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Karyological comparisonof water frog (Rana cf.ridibunda) populations fromBahrain, eastern Saudi Arabiaand EgyptSaeed A. Mohammed a , Abd El-Kader Gamal El-Din a , Helmy El-Dawy b , Haifa A. Al-Maskati a &Moustafa Selah ba Biology Department, College of Science,University of Bahrain, P.O. Box 32038, Isa Town,State of Bahrainb Zoology Department, Faculty of Science, Al-Azhar University, Cairo, EgyptVersion of record first published: 28 Feb 2013.

To cite this article: Saeed A. Mohammed , Abd El-Kader Gamal El-Din , Helmy El-Dawy , Haifa A. Al-Maskati & Moustafa Selah (1997): Karyological comparison ofwater frog (Rana cf. ridibunda) populations from Bahrain, eastern Saudi Arabia andEgypt, Zoology in the Middle East, 15:1, 41-49

To link to this article: http://dx.doi.org/10.1080/09397140.1997.10637738

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Arnphibia

Karyological comparison of water frog(Rana cf. ridibunda) populations from

Bahrain, eastern Saudi Arabia and Egypt

by Saeed A. Mohammed, Abd EI-Kader Gamal El-Din, Helmy EI-Dawy,

Haifa A. A1-Maskati and Moustafa Saleh

Abstract: Karyological analysis was carried out on three separate populations of the Water FrogRana cf. rldibunda from Bahrain, Saudi Arabia, and Egypt Chromosome preparations madefrom bone marrow cells showed that the three populations possess a karyotype of 26chromosomes. Variation in the relative arm lengths of the chromosomes 3, 5 and 11 among thethree populations was recorded. No cytological evidence of sex dimorphism was recorded in thethree populations. The present study failed to recognise any secondary constriction in thekaryotypes of the three populations. The karyological studies do not indicate that the threepopulations belong to different species.

Kurzfassung: Drei Wasserfrosch-Populationen (Rana cf. rldlbunda) in Bahrain, Saudi-Arabienund Agypten wurden in karyologischer Hinsicht verglichen. Chromosomenprllparate aus Kno­chenmarkzcllen ergaben bei alien drei Populationen 26 Chromosomen. VariabilitJIt der relativenLange der Arme wurde bei den Chromosomen 3, 5 und 11 festgestellt Es ergab sich keinHinweis auf Sexualdimorphismus der Chromosomen. Eine sekundllre EinschnUrung wurde inkeiner der drei Populationen gefunden. Die karyologischen Ergebnisse geben keinen Hinweisdarauf, daB die drei Populationen unterschiedlichen Arten zuzordnen sind.

Key words: Bahrain, Egypt, Saudi Arabia, karyology, Rana, water frog.

41

IntroductionRana ridibunda is widely distributed over large areas in the Middle East including theArabian Peninsula and the Nile Valley (ANDERSON 1898, GALLAGHER 1971, BALLETIO et al.1985). However, the status ofRana ridibunda has been revised during the last decade. It hasbeen found that R. ridibunda occurs mainly in Europe, and some populations which werepreviously considered to be R. ridibunda have been separated as new species (SCHNEIDER etal. 1984, BEERLI et al. 1994). The populations in Western Turkey, Israel and Egypt havebeen established as Rana levantina (e.g. AKEF & SCHNEIDER 1989, NEVO & SCHNEIDER1983, SCHNEIDER et al. 1992, TAWFIK et al. 1994).

This study examines the karyological status of three populations in Egypt, Bahrain andeastern Saudi Arabia in order to assess their phylogenetic interrelationships. In the last threedecades many karyological studies have been conducted on the genus Rana (e.g.MORESCHALCHI 1967, GRAY & NAGL 1982, KOREFF-SANTIBANEZ & GONTHER 1980,ZOWAlL 1986, ODIERNA 1989, Luo 1993, NISHIOKA et al. 1993). The three populations of

Zoology in the Middle East 15,1997: 41-49.. . .

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42 Zoology in the Middle East 15, 1997

•Egypt Bahrain

Saudi Arabia

•50 E

Fig. 1. Map showing the localities in Bahrain, Eastern Province ofSaudi Arabia, and Giza (Egypt)where specimens of the three water frog populations were collected.

water frogs in Arabia and Egypt are geographically isolated. The taxonomic status of thepopulations covered in the present study is not clear and needs to be seriously examined.The term Rana cf. ridibunda is used in this study. It also includes R. levantina from Egypt.A comprehensive research programme on the water frog in this region is currently inprogress.

