THE DIET OF WATER FROGS ( PELOPHYLAX ESCULENTUS " COMPLEX " ) FROM THE PETROVARADINSKI RIT MARSH ( SERBIA )

In the Petrovaradinski Rit marsh region, the composition, dynamics and importance of the food components of three syntopic anuran species of the genus Pelophylax were analyzed through all the seasons of their activity in order to determine the vulnerability of each species in the trophic competition. The results of the studies have shown that the trophic niche breadth, according to the importance index of contents, was the smallest for the species Pelophylax kl. esculentus and largest for the species Pelophylax ridibundus, meaning that the species Pelophylax kl. esculentus has the most uniform diet and is therefore most vulnerable to competition by other species.


INTRODUCTION
The food of anurans includes a large number of invertebrates and occasionally a smaller vertebrate species, so they have an important role as regulators of the density of prey species.Anurans are the hosts and carriers of various parasites and for that reason, the data about the diet of anurans have exceptional importance in explaining invasion pathways.It gives insight into the conditions and characteristics of habitats, the species living in the area, seasonal changes and other important components of particular biocenoses (Šimić et al., 1992b).The composition, dynamics and importance in the diet of 270 specimens of the Pelophylax esculentus species "complex" were determined in order to pinpoint any specificity that would be characteristic of Petrovaradinski Rit.
The quantitative indicators were calculated in order to determine which species is most vulnerable to competition.At the beginning of the study, niche breadths were determined, as well as the quotients of their overlap, both for the whole year and for all three seasons of activity.

Study site
The Petrovaradinski Rit marsh -(UTM DR 10, DR 11), which is periodically flooded by the river Danube, is a protected area, and together with the Koviljski Rit swamp is classified under IUCN category IV.Petrovaradinski Rit is situated in vicinity of Novi Sad and it is under great impact from anthropogenous factors.The length of the marsh is 12 km, and its width is on average 0.7 km.The altitude ranges from 74 to 94 m.The hydrographical characteristics, fauna and vegetation provide suitable conditions for the life and reproduction of anurans.In contrast to the greater part of its range where the hybrid Pelophylax kl.esculentus lives together with only one parental form (in most cases Pelophylax lessonae (Uzzel et Berger 1975)) in this locality it lives together with both parental forms, resulting in back-crossing breeding.Therefore, the studied locality offers an extraordinary opportunity to compare the diet of the species in the Pelophylax esculentus "complex".

Calculation of indices
Material, i.e. the content of the digestive tracts of anurans, was collected during the period 1994-1997 in the area of Petrovaradinski Rit during spring, summer and autumn.A total of 270 specimens of the genus Pelophylax were collected, 30 specimens per season per investigated species.During the four years material collection was by standard methods and according to the principle of random sample.Immediately after collection, the animals were processed in the laboratory, biometric data were collected and the sex of the specimens was determined.The material originates from animals that had already been killed by chloroform (CHCl 3 ) for an endoparasitological examination (Bjelić and Horvat 1999).Afterwards, the bodies are conserved and deposited in the Amphibian Collection of the Natural History Museum in Belgrade.
In this paper various quantitative indices were calculated, with emphasis on the importance index and the methods previously used by Obrtel and Holišova (1974) (Vide Krsmanović 1978).
The total content of the digestive tract represented 100% volume, and particular separated parts of the content were expressed as the relative volume, shown as 5, 10, ..., 95%.If certain parts of content were present in less than 5%, their presence was marked by the sign "+".
After identification, in order to analyze the results, the components of animal origin were grouped into various classes or lower taxa, while the components of plant origin were discussed as an all-including unit.
Besides the identification of the various components, we also calculated the quantitative indices that further convey their role and importance: Frequency of the component (f): represents the number of digestive tracts that included a particular component.
Relative frequency (f %): represents the percentage of digestive tracts that included a particular component when compared with the total number of digestive tracts.
Medium frequency (MF = ∑ f / N): represents the average number of appearances of components in a single digestive tract.
Relative presence (100f / ∑ f): represents the percentage of appearance of a particular component (f) when compared with sum of appearances of all components.
Total volume (t.v.) of particular component: sum of all relative volumes occupied by that particular component in all surveyed digestive tracts.
Relative volume (%v = 100 t.v./ ∑ v): percentage of total volume of a particular component when compared with the total volume of all digestive tracts.
Importance index [ I = (100f / Σf + 100 t.v./ Σv) / 2] of a particular component: the average value from relative presence and relative volume is used as an indicator of the role and importance of particular food sources in total diet.Hurlbert's standardized niche breadth (B A = B -1 / n -1): modification of Levin's formula for calculating niche breadth (B), where B = 1 / Σp i 2 , while p i is the proportion of individuals recorded in the resource or using the component i, while n is the number of possible components.The value of p i may be represented by one of the quantitative indices of the components.The values of B A are exclusively within the scale 0-1, in contrast to the niche breadth by Levin, where the values range from 1 to n (n represents the number of components).The standardized niche breadth enables a relatively easy and clear-cut comparison for the various species of anurans.B A is maximal (B A =1) when the same number of individuals is 2 ) 1/2 : this measures the overlaps between the niches of two species, where p i1 and p i2 represent the proportion of resource i within the total resource pool used by species 1 and 2, or the qualitative parameters of the components.The values of the quotients range from 0 (where there are no common components) to 1 (complete overlap).

