Life Cycle of Tree Frogs ( Hyla savygnyi ) in Semi-Arid Habitats in Northern Israel

The life cycle of tree frogs (Hyla savygnyi) in localities of various habitats in northern Israel in the Upper Galilee and Golan Heights (annual rainfall range of 500-1,000 mm) and the Hula Valley, ranging from 212 to 740 m above sea level (ASL), was studied. Tree frogs were observed around winter rain pools, springs and streams. Fifty-one different breeding places were monitored. Only ponds and springs having stable non-flowing water are used by tree frogs for reproduction and are where larvae can metamorphose. The male call in the breeding places attracts the female, who then comes to the male, and breeding occurs underwater. Breeding time in Israel varies according to location and geographical and ecological conditions. It takes place in March-April in northern Israel, the Upper Galilee, the Golan Heights and the Hula Valley. In various breeding places, larvae grow between April and June. Apparently tree frog larvae adapt to breeding places where stable water is found, such as in ponds or springs, but not to breeding places with flowing water such as streams or rivers. Various Anuran and Urodela larvae were found in the same breeding sites where the larvae of H. savygnyi were found, including Pseudepidalea Viridis, Rana bedriagae, Pelobates syriacus, Salamandra infraimmaculata and Triturs vittatus. Following metamorphosis, H. savygnyi adapt to terrestrial life in semi-arid habitats, and is found on plants or in hiding places under rocks and in holes to prevent dehydration particularly during hot and dry weather. The ability to change color helped the frogs to hide in various substrates. The rate of water loss of terrestrial H. savygnyi during dehydration is around 50% of body weight. Plasma osmolality increased from 200 mOsm/kg to about 450 mOsm/kg, which helped the tree frog to survive in semi-arid habitats.


Introduction
The distribution, morphology, ecology and taxonomy of tree frogs (Hyla savygnyi) in the Middle East have been studied quite extensively (Degani, Nagar, & Yom-Din, 2012;Gomez, Gauthier, & Lengagne, 2011;Grach, 2007;Gvoždík, 2010;Stock, 2008).The high genetic diversity in relatively small areas (Degani, Nagar, & Yom-Din, 2012) is making it difficult to study its phylogeography and taxa.Since it is very interesting to study this species and its adaption to various habitats and large range of distribution, the location of tree frogs must be defined in detail and might vary in met population.Nevertheless (Gvoždík, 2010 ), who carried out extensive mtDNA studies, suggested that H. savygnyi can be found in Israel and the Golan Heights, and that their geographic distribution also includes Jordan, Syria and Lebanon.However, other species might also be found in Israel, e.g., H. felixarabica sp.nov.(Gvoždík, 2010).Based on morphological characterizations, coloration and call structure, H. heinzsteinitzi is located at three sites in a small area in the Judean Hills within a 6x13 km range at altitudes of 730-895 m above sea level (ASL) (Grach, 2007).The Hyla genus represents various color changes (Degani, 2013;Nielsen, 1980).Most of the work dealing with color changes of the Hyla genus has been done on the European species, H. arborea (Nielsen, 1980) and more information has been published compared to H. savygnyi.Many aspects of the life cycle of the Hyla genus especially in Europe have been studied and published.The species belonging to the Hyla genus is nocturnal whereby the males display a vocal sac and dark flank stripe, which both vary in coloration (Gomez et al., 2009;Grach, 2007).The life cycle of H. arborea was described in detail in its wide distribution throughout Europe (Denmark [Aarhus and Jutland]), Sweden (Scania) and the southern shore of the Baltic Sea in Lithuania (Vilnius City), through Byelorussia (approximately on the line of the towns Oshmyany-Uzda-Slutsk) to southern Russia.From there, the margin runs south and south-eastwards approximately along the line of the Bryansk Province-Kursk Province west of the Byelgorod Province (Shebekino District), then southwards in the Ukraine from Kharkov City to the Dnepropetrovsk Province and the Donetsk Province.In Crimea, the frog is found along the southern shores and extreme northwest of the peninsula.The frog (H.arborea) is active primarily in the evening and at night, when it descends to the ground from vegetation to forage and rehydrate.During the autumn, it migrates to hibernate in Europe.Hibernation in Europe takes place from September-December to February-early May on land (in soil, burrows, heaps of stones and holes in trees).Reproduction occurs in places at different times in Europe from April to May, but sometimes in March, June or even late July.As a rule, more males are found in breeding pools than females (Tarkhnishvili, 1999).Females enter pools after the males and leave immediately after breeding.There is varying information on the number of eggs laid per female.A clutch contains about 200-1,000 eggs deposited in portions, usually in the form of small rounded clumps containing from 3 to 100 eggs.The H. arborea clutch contains 200-1,000 eggs deposited in a few clumps of 270-315 eggs over several hours, after which the female leaves the pool (Tarkhnishvili, 1999).Metamorphosis occurs from June to September, depending on the geographic position of a locality.In some cases, the larvae overwinter and complete transformation the following summer.H. arborea preys on various invertebrates, mainly insects.Its ability for long leaps makes it possible to forage on fast-flying insects, which comprise a considerable proportion of its food.The frogs forage on land.During the breeding season, adults forage periodically on the shore and on high plant stems above the water surface.
The current study examines the life cycle and adaption of different breeding sites to the terrestrial environment of the tree frog (H.savygnyi) and provides more information about the adaption of this species to semi-arid conditions.

