© 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 2n 26 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 2n 26 chromosomes with FN 52 (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 (mean SD). Abbreviation m, sm, and st indicate metacentric, submetacentric, and subtelocentric, respectively, and asterisks show chromosomes with C-positive satellite region
Species 123456
Polypedates RL 14.14 0.39 12.34 0.17 11.26 0.18 10.43 0.18 10.03 0.02 7.44 0.13 maculatus CI 47.10 1.00 36.62 1.82 34.94 2.46 33.65 1.15 43.36 1.40 42.17 0.19 Typemsmsmsmm m
Rhacophorus RL 16.18 0.77 12.94 0.49 11.98 0.88 11.02 0.07 9.97 0.02 5.61 0.23 malabaricus CI 38.46 2.01 33.45 2.26 32.92 1.98 41.77 0.03 43.39 2.07 36.98 0.09 Type sm sm* sm* m* m sm
Pseudophilautus RL 15.48 0.19 12.93 0.16 11.83 0.49 11.37 0.26 10.20 0.28 6.05 0.49 wynaadensis CI 44.45 1.51 34.33 0.56 33.88 0.63 46.01 0.84 44.02 0.14 46.31 2.29 Type m sm sm m m m
Ramanella RL 14.44 1.09 11.74 0.42 11.32 0.44 10.59 0.73 9.11 0.24 6.28 0.23 montana CI 44.28 0.66 36.75 0.48 31.02 1.98 36.11 1.59 43.08 1.94 40.74 2.78 Typemsmsmsmm m
Microhyla RL 15.95 1.45 13.34 0.77 11.53 0.49 10.44 0.18 9.32 0.06 7.05 0.74 ornata CI 44.42 2.21 44.55 0.05 33.71 2.63 32.88 0.98 42.57 1.63 43.31 1.99 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 2n 26 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
6.73 0.14 5.94 0.74 5.80 0.69 5.43 0.40 5.13 0.66 4.24 0.84 3.81 0.41 43.49 2.13 45.59 3.05 43.74 1.05 45.50 0.91 44.27 2.11 43.74 1.14 46.14 2.14 m m mmmmm
5.41 0.38 5.10 0.22 4.99 0.11 4.60 0.11 4.32 0.33 4.02 0.38 3.83 0.44 44.56 3.20 43.17 1.38 46.74 0.93 44.81 2.42 44.53 2.05 42.41 1.89 45.89 0.09 m m mmmmm
5.85 0.24 5.27 0.16 4.90 0.12 4.71 0.14 4.18 0.33 3.95 0.98 3.20 0.04 47.63 1.91 47.21 1.63 33.78 3.36 45.43 2.02 34.45 3.22 44.13 1.79 45.06 1.40 mmsmmsmmm
6.01 0.21 5.66 0.45 5.13 0.36 5.11 0.17 4.81 0.19 4.60 0.14 4.19 0.28 44.19 0.22 44.80 1.24 46.74 2.21 46.51 2.71 23.19 0.54 45.84 0.27 44.67 2.47 mm mmstmm
5.90 0.04 5.61 0.06 5.03 0.31 4.79 0.50 4.33 0.67 3.94 0.36 3.31 0.26 33.03 0.15 44.60 2.46 31.65 3.18 47.23 0.84 46.13 3.67 43.72 3.58 36.59 0.46 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. 5 (lower right).
Remarkable differences are recognized in no. 11 of Ra. montana and nos. 7, 9 and 13 of M. ornata, which have distinctly smaller centromeric index (Table 1, Fig. 3B). Centromeric index of no. 2 differs considerably between the 2 species.
