Features of the karyotypes of Pelophylax ridibundus Pallas, 1771 and Rana macrocnemis Boulenger, 1885 (Amphibia: Ranidae) from Armenia

hromosomal complements of Pelophylax ridibundus Pallas, 1771 from 9 localities (Northern, Central and South Armenia) and Rana macrocnemis Boulenger, 1885 from one locality (North-West Armenia) have been analyzed. The chromosome sets of P. ridibundus collected from 8 localities showed 2n=26, (10m+12sm+4st; NF=52). A secondary constriction has been observed in all studied individuals on the 10-th chromosome pair showing NOR-positive reaction. C-positive heterochromatin blocks have been observed on long arms of the 2-nd and 10-th pairs of chromosomes (7 localities). In addition, C-heterochromatin blocks have been found on interstitial regions of short arms of the 12-th pairs, as well as in telomeric regions of long arms of the 9-th pairs and on short arms of the 5-th pair in the frogs from 2 localities. The karyotype of P. ridibundus from populations near Ejmiatsin differs from other popula- tions (2n=26, 12m+10sm+4st). Diploid number of chromosomes of R. macrocnemis was also 26 (8m+12sm+6st, NF=52). Blocks of C-positive heterochromatin have been revealed in telomeric parts of the 1-st, 2-nd (p), 3-rd (q), 4-th (q), 6-th, 9-th (p), 10-th (p,q) and 13-th (q) pairs, as well as in interstitial regions of the 1-st and 2-nd pairs of chromosomes. Intrapopulation and interpopulation geographic variations of karyo- types and C-heterochromatin banding patterns of P. ridibundus have been revealed. Karyotypically, morphotypes "macrocnemis" and "camerani" are closely related.


INTRODUCTION
In Armenia, the Marsh frog Pelophylax ridibundus (=Rana ridibunda) Pallas, 1771 inhabits all humid and freshwater biotopes at 850-2500 meters above sea level (Melkumyan, Sirunyan, 1988;Egiazaryan, 2008).The long-legged wood frog Rana macrocnemis Boulenger, 1885 demonstrates significant morphological variability, including two widespread morphotypes "macrocnemis" and "camerani" in the Caucasus Isthmus, in vari-ous parts of Anatolia and northern Iran (Tarkhnishvili et al., 2001;IUCN, 2008).Rana macrocnemis occurs in a very broad range of biotopes: in broadleaved and mixed forests, steppes, sub-alpine and alpine meadows at the altitude ranging from 1000-2500 meters above sea level in Armenia.(Melkumyan, Sirunyan, 1988;Egiazaryan, 2008).At the foot of the Jawajhet mountain range, where these frogs were caught, the "camerani" form was found (Tarkhnishvili et al., 2001).Few data about the biology of these two species of the genera Rana Linnaeus, 1758 and Pelophylax Fitzinger 1883 living in Armenia have been published so far.It is known that P. ridibundus is characterized by intraspecifi c polymorphism in some morphometric features, such as body size and dorsal colour patterns (Melkumyan, Sirunyan, 1988;Manukyan, 2002).The biology of the two above mentioned species and mating calls of P. ridibundus have also been studied (Egiazaryan, 2008).
Chromosome sets of brown frogs of the Rana macrocnemis complex have been described from populations from Russia and Georgia (Ivanov, Madyanov, 1973;Orlova et al., 1977;Birstein, 1984;Popov, Dimitrov, 1999).So far chromosome sets of Ranidae species from Armenia have not been studied at all.
The purpose of this study is to analyze the karyotypes and to evaluate the intraspecifi c and interspecifi c chromosome polymorphism of P. ridibundus and R. macrocnemis.

MATERIAL AND METHODS
Specimens were collected from 9 localities of Armenia (Table 1, Fig. 1) in 2003 and 2004.Chromosome slides were prepared from bone marrow and spleen according to Haertel et al. (1974) and MacGregor, Varley (1986).C-banding was made as described in Sumner (1972) with some modifi cations.Ag-banding was done following the technique of Howell, Black (1978).Chromosome smears were observed under a «NU-2E» (K.Zeiss, Germany) microscope, with the 1125 magnifi cation (90 x 12.5).Chromosome complements were analyzed in mitotic and meiotic stages of cell di-

Locality
Pelophylax ridibundus vision.Homologous chromosome pairs were identifi ed according to their relative length (RCL) and the centromeric index (Cen.Ind) (Levan et al., 1964).Statistical analysis of chromosome pairs was performed using the program Statistica 6.0.

