Pelophylax Sp.) from Guilan Province, Iran (Anura; Ranidae

Herpetology Notes, volume 10: 677-680 (2017) (published online on 28 November 2017)

Karyological study of the Marsh frog (Pelophylax sp.) from Guilan province, Iran (Anura; Ranidae)

Nasim Bashirichelkasari1 and Haji Gholi Kami1,*

Abstract. Karyological characteristics of the Marsh frog (Pelophylax sp.) in Guilan province in the northern part of Iran show that the chromosome number is 2n=26 and the arm number NF=52. The chromosomes divided into two groups: large chromosomes (pairs 1-5) and small ones (pairs 6-13) and consist of one metacentric (M) and 12 submetacentric (SM) chromosome pairs. The Centromeric Index ranges from 28.77 to 37.66, the arm ratio between 1.65 and 2.46, the relative length between 3.96 and 11.94, and the length variation between 4.97 and 15.81. Average total length of the chromosomes is 128.21µm. The largest chromosome in this species is a pair of the submetacentric chromosome. This is the first study on karyotype analysis in a population of Pelophylax sp. in Guilan province and adds basic useful information for further population studies.

Keywords: Karyotype, Marsh frog, Pelophylax sp., Guilan, Iran

Introduction sp. from Iran using bone marrow, liver and reproductive cells treated with colchicine in vivo, with the intention The Marsh frog (Pelophylax sp.) belongs to Order of increasing the knowledge of frog cytogenetic and Anura, family Ranidae (Baloutch and Kami, 1995). its characterization. According to the latest molecular Ranidae is distributed in the whole of Iran except studies (Pesarakloo et al., 2016), Pelophylax frogs Sistan and Baluchistan province and desert areas and from Guilan are different to Pelophylax ridibunda and has two genera and three species in Iran (Rastegar- Pelophylax bedriagae and may represent a new species. Pouyani et al., 2008; Bashiri et al., 2015). The latest The results of this study can help to clarify the taxonomic report of Iranian water frogs of the genus Pelophylax status of the Pelophylax sp. From Guilan. considers the Marsh frog as Pelophylax sp. (Pesarakloo et al., 2016; Vitt and Caldwell, 2014). In addition to the morphological, skeletal and biometric characteristics, karyological data is key to animal identification and classification (Fakharzadeh et al., 2009). 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 (Chiarelli and Capanna, 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. This is why amphibian chromosomes are relatively well studied (Fakharzadeh et al., 2009). In this paper, we describe the karyotype for Pelophylax

1 Golestan University, Department of Biology, Faculty of Sciences, Gorgan, Iran. Figure 1. Sampling localities of Pelophylax sp. in Guilan * Corresponding author e-mail: [email protected] province, Iran. 678 Nasim Bashirichelkasari & Haji Gholi Kami

and in decreasing length order within each class. Finally, the karyotype was constructed by first dividing the chromosome pairs into classes on the basis of centromere position, and then by arranging the homologous pairs in decreasing length order within each group. Also, the idiogram was arranged by Adobe Photoshop CS6 software to provide the standard features of the chromosomes of Pelophylax sp.

Results The counts of chromosome ranged from 23 to 28 per metaphases with a mode at 26, representing 80% of the metaphases. In 20 metaphases from the bone marrow and Figure 2. Metaphase spread from liver tissue of female liver cells of five Pelophylax sp. specimens, the diploid Pelophylax sp. in Guilan province, Iran, 2n=26 (photo by N. Bashiri). chromosome number were 2n=26 (Figure 1), which is valid over 80% of metaphases cells. Cells lacking a normal number of chromosome values (2n=23–28) were probably caused by losses during preparation or additions from nearby cells. Sex chromosome is Material and Methods showed in Figure 4. Homologous pairs of chromosomes Five Marsh frogs with a weight of 10-30 g and snout- were arranged in decreasing size and Centromeric vent length 50–80 mm were collected from Guilan indexes. The representative karyotype for Pelophylax province in the North of Iran, during spring 2014. The sp. is shown in Figure 2. It has one metacentric (M) frogs were transported alive to the laboratory and kept and 12 submetacentric (SM) chromosome pairs. in an aquarium before analysis. The procedure used for The number of chromosome arms was determined karyotype test in samples has been described previously NF=52 and chromosome formula can be expressed as by Yadollahvand et al. (2013). 2n=1m+12sm. The morphological and numerical data The chromosome pairs were classified following are summarized in Table 1. The Centromeric index, Levan et al. (1964) into metacentric (M), submetacentric arm ratio, relative length and length variation range (SM), subtelocentric (ST) and telocentric (T). The pair of chromosomes are between 28.77–37.66, 1.65–2.46, numbers were attributed following this classification 3.87–12.33 and 4.97–15.81 (μm), respectively. Total