Discussion Currently, about 30 species of Rhacophoridae have been karyotyped (King 1990, Kuramoto 1990), including the genera Buergeria, Chiromantis, Kurixalus, Polypedates, Pseudophilautus, Rhacophorus and Theroderma. All have 2n 26 chromosomes without exception and the karyotypes are mostly composed of 5 large and 8 small metacentric or submetacentric pairs. The 3 Indian species examined in the present study showed this general pattern of the karyotype of Rhacophoridae. Non-centromeric C-bands were not found in Po. maculatus as reported by Singh (1974). The 2011 Karyotypes of 5 Indian Frogs 115 karyotypes of Po. leucomystax from Malaysia (Matsui et al. 1986, Kuramoto and Yong 1992) and Po. megacephalus from Taiwan (Matsui et al. 1986) are characterized by the relatively large size of pair no. 6, obscuring the dichotomy of 5 large and 8 small chromosome pairs. It is an interesting problem to confirm whether Indian members of Polypedates have general rhacophorid karyotype and southeast Asiatic members have karyotypes with relatively large no. 6 pair. Since only 3 Polypedates species mentioned above have been karyotyped, this problem remains for future studies. Karyometric data of Rh. maximus from Darjeeling (Rai and Dey 1991) are available for comparison with those of Rh. malabaricus. Although the pair numbers of metacentric and submetacentric pairs differ between the 2 species, general pattern of the 2 karyotypes are similar. Pair no. 2 in Rh. maximus was argued to be heteromorphic in males (Rai and Dey 1991); one of the homologue has a secondary constriction in the short arm while the other lacks it. Because secondary constrictions are often observed in only 1 complement of the pair, this heteromorphism seems to need future confirmation. King (1990) summarized the sites of secondary constrictions in chromosomes of Rhacophorus. They have been observed in one of the small chromosome pairs; no. 12 in Rh. arboreus, no. 11 in Rh. chenfui, no. 7 in Rh. schlegelii, and no. 11 in Rh. taipeianus. The non-centromeric C-bands in Rh. malabaricus represent secondary constrictions, and this rhacophorid, and Rh. maximus, too, are exceptional in having secondary constrictions on large chromosome pairs. The karyotype of Ps. annandalii from Darjeeling (Rai and Dey 1991) differs remarkably from that of Ps. wynaadensis in having a telocentric pair no. 10 and a heteromorphic pair, possibly XY sex chromosomes, in males. Pseudophilautus is the largest genus in the Rhacophoridae involving 106 species (Frost 2010), but no other congeners have been karyotyped. Future studies are expected to reveal karyological divergence in this genus. Previously, above 2 Pseudophilautus species were allocated to the genus Philautus, together with Theroderma asperum and Chiromantis doriae. Theroderma asperum from Peninsular Malaysia (Kuramoto and Yong 1992, as Philautus asper) exhibited a distinct heterochromatic band on the proximal region of the short arm of pair no. 6. In 2n 26 karyotype of Chiromantis doriae from China (Tan 1987, as Philautus doriae), no. 12 pair has a satellite at the terminal of the long arm. These heterochromatic bands are absent in Ps. wynaadensis and Ps. annandahlii. The Microhylidae is a very diverse family consisting of 11 subfamilies (Frost 2010). Differing from the Rhacophoridae with rather similar karyotypes with 2n 26 bi-armed chromosomes, diploid numbers of Microhylidae range from 2n 22 to 2n 28, excepting aberrant numbers of 30, 32, 48 and 52 (King 1990, Kuramoto 1990). The 2n 48 and 2n 52 karyotypes are of polyploid species. Four Indian species of Microhylidae previously karyotyped, Ra. variegata (Bai 1956), M. rubra (Bai 1956), Uperodon globulosus (Chakrabarti 1979), and U. systoma (Olmo and Morescalchi 1978), belong to the subfamily Microhylinae. All of these species have 2n 26 chromosomes, and Ra. montana and M. ornata examined in the present study had the same karyotypic constitution. Because the 2n 26 karyotype dominates and is most widely distributed in the Microhylidae, this number seems to be the basic diploid number of this family. Ramanella montana had 2n 26 chromosomes as in Ra. variegata (Bai 1956), but the karyotypic comparisons are impossible since the karyometric data of the latter are lacking. Similarly, detailed karyological studies are needed to elucidate the cytogenetic comparisons of the genus Uperodon. The genus Microhyla occurs widely over southeastern Asia. Curiously, southeastern Asiatic members of the genus Microhyla have been reported to have 2n 24 or 2n 22 chromosomes (King 1990, Kuramoto 1990, Kuramoto and Yong 1992, Supaprom and Baimai 2002). Microhyla okinavensis from the Ryukyu Archipelago, Japan, and M. fissipes from China, with 2n 24 chromosomes, have long been confused with M. ornata, because the external morphology is very 116 S. H. Joshy and M. Kuramoto Cytologia 76(2) similar to each other. Microhyla berdmorei from Thailand (Supaprom and Baimai 2002), M. heymonsi from China and Malaysia (Gao et al. 1985, Guo and Dong 1987, Kuramoto and Yong 1992), and M. pulchra from China and Thailand (Zheng and Wu 1995, Supaprom and Baimai 2002) have 2n 24 chromosomes, and M. butleri from China, Thailand and Malaysia (Kuramoto and Yong 1992, Zheng and Wu 1995, Supaprom and Baimai 2002) has 2n 22 chromosomes. Apparently, the southeastern Asiatic members of the genus Microhyla have derived from Indian members with the basic 2n 26 karyotype of the Microhylidae. In the 2n 24 karyotype of Microhyla, the pair no. 6 is relatively large, suggesting the reduction of chromosome number resulted from chromosome fusion. Secondary constrictions are present on pair no. 8 in M. berdmorei, and no. 7 in M. pulchra (Supaprom and Baimai 2002), whereas we could not detect non-centromeric C-band in M. ornata. Reexamination on the karyotype of M. rubra is needed, because the previous work on this species (Bai 1956) was carried out using histological technique, and therefore detailed karyological comparisons are impossible.
Acknowledgments We thank Rev. Father Swebert D’Silva, Principal of St. Aloysius College, and Rev. Father Leo D’Souza, Director of Applied Biology Laboratory, for providing facilities. Thanks are due to the Mangalore Jesuit Educational Society for support and encouragement.
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