022 236119 I – Balad Spring 140 161200 224200 J – Gahar Stream

Herpetol. Rom, 6, 2012 Molecular DNA variation in Hyla felixarabica 55

Table 2. Primers used in this study.

PCR product Sequence 5’to 3’ Primer Genes length (bp) 300 TCATCCTTCATTGACCTCCC Hcytfor1 cytochrome b TAAGAACAGTAAGATAACTCC Hcytrev1 500 AAACTGGGATTAGATACCCCACTAT 12SA-L 12S GAG GGT GAC GGG CGG TGT GT 12SB-H

DNA sequence analysis Analysis of molecular variance (AMOVA) is a method for estimating population differentia- tion directly from molecular data and enables the testing of hypotheses regarding differen- tiation. Various molecular data, direct sequence data or phylogenetic trees based on such molecular data, may be analyzed using this method (Excoffier et al. 1992). The study of DNA variation was done in two steps. First, the variations within the popu- lations were examined. Subsequently, the samples were run on one gel to find the genetic variations among the populations. To assess the similarity between individuals, band shar- = Nab) / (Na + Nb), where BS) ׳ ing (BS) of the RAPD PCR products was calculated as: BS = 2 level of band sharing between individuals a and b, Nab = number of bands shared by indi- viduals, a and b, Na = total number of bands of individual a and Nb = total number of bands for individual b (Jeffreys & Morton 1987, Wetton et al. 1987). The PCR patterns were com- pared only among samples that had been run on a single gel. Differences in BS were exam- ined by the d-test to determine differences between proportions (Parker 1976). Multiple sequence alignments and phylogenetic cluster analysis were carried out using the MegAlign computer program (Windows32 MegAlign6.1, DNASTAR, Inc.). Phylogenetic trees were generated by the neighbour-joining method from distance matrices, which were based on differences in sequences found in the multiple sequence alignment.

RESULTS

The nucleotide sequences of the DNA fragments were determined from a 255 bp clone of cytochrome b (Fig. 2) and a 320 bp clone of 12S (Fig. 3). The cytochrome b fragment varied at the nucleotide sites, 64, 124, 143, 145 and 175 among popula- tions of various breeding sites (Table 3; GenBank accession numbers from FJ595179 to FJ595197). A similar situation was revealed when determining the cytochrome b gene variation among H. felixarabica populations from different breeding sites (Fig. 4). The analysis of nine sequences with Arlequin software demonstrated a high gene identity varying between 98.4%-100. According to the analysis of cytochrome b, the genetic variation of individual H. felixarabica was uniformly distributed among

Herpetol. Rom, 6, 2012 56 Degani, G. et al.

Figure 2. Alignment of the nucleotide sequences of the cytochrome b fragment of Hyla savignyi larvae from nine breeding sites in northern Israel. B – Fara Pond, C – Matityahu Pond, D – Navoraya Spring, E – Jauda Spring, F – Sasa Pond, G – Leshem Pond, H – Dir-Hanna Pond, I – Balad Spring, J – Gahar Spring.

Herpetol. Rom, 6, 2012 Molecular DNA variation in Hyla felixarabica 57

larvae from ten breeding sites breeding sites from ten larvae Hyla savignyi Hyla savignyi Pond, I – Balad Spring, J - GaharSpring, Pond, I Stream. J - – Balad fragment of

S 12 B – Fara Pond, C – Matityahu Pond, D – Navoraya Spring, E – Jauda Spring, Jauda E – – Navoraya Spring, Pond, D Pond, C – Matityahu Fara B – F – Sasa Pond, G – Leshem Pond,GF – Sasa Pond, H – Dir-Hanna – Leshem Alignment of the nucleotide sequences of the sequences of of the nucleotide Alignment in northern Israel. A – Elrom Pond, A – Elrom in northern Israel. Figure 3.

Herpetol. Rom, 6, 2012 58 Degani, G. et al.

Table 3. The variations in the nucleotide sequences and GenBank accession numbers of the cytochrome b fragment.