Material and methodsRepresentative numbers of adult male and female waters frogs were collected from (1) Duraz inBahrain. (2) Al Qatif in the Eastern Province of Saudi Arabia, and (3) Abu Rawash in Egypt(Fig. 1). It is worth mentioning that another population of water frogs is present in the high Asirarea of Western Saudi Arabia, but unfortunately it was not possible to include it in the presentstudy.

The collected living specimens were identified as water frogs on a morphological basisaccording to ANDERSON (1898) and BALLETIO et al. (1985). and maintained separately in thelaboratory. They were first injected intraperitoneally with colchicine solution 14-16 hours beforethey were killed. Chromosome preparations were made from femoral bone marrow cells using theair-drying technique as described by SCHMID (1978). with minor modifications. Giemsa-stainedand well-spread metaphase plates were photographed with a magnification of 3000x.Measurements were made using the photographs of 28 metaphase plates from individuals fromBahrain (4 females and 2 males). 25 metaphase plates from frogs from Saudi Arabia (3 femalesand 2 males), and 31 plates from frogs from Egypt (4 females and 3 males).

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Amphibia 43

I---------i( A )~----l

Hll 'UI •• 61\ JeM

(B)

Aft all ".

I---------{( A-)!------l

xl AK XI AI I.(8)A,.. •• AA

Figs. 2-4. Metaphase spread (a) and its karyotype (b) ofRana collected from Bahrain (Fig. 2, top), SaudiArabia (Fig. 3, middle), and Egypt (Fig. 4, bottom)

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44 Zoology in the Middle East 15, 1997

Tab. I. Comparison of the relative length of individual chromosomes of the three studied Ranakaryotypes. Relative length: expressed in % of haploid chromosome set. * = significant t value;

** =highly significant t value; B =Bahrain, S.A. = Saudi Arabia, E =Egypt.

chromosome Bahrain S. Arabia Egypt tvalue t value t valuenumber (mean±SD) (mean±SD) (mean±SD) (B-S.A.) (B-E) (E-S.A.)

I 18.95±0.85 18.15±0.95 18.09±0.67 3.24** 4.35" 0.272 13.61±0.74 14.17±1.75 13.3I±O.72 1.54 1.57 2.48*3 13.02±1.16 13.72±1.60 12.24±0.72 1.83 3.12** 4.60**4 11.60±0.90 12.93±O.75 11.18±0.61 5.80** 2.11 9.61**5 9.47±1.11 10.0I±O.90 9.58±0.78 1.92 0.44 1.916 5.33±O.90 5.76±1.11 5.85±1.17 1.56 1.90 0.297 5.15±0.69 4.47±O.71 5.32±0.95 3.56** 0.78 3.73**8 4.79±0.85 4.25±1.30 4.90±1.02 1.81 0.45 2.11*9 4.26±0.58 3.90±1.05 4.63±1.26 1.57 1.46 2.36*10 4.08±1.16 3.45±1.10 4.36±O.95 2.01* 1.01 3.32**11 3.73±0.64 3.36±1.50 4.15±1.28 1.19 1.56 2.12*12 3.55±0.74 3.28±0.80 3.67±1.26 1.27 0.44 1.3713 2.40±0.58 2.27±O.85 2.71±1.06 0.65 1.37 1.68

Tab. 2. Chromosome arm ratio and type of three Rana populations collected in Bahrain, easternSaudi Arabia and Egypt. m =metacentric (AR 1.0-1.7), sm = submetacentric (AR 1.7-3.0), st =

subtelocentric (AR 3.0-7.0).

chromosome Bahrain Saudi Arabia EgyptDumber arm ratio type arm ratio type arm ratio type

