New Karyological and Morphometric Data on Poorly Known Bufo surdus and Bufo luristanicus in comparison with Data of Diploid Green Toads of the Bufo viridis complex from South of Iran

Fatemeh FAKHARZAEH, Jamshid DARVISH Haji Gholi KAMI, Fereshteh GHASEMZADEHand Eskandar RASTEGAR-POUYANI

1Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran

2Department of Biology, Faculty of Sciences, Shahid chamran University of Ahvaz, Ahvaz, Iran

3Department of Biology, Faculty of Sciences, Golestan University, Gorgan, Iran

4Department of Biology, Faculty of Sciences, Hakim Sabzevari university, Sabzevar, Iran

New Karyological and Morphometric Data on Poorly Known Bufo surdus and Bufo luristanicus in comparison with Data of Diploid Green Toads of the Bufo viridis complex from South of Iran

Fatemeh FAKHARZAEH1,2*, Jamshid DARVISH1, Haji Gholi KAMI3, Fereshteh GHASEMZADEH1and Eskandar RASTEGAR-POUYANI4

1Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran

2Department of Biology, Faculty of Sciences, Shahid chamran University of Ahvaz, Ahvaz, Iran

3Department of Biology, Faculty of Sciences, Golestan University, Gorgan, Iran

4Department of Biology, Faculty of Sciences, Hakim Sabzevari university, Sabzevar, Iran

Previous studies on the Bufo viridis complex, which is distributed broadly across Iran, are incomplete and restricted to a few regions or a few samples. In this paper a new detailed study on the B. viridis complex in southern of Iran (from West to East) is presented. The analysis of 18 morphometric characters with univariate and multivariate methods reveals significant differences between three members of the B. viridis complex namely B. variabilis, B. luristanicus, and B. surdus distributed in southern part of Iran. Our result help to resolve an old taxonomic problem about B. surdus subgroup (taxa closely related to B. surdus) confirming that B. luristanicus and B. surdus are distinct species. Moreover, for the first time we report and describe karyotype details of B. luristanicus and B. surdus which confirmed that they are diploid. Karyological studies demonstrate that all toads from three mentioned species have 2n = 22 chromosomes. These chromosomes are arranged into two groups. First group has six large chromosomes and the second group is composed of five small chromosomes. These chromosomes are metacentric or submetacentric. The number of submetacentric chromosomes is different in three mentioned species of B. viridis complex. Neither sexual heteromorphism, nor secondary constriction was observed in any pairs of chromosomes.

Bufo viridis complex, taxonomy, Bufo surdus, Bufo luristanicus, Bufo variabilis, karyotype, morphometry, Iran

1. Introduction

Green toads (Bufo viridis complex) are one of the prominent anuran species complexes which are broadly spread in the Palearctic region. Since B. viridis was described by Laurenti in the 18th century, considerable morphological variation has been observed for green toads, resulting in the description of numerous forms as species and subspecies (St?ck et al., 2001a, 2005). The complexity of this species complex and variationin cytogenetic forms in Asia is more than Europe and Africa (Batista, 2006). So far, all three ploidy levels (diploid, triploid and tetraploid) have been reported from Asia (St?ck et al., 2001b). Recent cytogenetic studies show that in addition to the diploid forms that have been reported from Iran, B. oblongus species distributed in east of Iran is tetraploid (St?ck et al., 2001b, 2003, 2005, 2006).

Despite significant progress in this area, the taxonomy of the B. viridis complex is still unknown. Ploidy level of many populations is not recognized and the relationships among members of this group are uncertain and need further investigations (Borkin et al., 2000). It has been indicated before that there are three taxa closely related

to B. surdus, namely B. luristanicus, B. surdus surdus and B. surdus annulatus, which together are the B. surdus subgroup (e.g. Baloutch and Kami, 1995; St?ck et al., 2001a). These taxa occur in Iran and have many characters in common, including a relatively small body size, extremely small or absent visible tympana and nearly squared parotoid glands. Inger (1972) placed these taxa in the B. viridis complex while St?ck et al. (2001a) believe the B. surdus subgroup does not belong to the B. viridis complex. Taxonomic status of B. surdus subgroup is still unknown. Moreover, ploidy levels and karyotype details of members of B. surdus subgroup are unclear and need further study.