GenBank Nucleotide site of variation accession number 175 145 143 124 64 FJ595192 C A C C Y J – Gahar Spring FJ595195 Y R C Y Y C – Matityahu Pond FJ595193 C R C C Y E – Jauda Spring FJ595197 C R C C Y F – Sasa Pond FJ595196 C R C C Y D – Navoraya Spring FJ595194 C R Y C Y G – Leshem Pond FJ595190 C G C C T I – Balad Spring FJ595189 T G C T T H – Dir-Hanna Pond FJ595191 C G Y C T B – Fara Pond

B – Fara Pond, C – Matityahu Pond, D – Navoraya Spring, E – Jauda Spring, F – Sasa Pond, G – Leshem Pond, H – Dir-Hanna Pond, I – Balad Spring, J - Gahar Stream.

Figure 4. Unrooted phylogenetic tree of the partial cytochrome b fragment based on nucleotide sequences of the Hyla savignyi. The length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the number of substitution events. The phylogenetic tree was constructed using the MegAlign program (DNASTAR) by the CLUSTALW method. The branch length represents the evolutionary distance.

Herpetol. Rom, 6, 2012 Molecular DNA variation in Hyla felixarabica 59

populations. However, the variation among all of the populations was higher (69.74%), than the variation within the populations (30.-%), according to the mito- chondrial cytochrome b gene (AMOVA p<0.0.5). The 12S gene sequence varied at 14 nucleotide positions among populations of breeding sites in Israel (Fig. 3 and Table 4). The nucleotide substitutions and per- cent of identity of the 12S gene are presented in figure 5. The phylogenetic trees that were constructed from the 12S sequences show that Fara Pond (B1, B2 and B3), the most northwestern population, differed the most from the other populations (Figs 1 and 5), and Jauda Spring (E1, E2 and E5), a population found in the south- eastern region, and which was located at the lowest altitude, was a close second. However, some individuals (H3, G1 and D5), which belong to a population of rela- tively low variation, according to Arlequin software, were demonstrated to have a high variation. Based on 12S and according to the AMOVA test, the variation among the populations was 89.64% and within the populations was 10.36%. This difference among the populations was significant (p<0.05).

Table 4. Variations among the nucleotide sequences of the 12S fragment.

GenBank 12S nucleotide differences accession (nucleotide position) numbers 63 60 58 56 52 46 40 24 23 21 12 8 3 2 M T Y C A A G A Y - A M T A FJ595180 Dir-Hanna Pond A T C C A A G A C - A A T A FJ595181 Balad Spring A T T C A A G A C - A C T A FJ595182 Elrom Pond A C C T G G A T C G G A C T FJ595183 Fara Pond A T C C A A G A C - A A T A FJ595184 Gahar Stream A T Y C A A G A C - A C T A FJ595185 Jauda Spring A T Y C A A G A Y - A A T A FJ595186 Leshem Pond A T C C A A G A C - A A T A FJ595187 Matityahu Pond A T C C A A G A C - A A T A FJ595188 Navoraya Spring A T C C A A G A C - A A T A FJ595179 Sasa Pond

Legend for letters representing more than one nucleotide: M = A/C, R = A/G, W = A/T, S = C/G, Y = C/G, Y = C/T, D = A/G/T.

The DNA variation of H. felixarabica at various breeding sites was assessed us- ing the primers, OPA-16 and OPA-18, as presented in Figure 6. There were 5 - 14 identical bands when OPA-16 was used (Table 5) and 7 - 14 identical bands with

Herpetol. Rom, 6, 2012 60 Degani, G. et al.

OPA-18 (Table 5). When two populations were compared, as in Table 6, the num- ber of common bands varied between 0 - 7 when using the OPA-16 primer and be- tween 0 - 8, when using the OPA-18 primer. Some populations had a very low similarity between them as compared to the other populations, e.g., populations Leshem Pond (G) and Jauda Spring (E), when using OPA-16 (Fig. 6).

Figure 5. Unrooted phylogenetic tree of the partial 12S fragment based on nucleotide sequences of the Hyla savignyi. The length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the number of substitution events. The phylogenetic tree was constructed using the MegAlign program (DNASTAR) by the CLUSTALW method. The branch length represents the evolutionary distance.

Herpetol. Rom, 6, 2012 Molecular DNA variation in Hyla felixarabica 61

Figure 6. RAPD PCR results using primers a) OPA-16 and b) OPA-18. [A – Elrom Pond, B – Fara Pond, C – Matityahu Pond, D – Navoraya Spring, E – Jauda Spring, F – Sasa Pond, G – Leshem Pond, H – Dir-Hanna Pond, I – Balad Spring, J - Gahar Stream]

Herpetol. Rom, 6, 2012 62 Degani, G. et al.