RESULTS
Chromosome sets of the observed specimens are presented in Tables 2, 3

Pelophylax ridibundus
Diploid karyotypes of specimens from 8 lo-calities in Armenia were similar in their chromosome numbers and morphology and consist of 26 chromosomes (NF=52).Chromosomal complements include 5 pairs of metacentric (m), 6 pairs of submetacentric (sm) and 2 pairs of subtelocentric (st) chromosomes (Tables 2  and 3).The chromosome set of frogs from a population near Ejmiatsin differs from other populations and possesses karyotypic formula of 12m+10sm+4st.A secondary constriction was demonstrated by Ag-bandings on the long arm of the submetacentric pair of chromosomes (10-th) in both sexes in all studied populations (Fig. 3, a).Differences between chromosome sets of females and males have not been revealed.
C-positive heterochromatin blocks in karyotypes of frogs collected in Azat and Vohchaberd were revealed in the interstitial (i) regions of the long (q) arms of the 10-th chromosome pair and the short (p) arms of the 12th, chromosome pair and telomeric regions of the long arms of the 2-nd and 9-th pairs and on the short arms of the 5-th pair (Table 3; Fig. 3,  a).Blocks of C-heterochromatin in the chro-mosome sets of frogs collected from other 7 localities of Armenia were only observed on long arms of the 2-nd and 10-th pairs.Agbanding revealed an argentophilous body localized on the submetacentric pair of chromosomes (10-th) of all males and females from all mentioned localities (Fig. 3, a).

Rana macrocnemis
The karyotype of the form "camerani" also includes 26 bi-armed chromosomes (NF=52; 8m+12sm+6st) (Fig. 2, b).Blocks of C-posi-  tive heterochromatin were revealed in telomeric regions of the 1-st, 2-nd (p), 3-rd (q), 4th (q), 9-th (p), 10-th (p.q) and 13-th (q) pairs as well as in interstitial regions of the 2-nd pair of chromosomes (Fig. 3, b; Table 3).Weakly stained C-banding patterns were observed on the interstitial part of the arm of the 1-st pair, on the telomeric parts of the long arm of the 6-th pair and 9-th pair and, also, on the short arm of the 13-th pair (Fig. 3, b).The analysis of silver stained slides revealed NOR-bearing blokes in the 10-th chromosome pair (Fig. 3,  b).
The karyotype of P. ridibundus from different localities of Armenia has been examined for the fi rst time within the framework of this study.It was demonstrated that diploid chromosomal set also equals to 26 and includes metacentric, submetacentric and subtelocentric morphological elements.The use of conventional Giemsa staining did not always display the secondary constriction, whereas Ag-banding of chromosomes did reveal it.Homologues of the 10-th pair in all the specimens studied (males and females) possess a secondary constriction on their long arms (Figs 2,а;3,a).
Comparison of chromosome sets of P. ridibundus from Armenia (8 populations), Turkey, Saudi Arabia, Ukraine, Russia, Central and South Europe, and China shows both similarities and distinctions.All karyotypes of P. ridibundus from different geographic regions studied are similar in their diploid chromosome numbers; in morphology of seven chromosome pairs: 1-st (m), 2-nd (sm), 3-rd (sm), 5-th (m), 6-th (m), 8-th (st), 13-th (sm); in the pair of submetacentric chromosome, bearing the secondary constriction (9-th -Russia, Ukraine, Turkey and 10-th -other populations), and in the 12-th pair of subtelocentric chromosomes (Armenia, Turkey, Saudi Arabia) at more accurate measurements (Table 4).Despite this, the chromosomal complements of P. ridibundus from the compared regions were different.Thus, interpopulation variations in numbers and morphology of metacentric, submetacentric and subtelocentric chromosomes of P. ridibundus, were demonstrated (Table 4).In the karyotypes of P. ridibundus from Saudi Arabia, Central Europe and Kabardino-Balkaria some of middle and small size chromosomes can be regarded as subtelocentrics (CI=21.9;CI=24.7) on the basis of our measurements.The observed differentiation in morphology of the 7-th to the 12-th chromosome pairs (Table 4) can be a result of different stages of chromosome spiralization (from meta-to submetacentric, from submeta-to subtelocentric).Three pairs of subtelocentic chromosomes in some populations of the lake frog may emerge as a result of chromosomal rearrangements (translocations) under the infl uence of different ecological conditions.
The interpopulation variations in different patterns of positions of C-positive heterochromatin regions were also revealed.The majority of the C-blocks (eleven pairs of the chromosomes) were observed in the karyotypes of P. ridibundus from populations of Central Europe (Schmid, 1978) and Armenian Azat and Vohchaberd (fi ve pairs) (Fig. 3, b; Table 3).

