© 2011 The Japan Mendel Society Cytologia 76(2): 111–117, 2011 Karyological Studies on 5 Anuran Species (Rhacophoridae, Microhylidae) from the Western Ghats, Southwest India S. Hareesh Joshy1 and Mitsuru Kuramoto2* 1 Rondano Biodiversity Research Laboratory, St. Aloysius College, Mangalore 575 003, India 2 3–6–15 Hikarigaoka, Munakata, Fukuoka 811–3403, Japan Received December 21, 2010; accepted February 21, 2011 Summary Karyotypes of 5 species of Indian frogs belonging to 2 families Rhacophoridae (Polypedates maculatus, Rhacophorus malabaricus, and Pseudophilautus wynaadensis) and Microhylidae (Ramanella montana and Microhyla ornata) were described. All of the 5 species had 2nϭ26 bi-armed chromosomes with 5 large and 8 small pairs. In Rhacophorus malabaricus, 3 large chromosome pairs (nos. 2–4) possessed telomeric C-positive bands on the long arm, whereas C-positive bands were confined to the centromeric region of the chromosomes in the other 4 species. None of the species had identifiable heteromorphic sex chromosomes. Based on karyometric data and C-banding, the karyotype of each species was characterized and was compared with those of the related species. It became evident that the Indian species of the genus Microhyla differ karyologically from the congeners in southeastern Asia. Key words Karyotype, C-banding, Rhacophoridae, Microhylidae, India. In the past 6 decades, 24 species of Indian anurans were studied for their cytogenetic attributes. Of these, 3 species belong to Bufonidae (Asana and Mahabale 1941, Natarajan 1958b, Manna and Bhunya 1966, Chatterjee and Barik 1970, Singh et al. 1970, Mittal and Sawhney 1971, Singh 1974), 9 to Dicroglossidae (Natarajan 1957b, 1958a, Singh et al. 1970, Mittal and Sawhney 1971, Singh 1974, Yadav 1974, Yadav and Pillai 1975, 1976, Chakrabarti et al. 1983, Joshy et al. 1999, Joshy and Kuramoto 2008), 1 to Nyctibatrachidae (Joshy et al. 1999), 1 to Ranixalidae (Joshy et al. 1999), 3 to Ranidae (Joshy et al. 1999, 2006), 3 to Rhacophoridae (Natarajan 1957a, Singh et al. 1970, Singh 1974, Rai and Dey 1991), and 4 to Microhylidae (Bai 1956, Chakrabarti 1979, Olmo and Morescalchi 1978). Most of the previous studies, however, lacked numerical data on size and shape of chromosomes, and this makes karyotype comparisons between taxa very difficult or even impossible. In the present study, we describe karyotypes of 3 species of Rhacophoridae and 2 species of Microhylidae from the Western Ghats. Of the 5 species examined, the karyotype of Polypedates maculatus was previously reported by Natarajan (1957a), Singh et al. (1970) and Singh (1974) without karyometric data. The karyotypes of the other 4 species are here reported for the first time. Materials and methods Three species belonging to Rhacophoridae, Polypedates maculatus, Rhacophorus malabaricus, and Pseudophilautus wynaadensis, were collected from Madikeri (altitude ca. 900 m) of Kodagu District, Karnataka, and 2 species belonging to Microhylidae, Ramanella montana and Microhyla ornata, were