© 2008 The Japan Mendel Society Cytologia 73(3): 243–250, 2008

Comparative Chromosome Studies of Five of the Fejervarya (Anura: Ranidae) from South

S. Hareesh Joshy1,*, and Mitsuru Kuramoto2

1 Rondano Research Laboratory, St. Aloysius College, Mangalore-575 003, India 2 3-6-15 Hikarigaoka, Munakata, Fukuoka 811-3403, Japan

Received December 26, 2007; accepted July 15, 2008

Summary The karyotypic and C-banding analysis of five species of Indian belonging to the genus Fejervarya (F. cf. limnocharis, F. cf. brevipalmata, F. cf. keralensis, F. rufescens and F. sahyadris), which are distributed in the , Southwestern India, was carried out. All species had 2n26 chromosomes with five large and eight small pairs. Most chromosomes were metacentric or submetacentric, and only no. 9 pair of F. cf. brevipalmata was subtelocentric chromo- some. Nos. 1, 12 and 13 were metacentric and no. 3 was submetacentric in all the species analyzed. In F. cf. brevipalmata, an achromatic gap was present in the pericentric region of no. 6 chromosome. The chromosome pair no. 11 of F. cf. keralensis had a secondary constriction with a prominent satel- lite in the short arm. In three of the five species, C-positive regions were confined to the centromere of each chromosome, but F. rufescens and F. sahyadris showed non-centromeric C-bands on both ends of three pairs of large chromosomes, in addition to the centromeric C-bands. None of the species had identifiable sex chromosomes. The results are compared with karyotypes of the other Fe- jervarya species and the possible causes for the inter-specific karyological divergence are discussed.

Key words Fejervarya, Karyotype, C-banding, Western Ghats

The Western Ghats region of southwestern India is considered as one of the 25 “hottest hotspots” of biodiversity in the world (Myers et al. 2000). Among 224 species of known from India, 117 occur in the Western Ghats, 89 being endemic to this region (Daniels 1992). Biju (2001) suggested the existence of many undescribed species in the Western Ghats. Cytogenetic studies of anurans are interesting because of the apparent karyotypic conservation and divergence, but cytogenetic information on the anurans of the Western Ghats is extremely scanty. Among the Indian frogs of Ranidae (sensu Duellman 2003), only 12 species have been cyto- genetically investigated. The karyotype of tigerinus (as tigrina) was studied by Asana and Kharodi (1937), Sharma et al. (1966), Yadav and Pillai (1975), and Chakrabarti et al. (1983). The karyotype of cyanophlyctis (as Rana cyanophlyctis) was described from South India (Natarajan 1958), from Punjab (Mittal and Sawhney 1971) and from Chandigarh (Yadav and Pillai 1975), and the karyotype of E. hexadactylus (as Rana hexadactyla) was described by Natarajan (1958). The karyotypes of Rana temporalis, R. curtipes, R. malabarica, Fejervarya cf. brevipalmata (as Rana brevipalmata), F. cf. keralensis (as Rana keralensis), beddomii (as Rana beddomii) and major were reported by Joshy et al. (1999, 2006). Vences et al. (2000) reported the karyotypes of Indirana sp. and Indirana cf. leptodactyla. Since only a few cytogenetic studies have been performed on the genus Fejervarya, a compar- ative karyological study of five species from the Western Ghats was carried out in the present study. C-banding data for Fejervarya species are here reported for the first time. The genus Fejervarya is distributed widely from India to and Japan. Although 11

* Corresponding author, e-mail: [email protected] 244 S. H. Joshy and M. Kuramoto Cytologia 73(3)

Fejervarya species were reported to occur in the Western Ghats (Frost 2007), it is almost certain that many undescribed species exist there. We collected frogs similar to F. brevipalmata and F. keralensis but our detailed comparisons with museum specimens revealed considerable differences between them. Indian “F. limnocharis” was proved to be a different taxon from F. limnocharis from Java (type locality of Rana limnocharis) (Biju 2001, Kurabayashi et al. 2005, Djong et al. 2007). Thus, we use the names F. cf. brevipalmata, F. cf. keralensis and F. cf. limnocharis for these taxa, respectively. Dubois et al.. (2001) described Minervarya sahyadris from the Western Ghats. Be- cause this taxon was completely involved within the genus Fejervarya by our unpublished mtDNA comparisons, we treat this species as a member of Fejervarya.

Materials and methods Fejervarya cf. limnocharis and F. cf. brevipalmata were collected in Sullia, Dakshina Kannada District, F. cf. keralensis in Madikeri, Kodagu District, and F. rufescens and F. sahyadris in Sagar, Shimoga District, all in Karnataka State. Metaphase chromosome spreads were prepared from bone marrow cells by conventional air- dry method of Omura (1967) with slight modifications. The individual frogs were intraperitoneally injected with 0.2 ml to 1.5 ml of 0.3% colchicine and were sacrificed after 14–16 h. For the hypo- tonic treatment, dilute KCl solution (0.4%) was applied for 30 to 40 min. The method of Sumner (1972) was followed for C-band localization. The diploid chromosome number was determined by observing more than 100 metaphase plates for each species. Chromosomes were measured with a digitizer on enlarged photomicrographs. Chromosome pairs were numbered in the order of decreas- ing mean relative length, and centromeric position was designated based on Levan et al. (1964).

Results Fejervarya cf. limnocharis The diploid number was 2n26 (Fig. 1A). All chromosomes were bi-armed with the FN of 52. Chromosome pairs were clearly divided into two size groups, five pairs of large and eight pairs of small chromosomes. The chromosome complement consisted of eight pairs of metacentric (nos. 1, 5, 6, 7, 8, 9, 12 and 13) and five pairs of submetacentric (nos. 2, 3, 4, 10 and 11) chromosomes (Table 1). The centromeric regions of all the chromosomes were stained C-positively (Fig. 2A). No sex chromosomes were detected.

