Abstract: In addition to the morphological, skeletal and biometric characteristics, karyological study is the key to the animal identification and classification. Due to the insufficient studies on amphibians in Khorasan Province (Iran), this research studies five areas of this province, namely Mashhad, Sarakhs, Bojnoord, Shirvan and Tibad. Totally, 100 samples were collected in these regions from April to November, which were transferred alive to the lab. In the laboratory, the vimnoblastin was injected to the samples proportional to their weights and consequently, the bone marrows were extracted. The obtained solution was dropped from a height of 60 to 70 cm on the frozen slides placed on an inclined surface. Prepared slides were stained and photographed to study the chromosomes. Relative length and arms ratio of the chromosomes were used to recognize the centromere position and chromosomal type. Finally, based on the above studies the related idiograms were derived. This research illustrates that all frogs have a set of 26 (2n = 26) chromosomes and all toads have a set of 22 chromosomes. It is concluded that all collected frogs and toads from the different stations are Rana ridibunda and Bufo viridis species, respectively. This research shows no sex dimorphism in all samples.
INTRODUCTION
Morphology of karyotype, which includes number, shape and size of chromosomes, forms one class of the phenotypic characteristics, which is usually independent of the genotype (Chiraelli and Capana, 1973; Green and Sessions, 1991). Most of the amphibians do not have a large number of chromosomes. Nevertheless, their chromosomes are relatively large in size and easy to obtain, so the amphibians have an excellent position in chromosomal research.
So far, the karyological studies on Anura in Great Khorasan (Fig. 1), are limited to a few cases (Nehmati, 1998). Kami and Baluch (1994) has done an extensive investigation on Iran Amphibian and recognized various species of Amphibian in Iran. Some other studies have been conducted on Amphibians in the regions close to Iran. For example, Balletto et al. (1985) have studied the Amphibians of the Arabian Peninsula. In Turkey, a research was carried out on the cytogenetic study of the pelobates syriacus (Amphibia, Anura) which examined cytological aspects of the chromosomes (Ugurtas et al., 2001).
| Fig. 1: | Great Khorasan map and the studied areas |
Their method is similar to the method described in this study. Moreover, Al-Shehri and Al-saleh (2005) have presented a comprehensive research on karyotype of amphian specially Anura in Saudi Arabia.
Particularly, a few researches on Bufo and Rana can be found in the study. Spasic-Boskovic et al. (1999) have studied population composition and genetic variation of water frogs (Anura Ranidae) from Yougslovi. Also, Spasic-Boskovic et al. (2000) have investigated on karyotype of two species of Bufo from Yougslovi. Another research is devoted to karyological comparison of water frog (Rana cf. ridibunda) population from Bahrain, Eastern Saudi Arabia and Egypt that has been studied by Saeed (1997). Dujsebayeva et al. (1997) have investigated on diploid and tetraploid of Bufo viridis complex (Anura: Bufonidae) in Southern Kazakhstan. Also Stock (2005) have studied on multiple origin of tetraploid taxa in Eurasian Bufo viridis subgroup. Finally, Mindrescu and Ghiorghita (2008) have done a cytogenetic investigation on Rana idibunda. and Rana esculenta fom the North of Moldavia.
In this study, the karyological and morphological research on 100 samples of Anura collected from five areas in Khorasan Province (Mashhad, Sarakhs, Bojnoord, Shirvan and Tibad) is studied. These areas are shown in Fig. 1.
MATERIALS AND METHODS
Morphological Studies
In this research, 16 morphological characters, such as absence or presence
of paratoid glands, vomerian teeth, dorsal fold, temporal spot, locomotion organs
and etc. (Kami and Baluch, 1994; Balletto
et al., 1985), were used to study all collected samples.
Karyological Studies
For this research, all samples were transferred alive to the Bio-systematic
lab of Ferdowsi University of Mashahd, where this research was conducted from
April 2003 to December 2006. The number of the collected samples from each area
in Khorasan (Fig. 1), is given in Table 1.
Totally, 100 samples were collected in these regions from April to November
of 2004, 2005 and 2006.
| Table 1: | Number of samples of each area |
| Fig. 2: | Karyotype of Rana ridibunda (A) Bojnoord, (B) Mashhad and (C) Shirvan |
| Fig. 3: | Karyotype of Bufo viridis (A) Sarakhs and (B) Tibad |
At first for each gram of the sample weight 0.01 mL vimnoblastine was injected under the stomach skin. About 1 h later, the bone marrow was extracted and put it in KCl. Then the solution was kept for 15 to 20 min in incubator (37°C) and centrifuged three times. Before each centrifuge a fixator containing methanol and acetic acid (3:1) was used (Schmid, 1978a, b).
This solution was dropped from a height of 60 to 70 cm distance on the frozen slides placed on an inclined surface. After staining the prepared slides, they were studied using microscope and then photographed (Fig. 2, 3). Each chromosome was separated and then the length of short and long arms and total length of chromosome were measured. Based on Levan (1964) classification, chromosomes types were determined and arranged (according to their length) to obtain a chromosomal set.
RESULTS
Morphology and Identification of Species
The mentioned morphological characters were divided into three categories.
The first category included those characters that were more important in species
identification and amphibian classification, namely absence or presence of dorsal
fold, temporal spot, vocalization organs, hind locomotion organs position and
etc. (Kami and Baluch, 1994). On the other hand, some
of them such as vertebral line and color diversity, especially at body dorsal,
were different between populations of one area and probably had influenced by
environmental factors. The third category included those characters used to
recognize animal at supra species, namely absence or presence of vomerian teeth,
head shape, tongue shape and disk plates at the end of fingers. Based on the
morphological characters used in this study, it was concluded that all of toads
are from Bufo viridis species and all of collected frogs are from Rana
ridibunda species.