Bufo luristanicus, B. surdus (with two subspecies) and B. variabilis are three members of B. viridis complex distributed in south, south west and south east of Iran. B. luristanicus is endemic to Iran and inhabits the Zagrous Mountains. B. surdus surdus exists in south and south east of Iran and southwest of Pakistan. B. surdus annulatus is exclusively found in its type locality in southwestern of Iran, 80 km south of Shiraz, Fars Province (Schmidtler and Schmidtler, 1969). B. variabilis is found in the western and central parts of Iran.

Recent studies of the green toad complex in the Palearctic include only a few samples from limited parts of the vast territory of Iran (St?ck et al., 2001a, 2001b, 2005, 2006; Borkin et al., 2000; Litvinchuk et al.,2007; Javari and Torki, 2009; Degani et al., 2013). As a result, the available data on B. viridis complex in Iran is incomplete, and the taxonomic status of such species is unknown or uncertain.

The aim of this paper is to present a new detailed study of the B. viridis complex in the southern region of Iran and provide helpful information on distribution, morphology, morphometry and karyotype of B. viridis complex, so that more precise taxonomic decisions can be adopted.

2. Materials and Methods

2.1 SamplingA total of 47 specimens of B. viridis complex were collected from various regions in the southern part of Iran, from March 2012 to April 2013. Sampling information is given in Table 1 and Figure 1.

Figure 1 Map of Iran showing sampling localities of Bufo viridis complex. ●: B. variabilis; ▲: B. luristanicus; ★: B. surdus.

Table 1 Localities of toads studied in this work.

2.2 MorphometryEighteen morphometric characters were measured (Baloutch and Kami, 1995; St?ck et al., 2001a) using a digital caliper with an accuracy of 0.01mm. SPSS 16 was used for statistical analysis. Note that only adult toads were included in this study. The measured characters are as follows: 1- Snout-vent length (SVL), 2- head length (HL), 3-distance between tip of snout and anterior corner of eye (ESD), 4- internarial distance (IND), 5- upper eyelids distance (UED), 6- width of upper eyelid (UEW), 7-horizontal diameter of eye (ED), 8- tympanic diameter (DT), 9- thigh length (THL), 10- length of tibia (TL), 11-tarsus length: from the heel to the proximal edge of the inner metatarsal tubercle (TAL), 12- foot length: from proximal edge of the inner metatarsal tubercle to tip of 4th toe (FL), 13- length of first toe (LFT), 14- length of inner metatarsal tubercle (LMT), 15- length of parotoid gland (PL), 16- width of parotoid gland (PW), 17- distance between parotoid glands (PD), and 18- distance between nostril and anterior corner of eye (NED).

2.3 chromosome preparationSpecimens were transferred to lab alive. Toads were injected intraperitoneally with 0.3% colchicine solution for 12–16 hours before being sacrificed. Bone marrow was extracted and placed in 0.075 M KcL hypotonic solution. Then the solution was kept in room temperature for 45–55 minutes and the procedure described in Schmid (1978) was followed. Slides were stained with 4% Giemsa for 10–15 minutes, were studied with a microscope and well spread metaphase was photographed. Based on Levan et al. (1964) classification, chromosome types were determined and arranged to obtain a chromosomal set. Karyotype Analysis 1.2 software was used for morphometric measurements of each chromosome.

3. Results

3.1 Morphometric analysis

Univariate statisticsThe mean values and standard deviations of all morphometric characters were significantly different in the three taxa of B. viridis complex.

Our results (Table2) show that specimens of B. variabilis have the largest body length (SVL= 70.48 ± 8.73 mm) and B. luristanicus has the smallest body length (50.70 ± 3.36 mm) among the three taxa. B. variabilis has the largest values for all other morphometric characters except UED. B. surdus has the largest values For UED. Similarly, B. luristanicus owns the smallest amount in all morphometric characters except DT and LFT. DT was not observed for B. surdus. LFT of B. luristanicus was larger than that of B. surdus (3.52 ± 0.40 mm compared to 3.23 ± 0.41 mm). In this analysis, B. surdus stands in between the other two groups (Table 2) for all morphometric characters (except DT and LFT). Also analysis of variance (ANOVA) showed significant differences between the three groups in all measured characters (P＜0.05). character variation range in B. variabilis was considerably higher than the other groups (For example character variation range or Min-Max of SVL in B. variabilis (41.08 mm–85.21 mm) was considerably higher than B. surdus (56.4 mm–66.04 mm) and B. luristanicus (46.22 mm–58.65 mm)).