RESULTS
The qualitative and quantitative analysis of the diet in the species of the genus Pelophylax has shown it to be mostly composed of invertebrate species.In all studied anurans, there was a pronounced domination of the class Insecta during the whole activity period (see Table 1).
When analyzing the adult and larval stages of invertebrates and vertebrates in the content of the digestive tracts of the studied frog species, it is obvious that they are always dominated by adults in all three seasons of activity (see Table 2).

Quantitative indicators of the role and importance of diet components
Comparison of the relative frequencies of components in the studied anuran species shows a large percent representation of certain taxa: Coleoptera, Hymenoptera, Gastropoda, Diptera (see Table 3).
According to the average number of components per single digestive tract (the medium frequency), a species' diet may be considered larger or smaller.In the species Pelophylax ridibundus there were, on average, slightly more than three components MF = 3.24.In the digestive tracts of species Pelophylax lessonae there was, on average, somewhat less than three and a half components MF= 3.44, and in the species Pelophylax kl.esculentus there were, on average, about three and a half components MF = 3.52.
When we compare the ratio of relative volumes of components in the three species of anurans throughout the year, the results of this paper indicate a large percent representation of Coleoptera and Hymenoptera, Lepidoptera, Gastropoda and Diptera (see Table 3).

Seasonal variation in the importance index of food components Pelophylax ridibundus
Remains of Coleoptera were evidently the most important contents of diet in the species Pelophylax ridibundus in spring (I = 24.09)and summer (I = 23.36),while during autumn the dominant prey belonged to the order Diptera (I = 24.76).In second place by importance in spring were representatives of Gastropoda (I = 14.

Pelophylax kl. esculentus
The remains of Coleoptera are the most important contents of the diet of the species Pelophylax kl.

Niche breadth and quotient of overlap of feeding niches of analyzed species
The seasonal variations in the breadth of feeding niche are presented in Table 4.
The seasonal changes in the overlap quotient according to the importance index are shown in Table 5.
In spring and summer, the greatest overlaps were recorded in the species Pelophylax ridibundus and Pelophylax kl.esculentus, while in autumn a slightly larger overlap was recorded in Pelophylax lessonae x Pelophylax kl.esculentus.