Table 5. Band sharing among populations - OPA-16 and OPA-18.

Number of Bands Band sharing (BS) Specific to this Common to this and Breeding between this and Total population other populations sites other populations OPA-16 1.00 0 7 7 A 0.88 1 4 5 B 1.00 0 8 8 C 0.92 2 12 14 D 1.00 0 6 6 E 1.00 0 10 10 F 1.00 0 6 6 G 0.93 1 7 8 H 0.88 2 8 10 I 1.00 0 6 6 J OPA-18 1.00 0 7 7 A 1.00 0 4 6 B 1.00 0 8 10 C 1.00 0 12 8 D 1.00 0 6 10 E 1.00 0 10 8 F 1.00 0 6 8 G 0.66 2 7 14 H 1.00 0 8 10 I 1.00 0 6 10 J

A – Elrom Pond, B – Fara Pond, C – Matityahu Pond, D – Navoraya Spring, E – Jauda Spring, F – Sasa Pond, G – Leshem Pond, H – Dir-Hanna Pond, I – Balad Spring, J - Gahar Stream.

DISCUSSION

An analysis was carried out on the genetic variation among ten populations in a small area in northern Israel, using different genetic markers of molecular mito- chondrial DNA, cytochrome b and 12S, as well as nuclear DNA. RAPD PCR showed different results with each marker. Cytochrome b is a more conservative marker than 12S, as demonstrated by the very high variation observed in the RAPD PCR obtained with 12S. This pattern seems to be mainly attributed to differ- ences among breeding site populations. According to this analysis, a very high similarity (98.4%-100) was found among the populations. Since the other popula-

Herpetol. Rom, 6, 2012

Table 6/A. The common bands and band sharing (BS) between two paired populations according to RAPD PCR with the primers OPA-16.

B C D E F G H I J 0 4 4 4 5 2 4 3 4 A BS =0 BS=0.66 BS=0.38 BS=0.62 BS=0.59 BS=0.31 BS=0.53 BS=0.35 BS=0.62 1 4 2 1 2 0 1 1 B BS=0.08 BS=0.62 BS=0.36 BS=0.13 BS=0.63 BS=0 BS=1.3 BS=0.18 6 3 6 2 4 2 3 C BS=0.55 BS=0.43 BS=0.67 BS=0.12 BS=0.50 BS=0.22 BS=0.43 5 7 3 4 4 4 D BS=0.50 BS=0.58 BS=0.30 BS=0.63 BS=0.33 BS=0.40 4 3 3 3 4 E BS=0.50 BS=0.50 BS=0.43 BS=0.38 BS=0.67 4 4 4 4 F BS=0.50 BS=0.44 BS=0.40 BS=0.50 2 4 3 G BS=0.23 BS=0.50 BS=0.50 4 4 H BS=0.44 BS=0.57 4 I BS=0.5

B – Fara Pond, C – Matityahu Pond, D – Navoraya Spring, E – Jauda Spring, F – Sasa Pond, G – Leshem Pond, H – Dir-Hanna Pond, I – Balad Spring, J - Gahar Stream.

Table 6/B. The common bands and band sharing (BS) between two paired populations according to RAPD PCR with the primer OPA-18.

B C D E F G H I J 2 6 3 3 3 3 6 5 3 A BS=0.32 BS=0.71 BS=0.40 BS=O.35 BS=0.40 BS=0.40 BS=0.29 BS=0.58 BS=0.35 1 0 2 0 0 4 1 3 B BS=0.13 BS=0 BS=0.25 BS=0 BS=0 BS=0.40 BS=0.23 BS=0.38 5 6 6 6 7 7 5 C BS=0.56 BS=0.6 BS=0.86 BS=0.67 BS=0.58 BS=0.70 BS=0.50 6 7 5 7 7 5 D BS=0.43 BS=0.86 BS=0.63 BS=0.64 BS=0.78 BS=0.56 6 6 7 7 5 E BS=0.67 BS=0.67 BS=0.58 BS=0.70 BS=0.50 5 6 6 4 F BS=0.63 BS=0.55 BS=0.67 BS=0.44 4 5 5 G BS=0.63 BS=0.56 BS=0.56 9 7 H BS=0.75 BS=0.58 8 I BS=0.80

B – Fara Pond, C – Matityahu Pond, D – Navoraya Spring, E – Jauda Spring, F – Sasa Pond, G – Leshem Pond, H – Dir-Hanna Pond, I – Balad Spring, J - Gahar Stream.