It is notable that tympanic membrane was not visible in all collected samples belonging to B. surdus in this work , while all B. luristanicus samples had externally visible tympanic membrane. This membrane in B. luristanicus was smaller than B. variabilis (Table 2).

Multivariate statisticsDiscriminant analysis was used to describe functions that maximize the probability of correct classification of specimens to their original population. A stepwise discriminate analysis with eighteen morphometric traits reclassified 100% of all samples correctly into the three groups; B. variabilis, B. luristanicus and B. surdus. Each species was placed 100% in its expected group (see Table 3).

Based on Wilks’ Lambda, both functions are significant (P ＜ 0.05). First function represented 75.7% of variance and the second one showed 24.3% of variance.

Table 2 Morphometric characters of Bufo viridis complex (measurments in mm).

Table 3 classification results of discriminate analysis with eighteen morphometric characters.

Five of eighteen morphometric characters had the highest values of standardized canonical discriminant coefficients (Table 4).

Also Scatter plot of canonical functions show a clear separation between these three groups (Figure 2).

3.2 Karyotype analysisIn this study, we reported and described karyotype of B. luristanicus for the first time. This species was diploid and had 22 chromosomes (Figures 3 and 4).

chromosomes were divided into two major groups. First group consists of 6 large chromosomes and the second group contains 5 small chromosomes. Morphometric data such as length of short arm, length of long arm and other parameters are demonstrated in Table 5. B. luristanicus karyotype consists of 8 pairs of metacentic and 3 pairs of submeatacentric chromosomes. The 4th, 5thand 7thpairs of chromosomes were submetacentric, therefore metacentic and submeatacentric chromosomes were observed in both major groups. Related idiogram is shown in Figure 5.

Result of karyotype of B. surdus showed a set of 2n= 22 chromosomes (Figures 6 and 7). These chromosomes were arranged into two groups. First group consisted of 6 large chromosomes and the second one had 5 small chromosomes. Morphometric data of B. surdus is shown in Table 6.

The 1st, 4th, 5thand 7thpairs of chromosomes were submetacentric and the remaining pairs were metacentric. Therefore, metacentic and submetacentric chromosomes were observed in both major groups. The idiogram of this species is demonstrated in Figure 8.

Karyotype of B. variabilis from four localities of south Iran showed that this subspecies was diploid and had 2n = 22 chromosomes (Figures 9 and 10). Two major groups could be distinguished. The first group consists of six large chromosomes and the second group has five small chromosomes.

Karyotype of samples belonging to Mehkuh, Shiraz and Ahvaz exhibited that 4thand 7thpairs of chromosomes were submetacentric but in samples from choqa-zanbil 4thand 9thpairs of chromosomes were submetacentric.Morphometric analysis of chromosomes of B. variabilis samples is shown in Table 7 and Table 8.

Table 4 Standardized canonical discriminant function coefficients for Bufo viridis group. Five of eighteen characters were selected.

Figure 2 Scatter plot of canonical discriminant functions of eighteen morphometric traits of Bufo viridis complex

Submetacentric chromosomes exist in both major groups. Related idiograms of B. variabilis belonging to four mentioned regions are presented in Figures 11 and 12.

Other data from fieldUnfortunately, the attempts to get B. surdus annulatus subspecies from its type locality (80 km south of Shiraz, 5 km north of Mehkuh, Fars Province) in spring and summer seasons failed. This region is the only place for this subspecies that has been identified so far. Amphibians that were found in this area were Pelophylax ridibundus, Hyla savignyi and B. variabilis. Therefore, only B. surdus surdus subspecies were included in this research. Unfortunately, our efforts to obtain more specimens of B. surdus surdus to increase the sample size were not successful. One reason was that Sistan and Baluchestan (which is a large part of distribution areas of this subspecies) is an unsafe place to work specially in the border areas with the two neighboring countries and the other reason is that distribution range of this subspecies might have changed over time. For example, previously B. surdus surdus had been reported from Rudan (Deh Barez) in Hormozgan Province (St?ck et al., 2006), but we could not get this subspecies from Rudan. The area was occupied with an oriental species named Duttaphrynus olivaceus. Similarly, Zareian et al. (2012) found only one sample of B. surdus surdus from Gorm Mountain in Fars, south west of Iran.

