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International Journal of Zoology and Research (IJZR) ISSN 22788816 Vol. 3, Issue 3, Aug 2013, 21-28 TJPRC Pvt.

. Ltd.

KARYOTYPIC STUDY OF TWO TOAD SPECIES- DUTTAPHRYNUS STOMATICUS AND PSEUDEPIDALEA VIRIDIS (ANURA, AMPHIBIA) FROM JAMMU AND KASHMIR
NEELAM SABA & N. K. TRIPATHI Department of Zoology, University of Jammu, Jammu & Kashmir, India

ABSTRACT
Karyotypic studies of two bufonids (toads)- Duttaphrynus stomaticus and Pseudepidalea viridis, were carried out using the conventional Giemsa staining protocol. The karyotypes of the two genera revealed typical bufonid characters including diploid number 2n=22 and fundamental arm number FN=44. All the biarmed chromosomes were placed into two groups- Group 1 of larger chromosomes and Group 2 of smaller chromosomes. Chromosomes were either metacentric or submetacentric in nature. No acrocentric and telocentric chromosomes were observed. Only one pair (4th) was metacentric in P. viridis whereas two pairs (3rd and 5th) were submetacentric in D. stomaticus. All other chromosomes were metacentric in both the species. Ag-NOR banding showed a pair of conspicuous secondary constrictions corresponding to the nucleolar organizer regions (NORs) on seventh pair of chromosomes, but on long arm in P. viridis and short arm in D. stomaticus.

KEYWORDS: Karyotype, Giemsa, Chromosomes, Diploid Number, Fundamental Arm Number, NOR INTRODUCTION
The family Bufonidae (order Anura) is largest anuran family and currently comprises of 585 species in 50 genera (Frost, 2013; an online reference accessed on June 13, 2013). Bufonids are the toads distributed widely on the globe. Bufonidae is a worldwide hyloid clade of noncontroversial monophyly. Amphibian taxonomy was relatively stable for decades, as summarized by Frost (1985) and Duellman and Trueb (1986). The advent of direct DNA sequencing methods in the early 1990's enabled tests of previous hypotheses of phylogenetic relationships. In turn, new understandings of phylogeny led to suggestions for taxonomic revision. The first large-scale taxonomic treatment for all of living amphibians (Frost et al., 2006) proposed radical changes and additional publications have made further taxonomic revisions. The two species under investigation were placed under one genus named Bufo as B. stomaticus and B. viridis. According to the current revision (Frost, 2013), these two are placed in two genera and named as Duttaphrynus stomaticus and Pseudepidalea viridis respectively. The toads are cosmopolitan and attract the herpetologists from far and wide. Duttaphrynus stomaticus also called as the Indus Valley Toad, is distributed in the Indo-Pakistan subcontinent while Pseudepidalea viridis also called as the Green toad, is distributed in all the Palaearctic countries (Sahi, 1979; Gruber, 1981; Dinesh et al., 2009; Frost, 2013). The green toad is one of the polytypic amphibians. The amphibian species of Jammu and Kashmir have not received much attention of cytogeneticists and molecular biologists till now (Duda and Kaul, 1971 and 1973; Saba and Tripathi, 2012). It is an attempt to describe the karyotypic details of the species from the state so that their taxonomic relationships can be drawn. This preliminary chromosomal study will surely pave the way for further cytogenetic and molecular studies in future. Moreover the extended molecular studies after this basic step would be of immense help to establish taxonomic status of the specimen populations and study the species and subspecies which are currently inhabiting the state.