DISCUSSION
The available literature shows the representation of larvae in the diet to be between 0% and 30% (Popović et al., 1992, Török et Csörgo 1992, Šimić et al., 1992b, Šimić et al., 1995a, Medvedev 1974, Sczerbak et Sczerban 1980, Cogalniceanu et al., 1996, Gutowski et Krzysztofiak 1988, Bosman et al., 1988).However, none of these authors determined the ratio of adult and larval forms in a systematic fashion according to seasons, so comparison with results of

Graph. 2. Seasonal variation in importance index of food components in the diet of Pelophylax lessonae
Graph 1. Seasonal variation in importance index of food components in the diet of Pelophylax ridibundus this study is difficult.All the results show that the adult forms are dominant in the diet of the studied species.In contrast to some cited works, in this paper the presence of larvae was determined in all seasons.
The prey specimens determined to genus or species level include a certain number of taxa that bear the anthropocentric attribute "pest" -Gryllotalpa gryllotalpa, Harpalus sp., Leptinotarsa decemlineata, species from the families Pentatomidae, Curculionidae, Culicidae, as well as certain species from the genera Homoptera, Hymenoptera and Lepidoptera.Also recorded was the presence of larvae of the species Erystalis tenax (Diptera: Syrphidae), a bioindicator of water pollution.Similar results were also obtained by other authors (Juszczyk 1950, Medvedev 1974, Sin et al., 1975, Kamminga et al., 1991, Šimić et al., 1992b, Šimić et al., 1995a), indicating that the studied species are important predators of so-called pests, and that the diet of anurans may contribute to knowledge on the pollution status of water ecosystems.
Components of non-animal origin were found in the digestive tracts of all the studied anurans of Petrovaradinski Rit.Most of it is plant material and there might be some mud, a few stones or some other components.Many authors believe that the plant material accidentally enters the digestive tract of anurans during the hunting and swallowing of prey.However, some of them do not (Juszczyk 1950, Beškov 1970, Cogalniceanu et al., 1996, Sczerbak et Sczerban 1980), and some of them mention it as a component of diet -as a food resource or prey taxa (Chiminello et Genarni 1992, Török et Csörgo 1992).According to the field studies, the plant material accidentally gets into the digestive tract while swallowing prey.Considering that the plant material in the digestive tract of anurans of Petrovaradinski Rit looked partly digested, it might have some nutrient value.Das (1996) considered that the consumption of vegetation of Rana hexadactyla is intended, but in that case the volume of the plant material was much bigger (79.5 %) than the volume (around 5%) in the diet of the examined water frogs of Petrovaradinski Rit.Sin et al., (1975) have determined that the most commonly present components in the diet of the species Pelophylax ridibundus are representatives of Homoptera, while Günther (1990) determined that these were Coleoptera and Aranea.Results of the studies performed at Petrovaradinski Rit are similar to results by Gutowsky et Krzysztofiak (1988), who analyzed the relative frequency of components in the species Pelophylax lessonae and Pelophylax esculentus.
The importance index is a parameter that can define the role and importance of particular food sources in the most adequate way.The clearly most important contents of diet in the species Pelophylax ridibundus and Pelophylax kl.esculentus were  Coleoptera (I = 24.09and I = 20.31,respectively).In both species, second place in importance was occupied by representatives of Lepidoptera (I = 12.44 and I = 12.70, respectively).In the species Pelophylax ridibundus this is followed by Diptera (I = 12.38), Gastropoda (I = 11.59) and Hymenoptera (I = 8.97), while in the species Pelophylax kl.esculentus it is followed by Gastropoda (I = 12.47), Hymenoptera (I = 10.09) and Diptera (I = 10.46).The other components have small index values, indicating their minor energy-bearing role in the diet of these two species.
In the diet of the species Pelophylax lessonae the dominant role is played by Gastropoda (I =18.62), while Coleoptera (I = 17.78) are in second place of importance.Hymenoptera (I = 12.71) and Lepidoptera (I =11.71) represented a good food source for the species Pelophylax lessonae.The other food sources were poorly utilized (see Table 3).
Niche breadth and quotient of overlap of feeding niches of analyzed species Horvat et al., (1995) determined the breadth of feeding niche for the species Pelophylax ridibundus, Pelophylax lessonae and Pelophylax kl.esculentus from the area of Apatin.The differences between the niche breadths in their study and the niche breadths determined by this study are relatively small, as these are similar habitats, at a relatively small distance and in the same climate zone.Horvat et al., (1995) calculated the overlap index for species in the Pelophylax esculentus "complex" throughout the year in area of Apatin.Their values are somewhat larger than those determined for the same species during the studies at Petrovaradinski Rit, meaning that all studied species from the genus Pelophylax in the Apatin area were forced to use more identical components in their diet.