Figure 3 Bufo luristanicus from Hosseiniye region of Andimeshk, Khuzestan Province. Photo by F. Fakharzadeh.

Figure 4 Karyotype of Bufo luristanicus from Hosseiniye region of Andimeshk, Khuzestan Province.

Figure 5 Idiogram of Bufo luristanicus based on centromere position.

Figure 6 Bufo surdus from Sangan region of Khash. Photo by F. Fakharzadeh.

Bufo luristanicus samples collected from a river near Hosseiniye region of Andimeshk, Khuzestan Province (a place near the type locality of this species) were sympatric with B. variabilis at the same region.

4. Discussion

4.1 Morphometry and morphologyThe univariate and multivariate analyses of the morphometric characters used in this study revealed that the morphometric traits can clearly and completely discriminate B. luristanicus, B. variabilis and B. surdus taxa from each other and confirmed that they are separate species. Moreover, as mentioned in this study (Section 3.1) tympanic membrane is a good morphological character to distinguish these taxa from each other. B. surdus subgroup is distributed in southern region of Iran, which includes B. surdus surdus, B. surdus annulatus, and B. luristanicus. They have many common characters such as small body size, lack of visible eardrum or it may be very tiny and also all have square parotoid glands.

Figure 7 karyotype of Bufo surdus from Khash, Sistan and Baluchestan Province.

Figure 8 Idiogram of Bufo surdus based on centromere position.

Table 5 Morphometric data of Bufo luristanicus chromosomes.

Table 6 Morphometric data of Bufo surdus chromosomes.

Schmidtler and Schmidtler (1969) introduced annulatus and luristanicus as subspecies of B.surdus, but Mertens (1971) introduced B. luristanicus as a separate species. St?ck et al. (2001a) considered these two species as a separate species, because of lack of visible eardrum in B. surdus and its existence in B. luristanicus. Also Anderson (1985) and Baloutch and Kami (1995) considered B. luristanicus as distinct species. Afrasiab and Ali (1988) reported B. surdus from Iraq, but it was probably B. luristanicus. Torki and Javari (2009) believed that B. luristanicus is a subspecies of B. surdus and rejected the existence of luristanicus as a separate species. The results of present research are in total agreement with Mertens (1971), St?ck et al. (2001a), Anderson (1985), Baloutch and Kami (1995) statements. Despite Torki and Javari (2009) that indicated the well-develop spiny tubercles on dorsal surface of B. luristanicus and lack of this trait in B. variabilis, our observation shows the existence of this trait in the majority of B. luristanicus, B. variabilis and B. surdus samples.

The results presented in this work can resolve an old taxonomic problem in B. surdus subgroup. This study confirms that B. surdus and B. luristanicus are separate species. Moreover, the existence of externally visible tympanic membrane in all B. luristanicus samples and its absence in all B. surdus samples verify it to be a proper morphological character to distinguish these two species.

Therefore, common morphological traits in luristanicus and surdus (such as parotoid glands shape) are illusive that can be misleading even for a specialist at first glance. Our data confirm the existence of two distinct green toads in Zagrous Mountains of Iran and this conclusion agrees with the statements of St?ck et al. (2001b) and Eiselt and Schmidtler (1973).

On the other hand, most of B. surdus surdus samples collected in this study belong to Khash, Sistan and Baluchestan, the nearest available location in Iran to the type locality of this subspecies, which is Baluchestan, Pakistan (Boulenger,1891). The holotype is an adult male specimen without visible tympanic membrane and SVL is 67 mm (St?ck et al., 2001a). Range of SVL for our samples (See Table 2) is 56.4 to 66.04 mm. Also they are without visible tympanic membrane. So, in comparison to the holotype, our collected samples of this subspecies are slightly smaller. B. surdus surdus is distributed in Kerman, Sistan and Baluchestan and Hormozgan (Haji Abad) provinces (Baloutch and Kami 1995; St?ck et al., 2001a). The type locality of B. surdus annulatus is 80 km south of Shiraz, 5 km north of Mehkuh), Fars province (Schmidtler and Schmidtler, 1969). So far, only one specimen of this subspecies has been found from type locality in 1968. As described earlier, our attempt to find B. surdus annulatus from its type locality failed.