Study Area
The study was carried out over four consecutive decades .Different aquatic habitats were dispersed over an area of approximately 1,400 km 2 in northern Israel, including northern and eastern Israel, the Golan Heights and the Hula Valley (Figure 1).The habitats included ponds, rock pool holes, springs and streams that were stable or flowing bodies, and in which water was available part or all year-round.The unpredictable breeding places such as ponds, which were flooded during the autumn when rainfall began, gradually dried out between the late winter months and early summer.

Sampling
The water body was examined by nets (pore size 450 μm) from a depth of approximately 10-30 cm, as previously described by (Degani & Kaplan, 1999).Rain pools and ponds in Israel that were filled by rain held the water for 3-4 months before drying up; large ponds dried up after a period of 6-12 years.The elevation of the breeding places is between 0 to 1,000 m above sea level.The breeding places that were examined are presented in Table 1 and  Figure 1.

Terrestrial Adaption
Adult H. savignyi were collected from the Sasa Pond (Degani, 1982) during the breeding period in February-March.The experiments were conducted in desiccators over dry silica gel (0-5 relative humidity [RH]) at 10ºC.Upper limits of water loss were determined when the frogs lost their coordination.Five adults were used in each experiment.The adults were weighed using Mettler H 315 balance until the frogs (10%, 20% and 30%) reached their upper limit of water loss.Twelve adult tree frogs were dehydrated to different percentages of body weight to establish the effect of dehydration on concentration of both plasma and muscle.Blood samples were taken from the dehydrated frogs by puncturing the heart using a 1 ml syringe washed with lithium heparin as previously described (Degani, 1984).The blood samples were centrifuged immediately and the plasma frozen for later analysis.Sodium, potassium and chloride concentrations (Degani, 1981) and urea in the plasma were determined as described by (Degani, Silanikove, & Shkolnik, 1984).The time for the larvae to grow and complete metamorphosis is presented in Figure 2. Tree frogs can breed several times during the warm season, usually when there is a drop in barometric pressure, two or more inches of rainfall, and in the darkness of night.The times where H. savingnyi larval species were in the different breeding places are affected by ecological conditions and water quality (Table 2).
In various breeding places, the larvae growth period is between April and June.
Most of the habitats where tree frog larvae growth occurs are ponds.Some springs in which water collected in the pool are also used by tree frogs (Figure 1).Apparently tree frogs do not adapt to flowing water such as in streams.
The range of water quality in the breeding places of tree frogs is presented in Table 3.According to the results, the temperature ranges from 10ºC to 25ºC and oxygen from 1 to 18 mg/L in green water, with different concentrations of chlorophyll a from 0.1 up to 122 mg/L.The main ions affecting plasma concentration during dehydration are Na + and Cl -.However, a low urea accumulation was found (Table 4).In general, when the frogs are found on trees or plants their color is green and changes to brown and black according to the substrate (Figure 5).