collected from Sagar (altitude ca. 560 m) of Shimoga District, Karnataka. Both * Corresponding author, e-mail: [email protected] 112 S. H. Joshy and M. Kuramoto Cytologia 76(2) collecting sites are situated in the Western Ghats, one of the hotspots of biodiversity in the world, and Rhacophorus malabaricus, Pseudophilautus wynaadensis and Ramanella montana are endemic to this mountainous region. Generic names Polypedates, Rhacophorus, Pseudophilautus, Ramanella and Microhyla are abbreviated below as Po., Rh., Ps., Ra. and M., respectively. Specimens were injected with 0.1% colchicine solution, killed after 17 h of injection, and chromosome spreads were prepared from bone marrow cells of femur. Chromosomes were stained with Giemsa’s solution, and C-bands on chromosomes were stained using the method of Sumner (1972). Chromosomes were measured using dial calipers on enlarged photomicrographs and chromosome pairs were arranged in the order of decreasing length. Types of chromosomes based on the position of centromeres followed Levan et al. (1964). Results The 3 rhacophorid species examined, Po. maculatus, Rh. malabaricus, and Ps. wynaadensis, had 2nϭ26 chromosomes with FNϭ52 (Figs. 1A, 1B and 1C). The chromosome complement consisted of 5 pairs of large (nos. 1–5) and 8 pairs of small sized chromosomes (nos. 6–13); the difference in relative length between 2 successive chromosome pairs was by far the largest between no. 5 and no. 6 in all 3 species. All chromosome pairs were either metacentric or submetacentric pairs; there were no subtelocentric nor telocentric pairs (Table 1). C-banding karyotype of Rh. malabaricus (Fig. 2B) revealed that, apart from centromeric bands, 3 pairs of large chromosomes (nos. 2–4) possessed telomeric C-bands on the long arm, whereas C-positive bands occurred only in the centromeric region of chromosomes in Po. maculatus and Ps. wynaadensis (Figs. 2A and 2C). No heteromorphic sex chromosomes were recognized in the 3 species examined. The karyotypes of 3 species of Rhacophoridae are very similar in pair nos. 2, 3 and 5, and in most of small pairs. Distinct differences are recognized in no. 4 of Po. maculatus, no. 6 of Rh. malabaricus, and nos. 9 and 11 of Ps. wynaadensis, which have much smaller centromeric index (Table 1, Fig. 3A). A considerable species difference in centromeric index is also recognized in pair no. 1. Table 1. Relative length (RL) and centromeric index (CI) of chromosomes in 5 anuran species (meanϮSD). Abbreviation m, sm, and st indicate metacentric, submetacentric, and subtelocentric, respectively, and asterisks show chromosomes with C-positive satellite region Species 123456 Polypedates RL 14.14Ϯ0.39 12.34Ϯ0.17 11.26Ϯ0.18 10.43Ϯ0.18 10.03Ϯ0.02 7.44Ϯ0.13 maculatus CI 47.10Ϯ1.00 36.62Ϯ1.82 34.94Ϯ2.46 33.65Ϯ1.15 43.36Ϯ1.40 42.17Ϯ0.19 Typemsmsmsmm m Rhacophorus RL 16.18Ϯ0.77 12.94Ϯ0.49 11.98Ϯ0.88 11.02Ϯ0.07 9.97Ϯ0.02 5.61Ϯ0.23 malabaricus CI 38.46Ϯ2.01 33.45Ϯ2.26 32.92Ϯ1.98 41.77Ϯ0.03 43.39Ϯ2.07 36.98Ϯ0.09 Type sm sm* sm* m* m sm