According to Schmidt (1955) the type locality of B. luristanicus is Shahbazan at kilometer 324 of the trans-

Iranian railway, Lorestan province. 52 km as the crow flies NE of Dizful. But our researches show that there are two localities in Iran which called Shahbazan. One of them is located in lorestan Province without any railway near it but the other one (Shahbazan station) is located near Andimeshk and in 52 km as the crow flies NE of Dizful in Khuzestan Province and there is a railway here. Therefore we concluded that probably the type locality of B. luristanicus is near Andimeshk and Dizful in Khuzestan Province not in Lorestan province. Most of the collected samples of this species are from Hosseiniye region of Andimeshk. So this region is very close to the type locality of B. luristanicus. The Holotype is a male that its SVL is 51.8 mm. In comparison, range of our samples for SVL is 46.22 to 58.65 mm (See Table 2). So far B.luristasnicus has been reported from Fars, Lorestan and Khuzestan provinces. B. variabilis is distributed in western half and central part of Iran. Type locality of B. variablis is far from Iran and located in Germany (Pallas, 1777).

Table 7 Morphometric data of Bufo variabilis chromosomes from two localities of Fars. A: Mehkuh; B: Shiraz.

Table 8 Morphometric data of Bufo variabilis chromosomes from two localities of Khuzestan. A: Ahvaz; B: choqa-zanbil.

Figure 9 Karyotype of Bufo variabilis from two localities of Fars province. A: Mehkuh; B: Shiraz.

Figure 10 Karyotype of Bufo variabilis from two localities of Khuzestan province. A: Ahvaz; B: choqa-zanbil.

Figure 11 Idiogram of Bufo variabilis based on centromere position from two localities of Fars. A: Mehkuh; B: Shiraz.

Figure 12 Idiogramof Bufo variabilis based on centromere position from two localities of Khuzestan. A: Ahvaz; B: choqa-zanbil.

4.2 KaryotypeFor the first time karyotypic data for B. luristanicus and B. surdus were reported. All collected green toads that belong to three mentioned species are diploid and have 22 chromosomes. chromosomes are arranged into two groups. First group has six large chromosomes and the second group composes of five small chromosomes. These chromosomes are metacentric or submetacentric. Such pattern is highly conserved in most species of genus B. (e.g. Borkin et al., 2007; Bogart, 1972). In the examined samples of southern region of Iran, polyploid toads were not found. So far polyploid toads have been reported only by St?ck et al. (2001b, 2005, 2006) from east of Iran. St?ck et al. (2001b) stated that the deserts of central Iran seem to separate the polyploids in the East of central Iran from the diploids in the West. Therefore if their conclusion is correct, polyploid toads must be restricted to Eastern part of Iran.

According to St?ck et al. (2001a) the results obtained from DNA flow cytometry show that both B. luristanicus and B. surdus are diploid. Our karyological studies that have been done on B. luristanicus and B. surdus also confirm this conclusion.

Our study shows no sexual heteromorphism in any pairs of chromosomes. The uniformity of sex chromosomes among toads is accepted and indicated in many papers (Miura, 1995; Kasahara et al., 1996; cavallo et al., 2002; Borkin et al., 2007; Siripivasing et al., 2008; Fakharzadeh et al., 2009; Al-Shehri and Al-Saleh, 2010).

The number of submetacentric chromosomes is different in three mentioned species of B. viridis complex. Karyotype of B. surdus contains 4 pairs of submetacentric chromosome (1st, 4th, 5thand 7thpairs of chromosomes) while in B. luristanicus 3 pairs of chromosomes (4th, 5thand 7thpairs of chromosomes) are submetacentric and B. variabilis from four mentioned localities has only 2 pairs of submetacetric chromosomes (4thand mostly 7thpairs of chromosomes).

Our findings on karyotype of B. variabilis species from four mentioned localities exhibited no secondary constriction. Same results are indicated in other studies that used Giemsa-stained method (e.g. Borkin et al., 2007; Masik et al., 1976; Borkin et al., 1986a; Roth and Ráb, 1986). In Giemsa-stained karyotype of diploid B. viridis from Kerman (St?ck et al., 2001a, 2005), the chromosome pair 6 had a secondary constriction on its long arms, which is in contrast to the present study.

