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Introduction

The genus Salamandra Garsaut, 1764, belonging to the terrestrial Urodeles, is widely distributed in Europe and reaches, at its southern border, North Africa and the Middle East [1] (Figure 1). The systematics of this genus has undergone many changes, from its division into subspecies to its generally agreed upon division into six species [2-4]. Due to the large variation in salamander types, classification into species and subspecies is complicated and ambiguous. Some of the six species of the genus Salamandra are defined as subspecies in their wide distribution in Europe, North Africa and Asia, including Asia Minor and Israel [4] (Figure 1)

Locations of the studied populations (study sites) appear on this map: Kibbutz Sasa (33°1′37″N 35°23′40″E, 910 m above sea level [M.A.S.L.]) [7], Kibbutz Yehiam (32°59'39"N53'13'19E, 634 M.A.S.L.), Tel Dan (33°14′25″N 35°39′11″E, 190 M.A.S.L.) [8].

 This division and the large variation in colored spot patterns on the backs of salamanders among populations sometimes make it difficult to determine the species or subspecies with any certainty [1]. Steinfartz et al. [2] suggested classifying the species in Europe, North Africa and The Levant into six groups based on their mitochondrial DNA: Salamandra salamandra, Salamandra infraimmaculata, Salamandra corsica, Salamandra atra, Salamandra lanzai and Salamandra algira. Accordingly, the fire salamander (S. infraimmaculata) is located in Israel and S. algira in North Africa [9,10]. The coloration patterns were then associated with habitat type and geographical area of distribution [11]. For the comparison, salamanders in Israel were taken from the Mount Carmel area. Those researchers gave a first description of the karyotype of the fire salamander in Israel, finding 12 pairs of chromosomes [11].
In Israel, where the salamanders are located on the southern diaspora border, there are isolated populations which have survived in the north of the country. Due to the different environmental conditions in their habitats, a comparison of these populations could be interesting [1,12,13], in terms of morphological [14] and physiological [15-17] variations, larval growth [18], and genetic variations under different environmental conditions [5,19-21].
S. infraimmaculata shows a fragmented distribution in various types of habitats, including mountain ranges that are 130 to more than 1000 m above sea level [1,22]. Due to polymorphic coloration and a diversity of reproductive modes, it is very difficult to separate the species according to these variables [5]. Genetic studies have focused mainly on tadpoles in the various populations. Samples of salamanders after metamorphosis usually consist of relatively small numbers and determining the relationship between the morphological variance and molecular variability of several markers is problematic [5,22-24]. The color pattern of S. salamandra has been relatively well-described in many articles (see for review [25]). The arrangement of the colored segments on their black back varies from two yellow lines on S. terrestris to variously shaped yellow spots on S. salamandra, and there is great variability among individual details, including combinations of these two models [5,25]. The differences between the color- pattern phenotypes in S. salamandra and S. terrestris are very large not only between species, but also among individuals. In Israel, where the species S. infraimmaculata lives under the most extreme conditions, and where the differences between habitats may affect the variability of the populations, morphological differences were studied in several areas based on a relatively small number of adult salamanders: the Tel Dan habitat, with constant conditions and water availability year round, the Upper Galilee, and the Western Galilee (Mount Meron, Kibbutz Sasa and Kibbutz Yehiam), with mountain habitats that are dry and warm in summer and cold and rainy in winter [1,3,14]. The body size of the salamanders from the Tel Dan population was significantly smaller than that of salamanders from the other areas. These differences were supported by studies of the genetic differences

 between the Tel Dan population and salamanders in other parts of Israel living under more extreme ecological conditions [1,21]. In studies on the pattern of yellow spots on the back of salamanders between different regions of Israel (Tel Dan, Upper Galilee, Western Galilee and Mount Carmel), there were no clear differences between the regions [3]. We hypothesized that the adaptation of salamanders to extreme environmental conditions will be reflected in the Kibbutz Sasa (Sasa) and Kibbutz Yehiam (Yehiam) populations by morphological and physiological changes compared to salamanders in the Tel Dan (Dan) population, which live under less extreme conditions [1,21]. In the present study, we examined the potential differences among the spot patterns of the three different populations of S. infraimmaculata. We describe the different color-pattern phenotypes on the black backs of S. infraimmaculata in the various habitats: Dan (stable conditions throughout the year), Sasa near the Mount Meron region, and Yehiam in the Western Galilee. We further compare the color- pattern phenotypes of S. infraimmaculata to those of other salamander species [5] (Figure 1).