I 1.13±O.11 m 1.28±O.13 m 1.27±O.11 m2 1.30±0.09 m 1.66±O.22 m 1.50±0.16 m3 1.82±0.12 srn l.58±0.29 m 1.88±0.17 sm4 1.28±0.08 m 1.43±O.18 m 1.63±O. 13 m5 1.13±0.06 m 1.83±O.17 sm 1.25±0.16 m6 1.17±0.11 m 1.17±O.31 m 1.20±0.16 m7 1.23±0.13 m 1.24±0.18 m 1.22±0.17 m8 2.24±0.17 sm 1.67±0.27 m-sm 1.71±0.19 sm9 • 2.43±0.19 sm 1.93±0.12 sm 2.00±0.16 sm10 2.14±O.20 sm 1.89±O.19 sm 2.28±0.23 sm11 2.50±0.16 srn 2.80±0.26 sm-st 3.33±O.19 st12 1.86±O.11 sm 1.96±O.13 sm 2.58±0.21 sm13 2.50±0.06 srn 2.05±O.22 sm 2.64±O.19 sm

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Arnphibia 45

.Bahraln

DSaudl Arabia

IiUEgypl

15 -

10

5 -

2 3 4 5 e 7 B 9

Chromosomes10 11 12 13

Fig. 5. Relative length of the chromosomes of the three Rana populations.

The mitotic metaphase chromosomes were counted, measured, classified and then ideogramswere drawn according to MORESCHALCHI (1973, 1978). As there were no apparent differencesbetween male and female preparations, the results for both sexes were pooled. The data ofrelative chromosome length were analysed statistically using the t-test in order to assess thevariation among the three frog populations.

ResultsMorphological measurements of individual chromosomes in the three populations did notindicate any differences between male and female karyotypes. In addition, a close inspectionof all chromosomes pairs failed to show the presence of secondary constrictions in anyspecimens.

The karyotypes of the three populations of water frogs, as shown in Figs. 2-4, reveal the samediploid number of chromosomes 2n = 26. This number is the same as the model number ofchromosomes characterising Rana species. The 26 chromosomes are divided into two groups (Aand B) according to size. Group A consists of 5 pairs of large chromosomes while group Bcontains 8 small pairs of chromosomes.

The quantitative data of the different measurements are listed in Tabs. 1-2 andideogrammed in Fig. 5. A clear gradual decrease in values of relative chromosome lengthscan be observed among the three karyotypes. This actually applies to chromosome pairsfrom number 1 to number 5 in group A, and from number 6 to 13 in group B. Chromosome

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46 Zoology in the Middle East 15, 1997

no. 1 of the population of Bahrain has the highest value (18.9±0.16), compared to the otherpopulations. The remaining large chromosomes of the population of eastern Saudi Arabia(2-5) exhibited the highest values of the three populations. Regarding the smallchromosomes (6-13), the population of Egypt showed the highest mean relative lengthvalues. The result also showed that all chromosomes of the Bahraini frogs (except no. I and6) have intermediate values compared to the other two populations. When the data werestatistically analysed, significant differences in the mean relative length of somechromosomes among the three populations were recorded (Tab. I).

Regarding chromosome arm ratio, Tab. 2 shows that the large chromosome pairs, no. 1,2,and 4, are metacentric in the three karyotypes studied. By contrast, the chromosome pairsno. 3 and 5 are different. Chromosome no. 3 is metacentric in the case of Saudi Arabianfrogs, while it is submetacentric in both Bahraini and Egyptian frogs. Chromosome no. 5 issubmetacentric in Saudi Arabian frogs jUld metacentric in both Bahraini and Egyptian frogs.Six of the eight short chromosome pairs are submetacentric, with pairs no. 6 and 7 beingmetacentric in all three populations. Chromosome no. II shows clear variations in the threekaryotypes. It is clearly subtelocentric in the sample from Egyptian frogs and seems to be soin the sample from Saudi Arabian, while it is submetacentric in the samples from Bahrain.

DiscussionRana ridibunda has been thought to have a wide distribution. However, it has recently beenshown that Western Turkey, Israel and the Nile Delta are inhabited by a separate specieswhich is called R. levantina, and that the water frogs occupying most of Greece belong to aspecies called R. balcanica (e.g. AKEF & SCHNEIDER 1989, SCHNEIDER & SINSCH 1992,SCHNEIDER et al. 1992, NEVO & FILIPPUCCI 1984, TAWFIK et al. 1994). The surroundings ofDamascus in Syria are inhabitated by water frogs which belong to R. bedriagae (SCHNEIDER1997).