5. conclusion

The results of this paper verify the existence of three independent species of B. viridis complex, namely B. luristanicus, B. surdus and B. variabilis, in southern region (south, southeast and southwest) of Iran. In the present study for the first time karyotype details of B. surdus and B. luristanicus were reported. It was confirmed that both species are diploid and have 22 chromosomes. Moreover, it was explored that the number of submetacentric chromosomes is different in the three taxa of B. viridis complex.

These results can resolve an old taxonomic problem about B. surdus subgroup (taxa closely related to B. surdus). In summary B. luristanicus and B. surdus are distinct species and common morphological traits in luristanicus and surdus are seductive.

AcknowledgementsThe authors are grateful to Dr. Matthias St?ck for his valuable comments, and Mr. Asghar Khajeh for his assistance in sampling from Sistan and Baluchestan province.

Afrasiab S. R., Ali H. A.1988. A new record of toad Bufo surdus, Boulenger (Amphibia, Bufonidae) from Iraq with preliminary key for Iraqi Amphibia. Bull Iraq Nat Mus, 8(1): 115–123

Al-Shehri A. H., Al-Saleh A. A.2010. First report of the karyotype of Bufo dhufarensis from Saudi Arabia. J King Saud Univ, Science , 22: 73–76

Anderson, S. c.1985. Amphibians. In Yarshater E. (Ed.), Encyclopedia Iranica. Vol. 1, Fascicle 9: 987–990

Baloutch M., Kami H. G.1995. Amphibians of Iran. Tehran, Iran:

Tehran University Press, 177 pp (In Persian)

Batista V. , carranza S.,carretero M. A., Harris D. J.2006 Genetic variation within Bufo viridis: Evidence from mitochondrial 12s and 16s rRNA DNA sequences. Bull Soc catalana Herpetol Barcelona, 17: 24–33

Bogart J. P.1972. Karyotypes. In Blair W. F. (Ed.), Evolution in the Genus Bufo. Austin: University of Texas Press, 171–195

Borkin L. J., caune I. A., Pisanetz E. M., Rozanov Y. M.1986. Karyotype and genome size in the Bufo viridis group. In Ro?ek Z. (Ed.), Studies in Herpetology, charles University, Prague, 137–141

Borkin L. J., Rozanov J. M., Litvinchuk S. N.2000. Nuclear DNA content in some green toads (Bufo viridis complex) of Turkey and Iran. Russ J Herpetol, 7(3): 171–180

Borkin L. J., Shabanov D. A., Brandler O. V., Kukushkin O. V., Litvinchuk S. N., Mazepa G. A., Rozanov J. M.(2007). A case of natural triploidy in European diploid green toad (Bufo viridis), with some distributional records of diploid and tetraploid toads. Russ J Herpetol, 14(2), 121–132

cavallo D., Devita R., Eleuteri P., Borkin L., Emechenko V., Odiema G., Balletto E. 2002. Karyological and flow cytometric evidence of triploid specimens in Bufo viridis (Amphibia Anura). Eur J Histochem, 16: 159–164

Degani G.,Goldberg T., Gasith A., Elron E., Nevo E.2013.DNA variations of the green toad Pseudepidalea viridis (syn. Bufo viridis) from various habitats. Zool Stud, 52: 1–15

Eiselt J., Schmidtler J. F.1971. Vorlaufige Mitteilung über zwei neue Subspecies von Amphibia salientia aus dem Iran. Ann Naturhist Mus Wien, 75: 383–385

Fakharzadeh F., Darvish J., Ghassemzadeh F., Kami H. G. 2009. Anuran karyological study of Khorasan province. Asian J Bio Sci, 2(3): 66–73

Inger R. F.1972. Bufo of Eurasia. In Blair W. F. (Ed.), Evolution in the genus Bufo. Austin: University Texas Press, 102–118

Javari M., Torki F.2009. Notes on morphology, ecology, behavior and systematics of Bufo luristanicus Schmidt, 1952. Herpetozoa, 21: 171–178

Kasahara S., Siva A. P., Haddad c. F.1996. chromosome banding in three species of Brazilian toads (Amphibia-Bufonidae). Rev Brasil Genet, 19: 237–242