Material and Methods

The study areas included Kibbutz Sasa and Kibbutz Yehiam, both semi-arid habitats in the region of one of the highest mountains in Israel, Mount Meron (Figure 1). The winter in this area is longer and rainier than in most of the rest of the country, with annual precipitation reaching 1000 mm.

The summer is comparatively dry, with no rain. The Tel Dan Nature Reserve is in the northeast of the country; it lies among hills and is bounded by mountains to the north, but is itself only 180 m above sea level. The area as a whole is characterized by numerous spring-fed streams with running water, and is the only such area in Israel.

Field observation, samples and photographs of salamanders for coloration pattern determination were from populations that have been intensively studied and for which all other parameters (body weight and length) have been well- described [1]. Of all of the salamanders that were photographed, only those that showed a clear difference between a black back and yellow spots were taken as samples for this study. Each salamander was individually inspected for its unique pattern of yellow spots on its head and back. Observations were conducted on rainy days when the salamanders are active on the surface in the three habitats and easy to find at night [7,26]. In the Dan population, 454 salamanders were photographed, of which 100 were sampled to measure the percentage of yellow and black coloring. In Sasa, 201 salamanders were photographed, and 62 sampled for color measurements. In Yehiam, 200 salamanders were photographed, of which 60 were sampled for color measurements. In all locations, about a third of the salamanders were photographed more than once. The number of spots on the head and back of each individual salamander was determined. The images were entered into the computer and the

ratio of black to yellow color was calculated as the percentage of yellow color using the formula [Yellow/ (Yellow + Black)] x 100. The photograph of the salamander was copied into Word Object design software, giving it a white background. The image was then copied to the software program Cool PHP Tools Image Color Extract, where the percentage of yellow on the salamander’s back was determined.

Statistical Analysis

All statistical processing was done using JAMOVI 2.0.0 software. To check for differences in the number of yellow spots on the salamanders’ back and in the black-to-yellow ratio among the various populations, we used several statistical tests. We used one-way analysis of variance (ANOVA) and post-hoc Student t-test for each pair of averages, without correction based on multiple tests.

ANOVA was used to examine the variation among all three populations in the number of yellow spots on the back and the black-to-yellow ratio. The t-test was used to compare between two populations. The number of salamanders with different color patterns in the various populations was estimated by Z-proportion analysis. The significance threshold was p < 0>

The color of the spots differed among individual salamanders in the same population, with various different shades of yellow. The patterns of the yellow spots on the backs of the salamanders in the different populations (Dan, Sasa and Yehiam) are shown in Figure 2: one row, two rows or scattered. The size and shape of the spots differed; some were round and others had different shapes. It was difficult to discern the differences in spot patterns between the different populations (Figure 2). A comparison of the patterns of yellow spots on the backs of the salamanders (one row, two rows and scattered) between populations (Dan, Sasa and Yehiam) is shown in Figure 3. Differences were not significant (t-test, Z-test and ANOVA, p > 0.05).

Figure 3: Comparison of the patterns of yellow spots on the backs of the salamanders (one row, two rows and scattered) between habitats (Dan, Sasa and Yehiam). Differences were not significant (Z-test, p > 0.05).

For two indices (proportion of yellow/black and number of spots on the head), the Dan population differed from the two other populations of salamanders (t-test, Z-test and ANOVA, p < 0>

populations, with the percentage of yellow on the back being significantly larger for the former (Figure 4) salamanders (t-test, Z-test and ANOVA, p < 0>

Various numbers of yellow spots were found on the head of the salamanders in the three populations, from 1 to 7 (Figure 5). In all populations, there were more salamanders with 4 spots than with any other number of spots. In the Dan population, there were significantly more salamanders with 1 to 3 spots than in Sasa or Yehiam. Here again, the spot pattern for the Dan population differed significantly (ANOVA, p < 0> 0.05).

The color of the spots varied from light yellow (yellow with white hue) to yellow with a red hue in all populations studied (Figure 6).

Salamandra that also appeared on S. infraimmaculata in present study [5,6,29]. The images were entered into the computer and the percentage of yellow color was calculated as [Yellow/(Yellow + Black)] x 100.