Molecular DNA variation in Hyla felixarabica 65

tions had a highly similar cytochrome b gene, this marker is less suitable to study the genetic variation among the populations of one geographical area. This is in agreement with previous studies (Faivovich et al. 2005). Much research has focused on the genetic variables of Hyla in different geographical regions (Canestrelli et al. 2007, Stock et al. 2008). Nuclear loci and a fragment of the mitochondrial cyto- chrome b gene have clearly shown the existence of a remarkable genetic structure in the Italian tree frog. These results show a difference only between two geo- graphically coherent groups of populations, one comprising those located north of the northern Apennines, and the other, in those located south of it. Those closest to the northern side of this mountain chain were genetically intermediate between the two groups (Canestrelli et al. 2007). Higher genetic variation was found among the various populations, when using the mtDNA 12S, as compared to cytochrome b, which is a more suitable genetic marker in RAPD PCR for the study of various populations in a small area. These results are in agreement with studies in other species of populations (e.g., (Degani et al. 1999, Mikulicek& Pialek 2003, Pearlson & Degani 2007, Weisrock et al. 2001). The area studied had a Mediterranean climate, which is a relatively dry, and has only six amphibian species. H. felixarabica (H. savignyi) is very common (Degani & Kaplan 1999). It inhabits different habitats and can be found in various types of breeding sites. In the present study, the H. felixarabica population was sighted at various altitudes, varying from140 - 1060 m ASL (Fig. 1). According to the 12S ge- netic marker, the populations that differed the most from the others were found at relatively high or low altitudes, e.g., Leshem Pond (G) (300 m ASL), Jauda Spring (E) (110 m ASL) and Elrom (A) (1060 m ASL). However, it seems that other factors influenced the genetic differences, e.g., ecological conditions (Degani et al., 1999, Pearlson & Degani 2007, Pearlson et al. 2007). Grach at al. (2007) found a local population of Hyla in Israel in a small area amongst three Judean Hills at altitudes of 730 - 895 m ASL. Based on morphological characteristics, coloration and call structure, he proposed it to be a new species named H. heinzsteinitzi (Grach at al. (2007). The high genetic variation among some of the H. felixarabica populations (e.g., according to RAPD PCR analysis with the primer OPA-16 - the populations Leshem Pond [G] and Jauda Spring [E] or according to the 12S gene, Leshem Pond [G], Jauda Spring [E] and Elrom Pond [A]), might support the hypothesis that the high variability of this species at the southern border of its distribution is affected by different conditions in a small geographical area. This explanation is supported by Nevo and Yang (1979), who studied the genetic variation of H. felix- arabica (H. savignyi). They examined the allozymic variation of the proteins, en- coded by 27 loci in eight populations from Israel, along two transects of increasing

Herpetol. Rom, 6, 2012 66 Degani, G. et al. aridity, from north to south. The results of this study demonstrated the association of polymorphisms with climatic patterns of increasing aridity, southwards and eastwards, and showed that central populations harbor more genetic variations than marginal populations. Nevo and Yang (1979) proposed that the genetic varia- tion, based on protein polymorphisms in H. felixarabica (H. savignyi), results from climatic selection rather than from stochastic processes or neutrality. They con- cluded that the environmental variation model seems to be the best predictor of genetic variation in this species.