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MATERIALS AND METHODS


Two male and one female specimen of Pseudepidalea viridis (Figure 1)whereas two male and two female specimens of Duttaphrynus stomaticus (Figure 6), were collected from District Doda (Bhaderwah- altitude 1558m) and District Jammu (altitude 300-4200m) respectively of Jammu and Kashmir during the monsoon breeding season. Before sacrificing the specimens were injected intramuscularly and intraperitoneally with 0.5% colchicine solution (@ 1ml per 100g body weight) for 3.5 hours. Then the animals were anaesthesized and dissected to take out the intestine, spleen and bone marrow. The tissues were hypotonised with 0.5% NaCl solution for 30 minutes at room temperature. Fixation of the tissue was done in 3:1 methanol-acetic acid fixative for 45 minutes changing the solution every 15 minutes. The material was then dabbed on clean slides, air-dried and stained with 2% giemsa stain (pH=7) for 30-35 minutes. Ag-NOR banding was done using Howell and Black (1980) protocol with slight modifications. Slides were scanned under Olympus research microscope and the best metaphase complements were photographed at 100X magnification. Morphometry was done using occulometer.

RESULTS
A total of 40-50 metaphase stages were selected to establish the diploid chromosome number and sexual heteromorphism. Both male spermatogonial metaphase and female somatic metaphase complements were used for karyological study but no sex chromosome heteromorphism was observed in any of the species. The basic chromosome number was found to be 2n=22 (Figure 2 and Table 1). Eleven pairs of chromosomes were placed into two groups comprising of Group A: six pairs of large chromosomes and Group B: five pairs of small chromosomes. All chromosomes in both the karyotypes were biarmed and of two types, that is, metacentric and submetacentric type (following Levan et al., 1964). In Pseudepidalea viridis, Pair no. 4 of group A was found to be submetacentric and all the other chromosomes in both the groups were metacentric type. Haploid formula for the complement was calculated as n=10M+1SM and the corresponding fundamental arm number was calculated as NF=44. Mean haploid length was 16.53m and total complement length was 33.06m. Ag-NOR banding showed two NORs on short arm of pair number 7 in group B (Figure 3). Morphometric data is given in the Table 1. Histogram (Figure 4) and ideogram (Figure 5) were prepared using morphometric data. In Duttaphrynus stomaticus, general karyotype composition being similar to that of P. viridis, Pair no. 2 and 5 of group A was found to be submetacentric while rest of all the chromosomes, in both the groups, were found to be metacentric type. Haploid formula for the complement was calculated as n=9M+2SM (Figure 7 and Table 2). A pair of NORs was on seventh pair on long arm (7q) (Figure 8). Morphometric data is given in Table 2 and histogram (Figure 9) and idiogram (Figure 10) was constructed using the data.

Figure 1: An Adult Green Toad- Pseudepidalea viridis

Karyotypic Study of Two Toad Species- Duttaphrynus stomaticus and Pseudepidalea viridis (Anura, Amphibia) from Jammu and Kashmir

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Figure 2: Karyotype of P. viridis Prepared from Spermatogonial Metaphase

Figure 3: NOR-Banded Karyotype of P.viridis Showing a Pair of NORs on Pair no. 7 (7p)

Figure 4: A Histogram Showing the Relative Length Percentage of Chromosomes of Male Pseudepidalea viridis

Figure 5: An Ideogram of Male Pseudepidalea viridis Constructed on the Basis of Chromosome Numbers and Position of the Centromere

Figure 6: An Adult Indus Valley Toad- Duttaphrynus stomaticus

Figure 7: Karyotype of D. stomaticus

Figure 8: NOR-Banded Karyotype of D. stomaticus Showing a Pair of NORs on Pair no. 7 (7q)

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Figure 9: A Histogram Showing the Relative Length Percentage of Chromosomes of Male Duttaphrynus stomaticus

Figure 10: An Idiogram of Male Duttaphrynus stomaticus Constructed on the Basis of Chromosome Numbers and Position of the Centromere