Karyology
Results of karyotype revealed that all samples of Rana ridibunda had a set
of 2n = 26 chromosomes clearly shown in Fig. 2. Table
2 shows arm ratio and chromosomal type of Rana ridibunda from Shirvan
area according to Levan (1964) classification. Related
idiogram was drawn which is shown in Fig. 4. It was observed
that karyotype of Rana ridibunda from Shirvan consisted of five long
pairs and eight short pairs of chromosome. Five long pairs were metacentric
or submetacentric and eight short pairs of chromosome were metacentric submetacentric
and acrocentric. Only one chromosome was acrocentric.
Table 3 also demonstrates the arm ratio and chromosomal type (based on Levan classification (1964)) of Rana ridibunda from Bojnoord area. The related idiogram is shown in Fig. 5. The karyotype of Rana ridibunda from Bojnoord consisted of five long pairs and eight short pairs of chromosome. Five long pairs were metacentric or submetacentric and eight short pairs of chromosome were metacentric submetacentric and acrocentric. Only one chromosome was acrocentric.
Table 4 shows the arm ratio and chromosomal type of Rana ridibunda from Mashhad. Accordingly, the related idiogram was drawn which is shown in Fig. 6. It was observed that karyotype of Rana ridibunda from Mashhad consisted of five long pairs and eight short pairs of chromosome.
| Table 2: | Arm ratios and type of centromeres of Rana ridibunda from Shirvan area |
| Fig. 4: | Idiogram of Rana ridibunda samples collected from Shirvan area in Khorasan |
| Table 3: | Arm ratios and type of centromeres of Rana ridibunda from Bojnoord area |
| Table 4: | Arm ratios and type of centromeres of Rana ridibunda from Mashhad area |
| Fig. 5: | Idiogram of Rana ridibunda samples collected from Bojnoord area in Khorasan |
| Fig. 6: | Idiogram of Rana ridibunda samples collected from Mashhad area in Khorasan |
Five long pairs were metacentric or submetacentric and eight short pairs of chromosome were metacentric submetacentricand acrocentric. Only one chromosome was acrocentric.
Karyotype of all samples of Bufo viridis showed a set of 2n = 22 chromosomes clearly recognized in Fig. 3. Table 5 shows the arm ratio and chromosomal type (based on Levan classification (1964)) of Bufo viridis from Sarakhs area. Figure 8 shows the related idiogram. It was concluded that karyotype of Bufo viridis from Sarakhs comprised of six long pairs and five short pairs of chromosome. The six long pairs were metacentric and the pairs were five short metacentric and submetacentric. No acrocentric chromosome was observed.
Table 6 also shows the arm ratio and chromosomal type of Bufo viridis from Tibad area. The related idiogram is shown in Fig. 7.
| Table 5: | Arm ratios and type of centromeres of Bufo viridis from Sarakhs area |
| Table 6: | Arm ratios and type of centromeres of Bufo viridis from Tibad area |
| Chor. No.Total lengthLong armShort armArm ratioType of centromere | |
| Fig. 7: | Idiogram of Bufo viridis samples collected from Tibad area in Khorasan |
| Fig. 8: | Idiogram of Bufo viridis samples collected from Sarakhs area in Khorasan |
It was seen that karyotype of Bufo viridis from Tibad consisted of six long pairs and five short pairs of chromosome, where the six long pairs were metacentric and the five short pairs were metacentric and submetacentric. There was no acrocentric choromosome.
DISCUSSION
As it was described earlier, all samples of Rana ridibunda had a set of 2n = 26 chromosomes (that was according to the general plan of karyotype for this speciation) including five long pairs and eight short pairs of chromosome. Karyotype of all samples of Bufo viridis showed a set of 2n = 22 chromosomes. In some papers such as Stock (2005) and Dujsebayeva (1997), the tetraploid form of Bufo viridis has been reported. However, in this investigation only diploid toads were found in the examined areas. Karyotype of all samples of Bufo viridis consisted of six long pairs and five short pairs of chromosomes, which was in total agreement with the results in Spasic-Boskovic et al. (1999, 2000). Collected frogs from different areas had Metacentric (M), Submetacentric (Sm) and Acrocentric (Ac) chromosomes. In this study among all samples of Rana ridibunda one chromosome was Acrocentric, but Mindrescu and Ghiorghita (2008) have pointed that in their study, water frogs (Rana ridibunda) have only Metacentric and Submetacentric chromosomes. They also mentioned that the chromosomes belonging to the 7th, 9th and 13th pairs of Rana ridibunda are more likely Acrocentric than meta- and submetacentric.
Collected toads had only Metacentric and Submetacentric chromosomes. Despite the results of Al-Shehri and Al-Saleh (2005), this study showed no sex dimorphism in all 100 samples, which was in total agreement with Saeed (1997).
This study demonstrated a thorough analysis of karyology of Anura in Khorasan provience. In conclusion, all samples of Rana ridibunda had a set of 2n = 26 chromosomes and Karyotype of all samples of Bufo viridis showed a set of 2n = 22 chromosomes Frogs had Metacentric (M), Submetacentric (Sm) and Acrocentric (Ac) chromosomes but toads had only Metacentric and Submetacentric chromosomes. For further investigation it is suggested that other areas of Khorasan Province (specially South of Khorasan that has never been studied) be investigated and compared with this study. Also, it is suggested to compare the species of Anura discussed here with those from Turkmenistan and Afghanistan, two countries nearby Khorasan province, to obtain precise results.