Discussion
The current study described the life cycle and adaption to terrestrial life of tree frogs (H.savygnyi) in northern Israel, adding information to studies carried out on this species of the Middle East tree frog (Degani, Nagar, & Yom-Din, 2012;Gomez, Gauthier, & Lengagne., 2011;Grach, 2007;Gvoždík, 2010;Stock, 2008).The results of the current study are in agreement with previous studies, whereby the breeding places of H. savygnyi include various stable water bodies, including winter pools in which water is available at least two or more months (Degani, 1999) and springs (Figure 1).This finding shows that no differences exist between breeding places of H. savygnyi and H. arborea (Tarkhnishvili, 1999) in which reproduction occurs in small stagnant water bodies (ponds and puddles, some of which are very small) and slowly flowing brooks with dense herbaceous and shrub vegetation (Kuzmin., 1999).This is a relatively short period of larvae growth and complete metamorphosis, representing adaption to xeric habits where many of the breeding places dry up after several months (Degani & Kaplan, 1999).
H. savygnyi lives in much drier landscapes than H. arborea schelkownikowi, including steppes, deserts and semideserts (Gvozdik, Moravec, Klutsch, & Kotlik, 2010).Therefore, its adaption to terrestrial life is very important.Following metamorphosis, H. savygnyi lives mainly near water bodies, in wet sites, oases, gardens, bushlands and mountain forests.However, during the summer time and on hot days, the tree frogs found hiding places under stones or in holes in the ground.During this time, H. savygnyi is in danger of dehydration.The frogs' relatively small body and skin that does not prevent water loss, as was found in this study of H. savygnyi, is in agreement with previous findings (Degani, 1984) whereby tree frogs have to find different breeding places to adapt to semiarid habitats.The ability to lose 50% of their body weight and increase plasma concentration, as described in the current study, are ways of helping them to survive in terrestrial life.The only amphibian species that is more adapted to terrestrial life than H. savygnyi is Pseudepidalea viridis (Degani, Silanikove, & Shkolnik, 1984).P. viridis has the ability to survive with high plasma osmolality by urea accumulation, which is not found in H. savygnyi.The high genetic and color diversity in relatively small areas (Degani, Nagar, & Yom-Din, 2012;Degani, 2013) might help this species to adapt to different environments found along the southern border of its distribution that includes Mediterranean semi-arid and arid environments.The results of this study along with a previous study (Degani, Nagar, & Yom-Din, 2012;Degani, 2013) support the hypothesis that the ability to change color and plant preference depends on adaptability to different habitats and different substrates.These phenomena help H. savygnyi to silence and prevent carnivores if they are found in the different substrates.Accordingly, H. savygnyi may even be found at long distances from water bodies in xeric environments, such as rocky slopes and in xerophytic bushes (Tarkhnishvili, 1999).
In conclusion, the life cycle of the tree frog (Hyla savygnyi) in semi-arid habitats is affected by various phenomena, e.g., unpredictable breeding places, short periods of larvae growth and complete metamorphosis, the ability to change color affected by various substrates, and high percentage of water loss, which help it survive in semi-arid habitats.

Figure 4 .
Figure 4. Plasma concentration during water body loss of Hyla savingnyi

Figure 5 .
Figure 5. Mature different colored H. savingnyi in various places in the habitats

Table 2 .
Overlap times when Hyla savingnyi larval species were in different habitats with other amphibian larvae.

Table 3 .
The range of abiotic parameters of water at various breeding sites where Hyla savingnyi larvae grow and complete metamorphosis H. savingnyi moves to terrestrial life in semi-arid habitats.The frogs are found on plants or in hiding places under rocks and in holes to prevent dehydration particularly during hot and dry weather.The rate of water loss of terrestrial H. savingnyi is presented in Figure4.At dehydration of around 50% body weight, plasma osmolality increases from 200 mOsm/kg to about 450 mOsm/kg.