Levan A., Fredga K., Sandberg A. A.1964. Nomenclature for centromeric position on chromosomes. Hereditas, 52: 201–220

Litvinchuk S. N., Rosanov J. M., Usmanova N. M., BorkinL. J.,Mazanaeva L. F., Kazakov V. I.2007. Variability of microsatellites BM224 and Bcal7 in populations of green toads (Bufo viridis complex) differing by nuclear DNA content and ploidy. cell Tissue Bio, 1 (1): 65–79

Masik E. J., Kadirova B. K., Toktosunov A. T.1976. Karyotype pattern in the green toad Bufo viridis in Kirghizia. Zool Zh, 55(11): 1740–1743 (In Russian with English summary)

Mertens R.1971. Zur Kennthis von Bufo luristanicus (Salientia Bufonidae) .Salamandra, 7: 83–84

Miura I. 1995. The late replication banding patterns of chromosome are highly conserved in genera Rana, Hyla and Bufo (Amphibia, Anura). chromosoma, 103: 567–574

Pallas P. S.,Baldinger E. G., Erxleben J. c. P.1777. Naturgeschichte merkwürdiger Thiere, in welcher vornehmlich neue und unbekannte Thierarten durch Kupferstiche, Beschreibungen und Erklarungen erlautert werden. Berlin and Stralsund: G. A. Lange

Roth P., Ráb P.1986. Karyotype analysis of the Bufo viridis group: Systematic implications. In Roèek Z. (Ed.), Studies in Herpetology. Prague: charles University, 131–135

Schmidt K. P.1955. Amphibians and reptiles from Iran. Vidensk Medd Dansk Naturh Foren, 117: 193–207

Schmid M.1978. chromosome banding in amphibia. chromosoma, 66(4): 361–388

Schmidtler J. J., Schmidtler J. F. 1969. über Bufo surdus; mit einem Schlüssel und Anmerkungen zu den übrigen Kr?ten Irans und West-Pakistans. Salamandra, 5: 113–123

Siripivasing P., chulalaksananukul W., Pariyanonth P., Kaewsri S., Sittigul, S., Seatung N., Tanomtong A.2008. The identification of the sex chromosome and karyotype of four toad species (genus Bufo) in Thailand bt T-lymphocyte cell culture. cytologia, 73(3): 229–241

St?ck M., Günther R., B?hme W.2001a. Progress towards a taxonomic revision of the Asian Bufo viridisgroup: current status of nominal taxa and unsolved problems (Amphibia: Anura: Bufonidae). Zool Abh Staatl Mus Tierkunde Dresden, 51: 253–319

St?ck M., Frynta, D., Grosse, W.-R., Steinlein, c., Schmid, M.,2001b. A review of the distribution of diploid, triploid and tetraploid green toads (Bufo viridis complex) in Asia including new data from Iran and Pakistan. Asiatic Herp Res, 9: 77–100

St?ck M., Grosse W. R.2003. Die Bufo viridis-Untergruppe in MIttel- und Zentralasien: Eine?bersichzuVerbreitung, Polyploidie, Paarungsrufen und Taxonomie. Mertensiella, 14:179–217

St?ck M., Steinlein c., Lamatsch D. K., Schartl M., Schmid M.2005. Multiple origins of tetraploid taxa in the Eurasian Bufo viridis subgroup. Genetica, 124: 255–272

St?ck M., Moritz c.,Hickerson M.,Frynta D., Dujsebayeva T., Eremchenko V., Macey J.R., Papenfuss T.J., Wake D.B.2006. Evolution of mitochondrial relationships and biogeography of Palearctic green toads (Bufo viridis subgroup) with insights in their genomic plasticity. Mol Phyl Evol, 41: 663–689

Zareian H., Gholamhosseini A., Esmaeili H.R., Teimori A. 2012. Vertebrates of Kuh-e-Gorm non-hunting area, Jahrom, Iran: Diversity, conservation and challenges. Iran J Anim Biosyst, 8(2): 169–182

*corresponding author: Fatemeh FAKHARZAEH, from Ferdowsi University of Mashhad, Mashhad, Iran, with her research focusing on phylogeny and taxonomy of Bufonidae in Iran.

E-mail: ffakharzadeh@yahoo.com

Received: 7 March 2014 Accepted: 22 August 2014