Species
According to the literature, C-heterochromatin is concentrated in the centromere region in many species of the family Ranidae (Schmid, 1978;Miura, 1995).Telomeric bands were seen in the short arm of the 3-rd and 5-th chromosomal pair of Pelophylax ridibundus, P. esculentus Linnaeus, 1758 and P. lessonae Camerano, 1882 (Heppich, 1978).The comparison of C-banding patterns in karyotypes of P. ridibundus from Armenia (our data) and Central Europe (Heppich, 1978;Schmid, 1978) showed that two pairs of chromosomes (5-th and 10-th) bear similar Cpositive blocks.It is known that constitutive heterochromatin in the genera Rana and Pelophylax reacts variably to alkaline pretreatment and Ba(OH) 2 treatment (Schmid, 1978).The fact that the C-heterochromatin blocks are to be detected only in telomeric regions of chromosomal arms (Armenia) or on one chromosome pair might be accounted for by the methodical features of the chromosome slide preparation.However, the case of the C-heterochromatin appearance on short arms of the 3-rd pair (Spasič-Bošković et al., 1999) could be explained by pericentric inversion.
In light of the analysis of chromosomal sets (Schmid, 1978;Mészarós, Bartos, 1978;Bucci et al., 1990;Spasič-Bošković et al., 1999) and modern notions on taxonomic status and zoogeographical distribution of species of the P. ridibundus complex, the slight differences between the karyotypes of frogs from Central and South-Western Europe (Serbia and Macedonia, Hungary) can be accounted by a different degree of chromosome spiralization (from meta-to submetacentric, from submetato subtelocentric) within the karyotype of R. fortis.Taking into consideration the fact that R. fortis inhabits Europe (Plötner, 2005;Plötner et al., 2008) and P. ridibundus inhabits Russia and Ukraine (Ananjeva et al., 1998), the karyological differences of these species in 2 pairs of chromosomes (m/sm) (Tables 3, 4) can be noted as interspecifi c.However, these chromosomal set distinctions at the morphological level (metacentric/ submetacentric chromosomes) can most likely be caused by the degree of chromosome spiralization rather than by chromosomal reorganizations.
According to Sinsch, Schneider (1999) P. ridibundus inhabits Armenia.The occurrence of P. bedriagae Camerano, 1882 in Armenia (Ananjeva et al., 1998) is doubtful, since authors (Ananjeva et al., 1998;Plötner, 2005) disagree on the frog's distribution.The analysis of chromosomal complements of P. ridibundus from Armenia and Ukraine showed the differences in morphology of the three pairs of chromosome (Table .4), which can be accounted by various degrees of chromosome spiralization (m/sm).These karyotype distinctions can be noted as intraspecifi c.
The comparison of the karyotypes of lake frogs from Armenia (P.ridibundus) and Eastern Turkey (Beyşehir and Izmir populations (Alpagut, Falakali, 1995)) (P.caralitanus) (Plötner, Ohst, 2001;Plötner, 2005;Ayaz et al., 2006) shows the differences in three chromosome pairs (m, sm, st) (Table . 4).These differences in the chromosome sets might be interspecifi c and can arise as a result of chromosomal arrangements in the karyotype of P. caralitanus.
The data on the distribution of P. bedriagae in Saudi Arabia (Sinsch, Schneider, 1999;Plötner, 2005) are questionable, since authors disagree on the frog's distribution.Based on abovementioned, the chromosome sets of lake frogs (P.ridibundus) inhabiting in Saudi Arabia (Sinsch, Schneider, 1999;Al-Shehri, Al-Salech, 2005) and Armenia differ in three pairs of chromosomes (Table 4).Only one pair (9th) among the differing pairs can be accounted for by the formation of chromosomal reorganizations (sm/st).Other pairs (7-th, 12-th) differ at the metacentric and submetacentric morphological elements level.Taking into account the fact that the same species (P.ridibundus) inhabits both in Armenia and Saudi Arabia, distinctions in chromosomal complements of P. ridibundus refers only to interpopulation chromosomal polymorphism.