DISCUSSIONS
Although the toad species share the conserved karyotypic characters of family Bufonidae but there are vivid intergeneric differences. The presence of Metacentric and submetacentric type chromosomes strongly depicts karyotypic conservatism as observed in most of bufonid toads. Chromosome numbers of bufonids have been found to be highly conserved over such huge period of evolutionary history. Basic chromosome number is 2n=22. Almost all the bufonids have 2n=22 (Beccari, 1926; Makino, 1932; Bianchi and Laguens, 1964; Manna and Bhunya, 1966; Ullerich, 1966; Volpe and Gebhardt, 1968; Roth and Rab, 1986; Stock et al., 2004 and 2005; Odierna et al., 2007; Al-Shehri and Al-Saleh, 2010; Baraquet et al., 2011; Yadav and Neeru, 2012; Saba and Tripathi, 2012) with only one exception of Bufo regularis in which 2n=20 (Beckert and Doyle, 1968 and 1970; Bogart, 1972; Al-Shehri and Al-Saleh, 2008). Chromosome form is also highly conserved with most of the karyotypes having symmetrically arranged biarmed chromosomes of metacentric, submetacentric and in few cases subtelocentric chromosomes (Natarajan, 1958; Bogart, 1968; Baraquet et al., 2011; Saba and Tripathi, 2012). Existence of NORs on 7th pair is yet another proof of karyotypic conservatism in these two species. Most of the toad species do have secondary constriction on 7 th pair (Schmid, 1978 and 1982). The species studied do possess the typical bufonid karyological characters. Since this is only preliminary

chromosomal investigation of toad species from our region, it must be treated as the basic step towards further cytogenetic and molecular studies in future, so that a full genetic profile of these and other anurans could be prepared for contributing to further taxonomic and evolutionary aspects of these species.

ACKNOWLEDGEMENTS
Authors are thankful to the Head, Department of Zoology, University of Jammu, for providing necessary lab facilities.

Karyotypic Study of Two Toad Species- Duttaphrynus stomaticus and Pseudepidalea viridis (Anura, Amphibia) from Jammu and Kashmir

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REFERENCES
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Lynch,J.D., Green,D.M., & Wheeler, W.C (2006). The Amphibian tree of life. Bull. Am. Nat. Hist., 297, 1-370. 15. Gruber, U. (1981). Notes on the Herpetofauna of Kashmir and Ladakh. Brit. J. Herpetol., 6, 145-150. 16. Levan, A., Fredga, K., & Sandberg, A.A. (1964). Nomenclature for centromeric position on chromosomes. Hereditas, 52, 201-220. 17. Makino, S. (1932). Notes on chromosomes of Rana temporaria L. and Bufo sachalinensis (Nikolskii). Proc. Imp. Acad. Tokyo, 8, 23-26.

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18. Manna, G.K., & Bhunya, S.P. (1966). A study of somatic chromosomes of both sexes of the common Indian toad, Bufo melanostictus Schneid. Caryologia, 19, 403-411. 19. Natarajan, R. (1958). Contribution to cytology of Indian anurans (Amphibians). A study of chromosomes in two sexes of Bufo melanostictus Schneider (Bufonidae). Proc. Indian Acad. Sci., 48, 297-301. 20. Odierna G, Aprea G., Capriglione T., Castellano S., & Balletto E. (2007). Cytological evidence for populationspecifi c sex chromosome heteromorphism in Palaearctic green toads (Amphibia, Anura). J. Biosci., 32(4), 763 768. Indian Academy of Sciences http://www.ias.ac.in/jbiosci 21. Roth P., & Rb P. (1986). Karyotype analysis of the Bufo viridis group: Systematic implications. In: Z. Rocek (ed.), Studies in herpetology. Proc. Ord. Gen. Meet. Soc. Europ. Herpetol., Prague, 131-136. 22. Saba, N., & Tripathi, N.K. (2012). Meiotic Chromosomes and Karyotype of Bufo viridis (Laurenti, 1768) from Jammu and Kashmir. Bull. Environ. Pharmacol. Life Sci. 1 (6), 21- 25. 23. Sahi, D.N. (1979). A contribution to herpetology of Jammu and Kashmir state (Unpublished Ph. D. thesis). Department of Biosciences, University of Jammu, Jammu. 24. Schmid, M. (1978). Chromosome Banding in Amphibia I. Constitutive Heterochromatin and Nucleolus Organizer Regions in Bufo and Hyla. Chromosoma (Berl.), 66, 361-388. 25. Schmid, M. (1982). Chromosome Banding in Amphibia VII. An alysis of the structure and variability of NORs in Anura. Chromosoma (Berl.), 87, 327-344. 26. Stock, M., Steinlein, C., Lamatsch, D., Schartl, M., & Schmid, M. (2005). Multiple origins of tetraploid taxa in Eurasian Bufo viridis subgroup. Genetica, 124, 255-272. 27. Ullerich F.M., (1966). Karyotype and DNA analysis of Bufo bufo, Bufo viridis and Bufo calamita (Amphibia, Anura), Chromosoma, 18, 316-342. 28. Volpe, E., & Gebhardt, B. (1968). Somatic chromosomes of marine toad, Bufo marinus (Linne). Copeia, 3, 570575. 29. Yadav A.S., & Neeru (2012). Karyological analysis of Indian toad, Bufo stomaticus Lutken, 1862 from Haryana (India). International Journal of Research in BioSciences, 1(1), 24-28. Available online at http://www.ijrbs.in