Pseudophilautus RL 15.48Ϯ0.19 12.93Ϯ0.16 11.83Ϯ0.49 11.37Ϯ0.26 10.20Ϯ0.28 6.05Ϯ0.49 wynaadensis CI 44.45Ϯ1.51 34.33Ϯ0.56 33.88Ϯ0.63 46.01Ϯ0.84 44.02Ϯ0.14 46.31Ϯ2.29 Type m sm sm m m m Ramanella RL 14.44Ϯ1.09 11.74Ϯ0.42 11.32Ϯ0.44 10.59Ϯ0.73 9.11Ϯ0.24 6.28Ϯ0.23 montana CI 44.28Ϯ0.66 36.75Ϯ0.48 31.02Ϯ1.98 36.11Ϯ1.59 43.08Ϯ1.94 40.74Ϯ2.78 Typemsmsmsmm m Microhyla RL 15.95Ϯ1.45 13.34Ϯ0.77 11.53Ϯ0.49 10.44Ϯ0.18 9.32Ϯ0.06 7.05Ϯ0.74 ornata CI 44.42Ϯ2.21 44.55Ϯ0.05 33.71Ϯ2.63 32.88Ϯ0.98 42.57Ϯ1.63 43.31Ϯ1.99 Type m m sm sm m m 2011 Karyotypes of 5 Indian Frogs 113 The 2 species of Microhylidae, Ra. montana and M. ornata, had 2nϭ26 chromosomes with 5 large and 8 small chromosome pairs (Figs. 1D and 1E). The difference in relative length between no. 5 and no. 6 was larger than any other 2 successive chromosome pairs, except that between no. 1 and no. 2 in M. ornata. Most chromosome pairs were metacentric or submetacentric, and only pair no. 11 of Ra. montana was subtelocentric (Table 1). C-band analysis indicated that only centromeric regions were C-positive (Figs. 2D and 2E). No heteromorphic sex chromosomes were recognized. The karyotypes of the 2 microhylids are similar in pair nos. 1, 3, 4, 5, 6, 8, 10 and 12. Fig. 1. Karyotypes of Polypedates maculatus (A), Rhacophorus malabaricus (B), Pseudophilautus wynaadensis (C), Ramanella montana (D), and Microhyla ornata (E). An arrow indicates subtelocentric pair. Table 1. (continued) 7 8 9 10111213 6.73Ϯ0.14 5.94Ϯ0.74 5.80Ϯ0.69 5.43Ϯ0.40 5.13Ϯ0.66 4.24Ϯ0.84 3.81Ϯ0.41 43.49Ϯ2.13 45.59Ϯ3.05 43.74Ϯ1.05 45.50Ϯ0.91 44.27Ϯ2.11 43.74Ϯ1.14 46.14Ϯ2.14 m m mmmmm 5.41Ϯ0.38 5.10Ϯ0.22 4.99Ϯ0.11 4.60Ϯ0.11 4.32Ϯ0.33 4.02Ϯ0.38 3.83Ϯ0.44 44.56Ϯ3.20 43.17Ϯ1.38 46.74Ϯ0.93 44.81Ϯ2.42 44.53Ϯ2.05 42.41Ϯ1.89 45.89Ϯ0.09 m m mmmmm 5.85Ϯ0.24 5.27Ϯ0.16 4.90Ϯ0.12 4.71Ϯ0.14 4.18Ϯ0.33 3.95Ϯ0.98 3.20Ϯ0.04 47.63Ϯ1.91 47.21Ϯ1.63 33.78Ϯ3.36 45.43Ϯ2.02 34.45Ϯ3.22 44.13Ϯ1.79 45.06Ϯ1.40 mmsmmsmmm 6.01Ϯ0.21 5.66Ϯ0.45 5.13Ϯ0.36 5.11Ϯ0.17 4.81Ϯ0.19 4.60Ϯ0.14 4.19Ϯ0.28 44.19Ϯ0.22 44.80Ϯ1.24 46.74Ϯ2.21 46.51Ϯ2.71 23.19Ϯ0.54 45.84Ϯ0.27 44.67Ϯ2.47 mm mmstmm 5.90Ϯ0.04 5.61Ϯ0.06 5.03Ϯ0.31 4.79Ϯ0.50 4.33Ϯ0.67 3.94Ϯ0.36 3.31Ϯ0.26 33.03Ϯ0.15 44.60Ϯ2.46 31.65Ϯ3.18 47.23Ϯ0.84 46.13Ϯ3.67 43.72Ϯ3.58 36.59Ϯ0.46 sm m sm m m m sm 114 S. H. Joshy and M. Kuramoto Cytologia 76(2) Fig. 2. C-banded karyotypes of Polypedates maculatus (A), Rhacophorus malabaricus (B), Pseudophilautus wynaadensis (C), Ramanella montana (D), and Microhyla ornata (E). Arrows indicate the chromosomes with non-centromeric C-positive band in B and subtelocentric pair in D. Fig. 3. Scatter diagrams showing karyotypic differences (A) between Polypedates maculatus (open square), Rhacophorus malabaricus (solid circle), and Pseudophilautus wynaadensis (open triangle), and (B) between Ramanella montana (open square) and Microhyla ornata (solid circle). Figures indicate pair numbers, and pairs encircled by lines in A are nos. 2 and 3 (left) and no. 5 (right) and those in B are no. 1 (upper right), nos. 3 and 4 (center), and no.