The analysis of the studied karyotypes of lake frogs inhabiting in Saudi Arabia (P.ridibundus) and in Western Turkey (P.caralitanus) also demonstrated the difference in four pairs of chromosomes (Table 4).Taking into account the taxonomic status of these frogs and chromosome sets differentiation (m/st pairs), the differences between karyotypes of lake frogs in Turkey and Saudi Arabia can also  be interspecifi c.Despite this, karyological differences in frogs of the P. ridibundus complex are not comparable with interspecifi c differences in other green frog species (Heppich, 1978;Schmid, 1978;Belcheva et al., 1985;Miura, 1995;Miura et al., 1997), as the morphological elements of the karyotype vary both among populations of P. ridibundus as well as among the species of P. ridibundus complex.It is known that the several techniques features, such as C-banding patterns, fl uorescence replication banding patterns (DAPI and FISH) are used for an accurate distinction of the species of green and brown frogs (Schmid, 1978;Miura, 1995;Miura et al., 1997;Picariello et al., 2002).Hence, those should also be used for the fi nal confi rmation of differences in the frog species within the P. ridibundus complex.
The comparative analysis of the karyotypes of frogs belonging to the complex of R. macrocnemis from Armenia (our data), Georgia (Ivanov, Madjanov, 1973), (Popov, Dimitrov, 1999) and form "macrocnemis" from Russia (Birstein, 1984;Ivanov, Madjanov, 1973) revealed their similarities on chromosomal pairs № 1, 2, 3, 4, 5, 6, 7 and 12 (Table 4).The cardinal differences of the chromosomal sets of "camerani" and "macrocnemis" forms relate to the chromosomal pairs № 8, 9, 10, 11 and 13 (Table 4).Interpopulation differentiations in number and morphology of the karyotypes elements of two forms of R. macrocnemis may relate to different degrees of chromosomes spiralization (m to sm) and, also, to be a result of chromosomal rearrangements (m to st; sm to st) under different ecological conditions.The analysis of the literature data of the external morphology, blood serum protein electrophoresis, allozymes and mtDNA, DNA sequence study (Ischenko, 1987;Tarkhnishvili et al., 2001;Veith et al., 2003;Çevik et al., 2006) of the frogs of the forms "camerani" and "mac-rocnemis" did not show signifi cant qualitative interspecifi c differences and led authors to consider "macrocnemis" and "camerani" as morphotypes.Thus, these brown frogs are chromosomally more closely related as well.
Comparison of the chromosome sets of Pelophylax ridibundus and Rana macrocnemis from Armenia detects the similarities in their diploid number of chromosomes ( 26), in the morphology of the chromosome pairs: 1-st, 2-nd, 3-rd, 5-th, and two pairs of subtelocentric chromosomes (8-th and 12-th/13-th) (Fig. 2 a, b; Table 4).Besides that, at comparison of distribution of C-banding patterns in the chromosome pairs of P. ridibundus (Schmid, 1978) and R. macrocnemis form "camerani" the similarities on the 1-st (p.t.), 2-nd (q.t.), 3rd (p.t.; p.i.) and 13-th (q.t.) pairs were shown (Fig. 3, a, b).The distinctions between the species are displayed in the different morphology of the several chromosome pairs (4-th, from 6-th to 11-th), in the secondary constrictionbearing chromosome pairs, in the morphology of NOR-positive regions bearing chromosome pairs and C-positive heterochromatin blocks localizations in the karyotype (Table 4).
Comparison of the chromosomal data of P. ridibundus with other species of the green frogs (Heppich, 1978;Schmid, 1978;Miura, 1995;Miura et al., 1997) and some species of brown frogs (Orlova et al., 1978;Birstein, 1984;Green, Borkin, 1993;Popov, Dimitrov, 1999) with data from this study confi rms the close similarity of several chromosome pairs by size and morphology.On the whole, the interspecifi c differences in karyotypes of species of Rana and Pelophylax are manifested in the distinctions of late replication banding patterns (Miura, 1995;Miura et al., 1997), fl uorescence replication banding patterns (Picariello et al., 2002) and in the locations of C-heterochromatin blocks (Schmid, 1978;Miura, 1995;Miura et al., 1997;present study).
Vicinity of the village Ranchpar Vicinity of Yerevan, canyon of the river Hrazdan Vicinity of the village Urcadzor Vicinity of the Ararat Vicinity of the village Shorzha, lakeside of Sevan Vicinity of the village Vohchaberd Vicinity of the village Garni, canyon of the river Azat Vicinity of the town Dilijan Vicinity of of the Jawajhet mountain range, vicinity of the Gukasyan and in Figures 2, 3.

Table 4 .
The chromosome features of Pelophylax ridibundus and Rana macrocnemis from different localities (• -NOR bearing chromosomes, sm* -pairs can mark out as subtelocentric).