REFERENCES

Andersen, L.W.., Fog, K., Damgaard, C. (2004): Habitat fragmentation causes bottlenecks and inbreeding in the European tree frog (Hyla arborea). Proceedings of the Royal Society London 271: 1293–1302. Canestrelli, D., Verardi, A., Nascetti, G. (2007): Genetic differentiation and history of populations of the Italian tree frog Hyla intermedia: lack of concordance between mitochondrial and nuclear markers. Genetica 130:241-55. Degani, G. (1982): Amphibian tadpole interaction in winter pond. Hydrobiologia 96: 3-7. Degani, G. (1986): Growth and behavior of six species of amphibian larvae in winter pond in Israel. Hydrobiologia 140: 5-10. Degani, G., Jackson, K., Dosoretz, C., Plotzky, Y. (1999): Molecular DNA variation in Salamandra s. infraimmaculata from different habitats. Israel Journal of Zoololy 44: 239-246. Degani, G., Kaplan, D. (1999): Distribution of amphibian larvae in Israeli habitats with changeable water availability. Hydrobiologia 405: 49-56. Excoffier, L., Smouse, P.E., Quattro, J.M. (1992): Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131: 479-491. Faivovich, J., Garcia, P.C., Ananias, F., Lanari, L., Basso, N.G., Wheeler, W.C. (2004): A molecular perspective on the phylogeny of the Hyla pulchella species group (Anura, Hylidae). Molecular Phylogenetics and Evolution 32: 938-950. Faivovich, J., Haddad, C.F.B., Garcia, P.C.A., Frost, D.R., Campbell, J.A,. Wheeler, W.C. (2005): Systematic review of the frog family Hylidae, with special reference to Hylinae: phylogenetic analysis and taxonomic revision. Bulletin of the American Museum of Natural History 294: 1–240. Grach, C., Plesser, Y., Werner, Y.L. (2007): A new, sibling, tree frog from Jerusalem (Amphibia: Anura: Hylidae. Journal of Natural History 41: 709-728. Gvoždík, V., Moravec, J., Klütsch, C., Kotlík, P. (2010): Phylogeography of the Middle Eastern tree frogs (Hyla, Hylidae, Amphibia) as inferred from nuclear and mitochondrial

Herpetol. Rom, 6, 2012 Molecular DNA variation in Hyla felixarabica 67

DNA variation, with a description of a new species. Molecular Phylogenetics and Evolution 55: 1146–1166. Jeffreys, A.J., Morton, D.B. (1987): DNA fingerprints of dogs and cats. Genetics 18: 1- 15. Mikulicek, P., Pialek, J. (2003): Molecular identification of three crested newt species (Triturus cristatus superspecies) by RAPD markers. Amphibia-Reptilia 24: 201-207. Nevo, E., Yang, S.Y. (1979): Genetic diversity and climatic determinants of tree frogs in Israel. Oecologia (Berlin) 41: 47-63. Parker, R. (1976): Introduction of Statistics for Biology. Camelot Press, Southampton, UK. Pearlson, O., Degani, G. (2007): Molecular DNA variations among Triturus vittatus vittatus (Urodela) from different breeding sites at the southern limit of its distribution. Acta Herpetologica 2: 69-77. Pearlson, O., Jackson, K., Degani, G. (2007): The gonadal cycle in males and females of Triturus vittatus vittatus (Urodela) from the southern limit of its distribution. Progressive Perspective of Veterinary Medicine 50: 227-233. Salducci, M.D., Marty, C., Fouquet, A., Gilles, A. (2005): Phylogenetic relationships and biodiversity in Hylids (Anura: Hylidae) from French Guiana. Comptes Rendus Biologies 328: 1009-1024. Stock, M., Dubey, S., Klutsch, C., Litvinchuk, S.N., Scheidt, U., Perrin, N. (2008): Mitochondrial and nuclear phylogeny of circum-Mediterranean tree frogs from the Hyla arborea group. Molecular Phylogenetic Evolution 49: 1019-1024. Veith, M., Degani, G., Seitz, A. (1992): High genetic homogeneity of Salamandra salamandra (L.) in Israel. Zoologische Anzeiger 229: 63–72. Weisrock, D.W., Macey, J.R., Ugurtas, I.H., Larson, A., Papenfuss, T.J. (2001): Molecular phylogenetics and historical biogeography among salamandrids of the "true" salamander clade: rapid branching of numerous highly divergent lineages in Mertensiella luschani associated with the rise of Anatolia. Molecular and Phylogenetic Evololution 18: 434-448. Wetton, J.H., Carter, R.E., Parkin, D.T., Walters, D. (1987): Demographic study of a wild house sparrow population by DNA fingerprinting. Nature 327:147-149.

Herpetol. Rom, 6, 2012