APPENDICES
Table 1: Chromosomal Morphometric Data of Male Pseudepidalea viridis (2n=22) from Spermatogonial Metaphase Complement Chromosome Number 1 2 3 4 5 6 7 8 Length of Short Arm p (m) 1.23 1.21 1.17 0.79 1.03 0.97 0.93 0.75 Length of Long Arm q (m) 1.53 1.39 1.27 1.47 1.20 1.05 1.02 0.90 Total Chromosome Length p+q(m) 2.76 2.60 2.44 2.26 2.23 2.02 1.95 1.65 Relative Length Percent 12.70 11.97 11.23 10.40 10.26 9.30 8.97 7.59 Arm Ratioq/p 1.24 1.14 1.08 1.86 1.11 1.08 1.09 1.20 Centromeric Index=p/p+q 0.44 0.46 0.45 0.34 0.46 o.48 0.47 0.45

Nomenclature Metacentric Metacentric Metacentric Sub Metacentric Metacentric Metacentric Metacentric Metacentric

Karyotypic Study of Two Toad Species- Duttaphrynus stomaticus and Pseudepidalea viridis (Anura, Amphibia) from Jammu and Kashmir

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9 10 11

0.69 0.63 0.40

0.80 0.69 0.60

Table 1:Contd., 1.49 6.86 1.32 6.07 1.00 4.60

1.15 1.09 1.50

0.46 0.47 0.40

Metacentric Metacentric Metacentric

Table 2: Chromosomal Morphometric Data of Male Duttaphrynus stomaticus (2n=22) from Spermatogonial Metaphase Complement Chromosome Number 1 2 3 4 5 6 7 8 9 10 11 Length of Short Arm p (m) 0.93 0.87 0.55 0.64 0.39 0.38 0.34 0.27 0.21 0.18 0.15 Length of Long Arm q (m) 1.02 0.96 0.96 0.84 0.78 0.54 0.39 0.36 0.31 0.27 0.25 Total Chromosome Length p+q(m) 1.95 1.83 1.51 1.48 1.18 0.92 0.73 0.63 0.52 0.45 0.40 Relative Length Percent 16.81 15.77 13.01 12.75 10.17 7.93 6.29 5.43 4.48 3.87 3.44 Arm Ratioq/p 1.09 1.10 1.74 1.31 2.00 1.42 1.14 1.33 1.47 1.50 1.66 Centromeric Index=p/p+q 0.47 0.48 0.36 0.43 0.33 0.41 0.46 0.42 0.40 0.40 0.37

Nomenclature Metacentric Metacentric Sub-metacentric Metacentric Sub-metacentric Metacentric Metacentric Metacentric Metacentric Metacentric Metacentric