CONCLUSIONS
The diet of all the studied anuran species during the whole period of activity is composed of easily available and abundant prey specimens -mostly or exclusively representatives of Invertebrata, dominated by representatives of the class Insecta.The presence of certain prey species in high numbers (anthill, spider nests and similar aggregations of Insecta and Invertebrata) or the presence of gregarious forms result in the increased presence in the diet of specimens of that prey type.This confirms the lack of species selection by predators and their opportunistic behavior in such cases.The hunting strategy of the studied species causes the dominance of terrestrial organisms in their diet.
The greatest seasonal oscillation in the average number of components per digestive tract was present in the species Pelophylax ridibundus, and the smallest was in the species Pelophylax kl.esculentus.
When the complete feeding period of the three studied species is analyzed, the difference in participation of larval stages of prey is insignificant; however, when analyzed by season it is very pronounced.The greatest percent participation of larval prey was recorded in the diet of the species Pelophylax ridibundus, and the smallest in Pelophylax lessonae.
According to the index of importance of components, the greatest importance throughout the year, combined for all three studied species of anurans, is held by representatives of the following taxa: Coleoptera, Hymenoptera, Diptera, Gastropo- The species Pelophylax kl.esculentus has the most uniform diet and is therefore most vulnerable to competition by other species.
The indicators of role and importance of diet components for the studied species of anurans from Petrovaradinski Rit show great similarity with the indicators from similar habitats and similar climates, such as Apatin, Mrtva Tisa (Vojvodina, Serbia) and the inundation zone of Danube in Romania.
11), followed by Diptera (I = 10.87) and Aranea (I = 8.54), while in the summer period there were Lepidoptera (I = 15.02) and Gastropoda (I = 12.81).During the autumn, Diptera are followed in this species' diet by Lepidoptera (I = 18.04),Coleoptera (I = 12.95) and Homoptera (I = 11.31).The other components have small values of index, indicating their minor energy role in the diet (see Graph 1).Pelophylax lessonaeGastropoda play a dominant role in spring (I = 24.00)and summer (I = 22.46) in the diet of the species Pelophylax lessonae.During the spring they are followed by Coleoptera (I = 23.67) and Hymenoptera (I = 11.17).During the summer, second place in importance is occupied by Lepidoptera (I = 17.11), followed by the remains of Coleoptera (I = 14.64).During the autumn, this species' diet is dominated by Hymenoptera (I =19.00), while Coleoptera (I = 15.14) are in second place of importance.The remains of Diptera (I = 13.83) and Lepidoptera (I = 13.50)represent a good food source for the members of this species.The esculentus in the spring (I = 22.48) and summer (I = 22.26) period.After Coleoptera, the most important groups in spring were Gastropoda (I = 18.61) and Aranea (I = 13.72), and during the summer Lepidoptera (I = 19.95) and Hymenoptera (I = 13.70).In the autumn, the first three places by importance in the diet of the species were: Lepidoptera (I = 16.88),Coleoptera (I = 15.95) and Diptera (I = 14.60).(see Graph 3).

Graph. 3 .
Seasonal variation in importance index of food components in the diet of Pelohylax kl.esculentus

Table 2 .
Participation of adult and larval stages in prey of three anuran species

Table 1 .
Diet composition of three studied species according to the seasons of activity S -Spring, Su -Summer, A -Autumn using various resources, meaning that the species does not differentiate between the resources, and it is minimal (B A =0) when all individuals exclusively use a single resource.The minimal niche breadth represents the maximal specialization.Pianka's standardized quotient of niche overlap

Table 3 .
Relative frequencies(%f) , relative volumes (%v) and importance indexes (I) of the prey through the year

Table 4 .
Niche breadth (BA) according to importance index of the components

Table 5 .
Feeding niche overlapping according to importance index of prey items during three seasons of activity