In Bahrain, the only amphibian species recorded is a water frog previously identified asRana ridibunda (GALLAGHER 1971). The nearest population of water frogs is present in theeastern province of Saudi Arabia, which is about 25 km from Bahrain. Based on somebiological and biometric criteria, the two groups were considered as representatives of thesame species (BALLETIO et al. 1985). The last time that Bahrain was completely inundatedwas during the early Pleistocene, some 0.5-1 million years ago (FAIRBRIDGE 1961).Accordingly, neither frogs nor any other terrestrial fauna were present at that period. The sealevel fluctuated greatly from the early Miocene, when the Gulf probably covered the Tigris­Euphrates-Karun rivers running to the Gulf of Oman. However, it is thought that animalsincluding Rana originally came to Bahrain from Saudi Arabia, during the late pluvials orearlier. The sea level in the Gulf began to rise again about 17,000 to 20,000 years RP., andreached its present level some 5,000 years ago, in a series of rapid advances separated bystillstands (PURSER 1973, DooRNKAMP et al. 1980).

In order to assess the actual phylogenetic relationship between frogs inhabiting Bahrainand those which occur in the eastern province of Saudi Arabia, a modem and reliabletechnique of chromosome karyotyping was implemented in the present study. Thisexperimental approach has been widely adopted as one method to provide reliableinformation for a comprehensive understanding of the phylogenetic status of Amphibia(KOREFF-SANTIBANEZ & GONTHER 1980, BELCHEVA et al. 1985, ODlERNA 1989). Water

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Amphibia 47

frogs from Egypt represent a population which is well established and has been completelyseparated from those of the Arabian Gulf region for a considerable time. This populationwas included in the present study to give a better understanding of the degree of variationbetween the other two close populations in the Gulf region. The total number ofchromosomes and their size in the three populations studied agrees with what has beenreported for other populations of R ridibunda (MORESCHALCHI 1967, 1973, GRIFFITH 1963,KOREFF-SANTIBANIZ & GONTHER 1980, LI et al. 1983, ZHENGAN & YIN 1983, BELCHEVA etal. 1985). The relative chromosome lengths of the three populations are within the ranger~ported by MEzARoS (1973), EBENDAL (1977) and KOREFF-SANTIBANEZ & GONTHER(1980) for Rana spp. As shown in Tab. 2, significant differences in the relative chromosomelength can be seen among the three frog populations. Regarding chromosome shape,dissimilarities between specimens from Bahrain and Saudi Arabia can be seen forchromosomes no. 3, 5 and 11. Such morphological differences in the chromosomes may beattributed to the geographical separation between the Bahraini and the Saudi Arabianpopulations.

In the present study, no cytological evidence was found for sex chromosome dimorphismin any specimens, which agrees with other reports on Rana (YONG 1975, ZHENGAN 1978, LIet al. 1983). However, studies on different species ofRana by ZOWAIL (1986) and NISHIOKAet al. (1993) have revealed the occurrence of sex chromosome dimorphism. Regardingsecondary constrictions, KOREFF-SANTIBANEZ & GONTHER (1980) and BELCHEVA et al.(1985) reported that such a constriction is located on the long arm of chromosome no. 10 inR ridibunda, while reports on other species of Rana showed the presence of the secondaryconstriction on other chromosome pairs (ODIERNA 1989, Luo 1993). However, a closeinspection of the present data has shown no sign of any secondary constriction in anykaryotypes.

However, it has been reported that karyotype evolution in amphibia does not necessarilyinclude morphological modifications of the chromosomes. Instead, intrachromosomalchanges are more likely to occur (MORESCHALCHI 1970, DUBOIS 1977). In our study, theresults show some differences between these three populations. These karyologicaldifferences probably reflect differences in their phylogenetic status. More investigations areneeded to answer the questions raised by this study.

Acknowledgement: We would like to express our thanks for Dr. G. HOAD and J. GASPARETTI for theirvaluable comments and remarks on the manuscript. The research was supported with a grant by theUniversity of Bahrain.

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Authors' addresses: Dr. Saeed A. Mohammed, Dr. Abd El-Kader Gamal El-Din and Dr. HaifaA. Al-Maskati, Biology Department, College of Science, University of Bahrain, P.O. Box 32038,Isa Town, State of Bahrain. - Dr. Helmy El Dawy and Dr. Moustafa Saleh, Zoology Department,Faculty of Science, Al-Azhar University, Cairo, Egypt.

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