Figure 3. Karyotype of liver tissue of female Pelophylax sp. in Guilan province, Iran, 2n=26 (photo by N. Bashiri). Karyological study of the Marsh frog from Guilan province, Iran 679

Figure 4. Haploid chromosomes of male Pelophylax sp. from testis tissue in Guilan province, Iran, n=13 (photo by N. Bashiri).

length of short arm, total length of long arm and total Discussion length of haploid chromosomes were 43.97, 84.23 and The karyotype of Pelophylax sp. is asymmetric 128.21 (μm) respectively. The largest chromosome is chromosomes consisting of a diploid number of 26 a pair of submetacentric chromosome. The idiogram chromosomes. This study represents for the first time of the Pelophylax sp. was made on the basis of the the karyotype of the population of Pelophylax sp. in this karyotype (Figure 5). area of Iran. The finding of 26 chromosomes diploid

Table 1. Summarized morphological and numerical data of Pelophylax sp. Table 1. Summarized morphological and numerical data of Pelophylax sp.. 1 Total length short arm Total length long arm Total length of haploid TableNF* 1. Summarized2n morphological and(ȝ m)numerical data of Pelophylax(ȝm) sp.. chromosome 1 (ȝm) 52 26 Total 43.97length short arm Total length84.23 long arm Total length128.21 of haploid NF* 2n (ȝm) (ȝm) chromosome * Arm ( ȝnumberm) 52 26 43.97 84.23 128.21 2 * Arm 3 number 2 Table 2. Numeral characteristics of the karyotype of Pelophylax sp. showing the mean values of 4 3 Table 2. Numeral characteristicsmeasurements from of theten bestkaryotype mitotic metaphases. of Pelophylax sp. showing the mean values of measurements5 from ten best Table 2. Numeral characteristics of the karyotype of showing the mean values of 4 mitotic metaphases. NO. TL (ȝm) Long arm Short arm ArmPelophylax CI sp. Relative length Classification measurements from ten (I( bestȝm) ) mitotic metaphases. (S( ȝm) ) ratio (%) 5 N O. NO. 1 TL15.81 (ȝm) Long10.66 arm Short5.15 arm Arm2.06 32.57 CI Relative12.33 length ClassificationSubmetacentric 2 14.63 9.90(I( ȝm) ) 4.73 (S( ȝm) ) ratio 2.09 32.33 11.41(%) Submetacentric 13 15.8113.12 10.668.44 5.154.68 2.061.80 32.5735.67 12.3310.23 SubmetacentricSubmetacentric 24 14.6312.66 9.90 8.20 4.73 4.46 2.09 1.83 32.33 35.22 11.41 9.87 Submetacentric Submetacentric 35 13.1211.65 8.447.74 4.683.91 1.801.97 35.6733.56 10.239.08 SubmetacentricSubmetacentric 46 12.6610.30 8.20 6.42 4.46 3.88 1.83 1.65 35.22 37.66 9.87 8.03 Submetacentric Metacentric 57 11.659.39 7.746.02 3.913.37 1.971.78 33.5635.88 9.087.32 SubmetacentricSubmetacentric 68 10.308.77 6.42 5.71 3.88 3.06 1.65 1.86 37.66 34.89 8.03 6.84 Submetacentric Metacentric 79 9.397.50 6.024.76 3.372.74 1.781.73 35.8836.53 7.325.84 SubmetacentricSubmetacentric 108 8.777.09 5.71 4.54 3.06 2.55 1.86 1.78 34.89 35.96 6.84 5.52 Submetacentric Submetacentric 119 7.506.69 4.764.32 2.742.37 1.731.82 36.5335.42 5.845.21 SubmetacentricSubmetacentric 1012 7.095.63 4.54 4.01 2.55 1.62 1.78 2.46 35.96 28.77 5.52 4.39 Submetacentric Submetacentric 1113 6.694.97 4.323.52 2.371.45 1.822.42 35.4229.17 5.213.87 SubmetacentricSubmetacentric 12 5.63 4.01 1.62 2.46 28.77 4.39 Submetacentric 6 13 4.97 3.52 1.45 2.42 29.17 3.87 Submetacentric 6 680 Nasim Bashirichelkasari & Haji Gholi Kami

Acknowledgments. We wish to thank Mr. R. Yadollahvand from Federal University of Paraíba, and Dr. F. Ganji from Golestan University for scientific and technical support.

References

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