Fejervarya cf. brevipalmata The diploid chromosome complement as determined from the somatic metaphase plates of both sexes was 2n26. Five pairs of large and eight pairs of small bi-armed chromosomes were clearly distinguished (Fig. 1B). Hence the FN52. In terms of centromeric position, nos. 1, 2, 4, 5, 7, 8, 11, 12 and 13 were metacentric, nos. 3, 6 and 10 were submetacentric, and no. 9 was subtelo- centric (Table 1). Either a heteromorphic or homomorphic achromatic gap was present close to the centromere in the long arm of no. 6 pair (Fig. 1B). The C-banded chromosome complements clear- ly showed that the C-positive bands occurred only in the centromeric region of each chromosome (Fig. 2B). No sex chromosomes were detected.

Fejervarya cf. keralensis The observation of metaphase chromosomes in both sexes of this species showed 2n26 chro- mosomes (Fig. 1C). All chromosomes were bi-armed and the FN was 52. The chromosomes were classified into two size groups. The first group consisted of five pairs of large chromosomes (nos. 1–5) and the second group consisted of eight pairs of small chromosomes (nos. 6–13). Chromo- 2008 Comparative Chromosome Studies of Five Species of the Genus Fejervarya 245 0.34 m 1.26 m 1.63 m 0.21 sm 1.63 m 0.54 m 1.68 sm 0.19 m 0.29 m 0.01 sm 0.21 sm 0.01 sm 0.54 m 2.17 m 47.29 2.51 m 44.86 0.56 sm 43.28 0.88 m 34.30 0.27 sm 44.44 3.07 m 47.44 2.24 m 35.39 0.69 m 48.06 0.25 sm 46.11 1.45 sm 37.11 0.78 sm 35.10 0.24 sm 36.36 0.23 m 42.86 rufescens sahyadris F. F. SD) 0.19 m 46.31 0.11 m 44.37 0.22 sm* 34.99 0.25 sm 46.33 2.8 sm 34.91 0.7 sm 43.16 1.8 m 38.44 1.8 m 43.85 0.54 m 36.22 2.80 m 34.68 0.99 sm 35.19 0.80 m 36.90 0.10 m 44.05 cf. F. ejervarya F with C-positive satellite region. with C-positive 1.09 m 46.02 2.78 m 44.12 0.29 m 36.12 1.19 sm 33.13 0.76 st* 34.22 1.63 m 34.77 1.94 m 46.22 1.85 sm* 43.15 0.61 m 46.05 0.31 m 44.31 0.13 sm 33.03 0.53 m 39.54 0.97 m 48.49 cf. F. 1.46 m 46.89 1.49 m 45.19 0.04 sm 46.95 0.62 sm 34.52 1.35 m 23.36 1.09 m 46.11 1.06 m 45.78 0.23 m 35.13 0.03 m 44.26 0.25 sm 40.54 0.05 sm 33.54 0.26 sm 41.80 1.10 m 43.21 cf. F. 0.45 46.56 0.58 45.57 0.42 36.95 0.52 35.34 0.46 44.57 0.35 45.69 0.37 42.48 0.66 44.35 0.44 44.02 0.42 36.32 0.24 37.10 0.57 35.83 0.58 46.49 0.33 4.14 0.23 4.39 0.23 4.95 0.32 5.12 0.43 5.38 0.38 5.60 0.39 5.84 0.59 6.77 0.48 10.05 0.47 10.81 0.43 11.52 0.16 12.48 0.79 12.80 rufescens sahyadris F. F. SD) (mean Centromeric index 0.21 4.03 0.17 4.24 0.33 4.61 0.33 5.04 0.27 5.40 0.29 5.65 0.29 6.27 0.23 7.03 0.48 9.56 0.62 10.48 0.14 11.63 0.65 11.96 0.68 14.01 Relative length and centromeric index of chromosomes in five species of the genus of chromosomes in five length and centromeric index Relative cf. F. Table 1. 0.42 3.85 0.16 4.17 0.21 4.56 0.27 4.69 0.33 5.06 0.17 5.37 0.09 5.65 0.15 6.52 0.39 9.89 0.25 10.82 0.49 11.53 0.49 12.57 0.47 15.48 cf. F. 0.24 3.96 0.30 4.44 0.27 4.73 0.24 4.96 0.37 5.16 0.29 5.47 0.25 5.65 0.24 5.91 0.34 9.62 0.32 10.37 0.28 11.15 0.59 12.86 0.81 15.77 cf. F. 5.02 5.34 5.67 6.06 10.40 10.94 11.57 13.14 15.31 limnocharis brevipalmata keralensis limnocharis brevipalmata keralensis 9 8 7 6 5 4 3 2 1 13 3.66 12 4.10 11 4.61 10 4.82 No. Abbreviations m, sm and st indicate metacentric, submetacentric and subtelocentric, respectively and asterisk shows chromosome and asterisk shows m, sm and st indicate metacentric, submetacentric subtelocentric, respectively Abbreviations Oaur length (mean Relative 246 S. H. Joshy and M. Kuramoto Cytologia 73(3)

Fig. 1. Non-banded karyotypes of Fejervarya cf. limnocharis (A), F. cf. brevipalmata (B), F. cf. keralensis (C), F. rufescens (D) and F. sahyadris (E). Arrows indicate the chromosomes with a secondary con- striction.

Fig. 2. C-banded karyotypes of Fejervarya cf. limnocharis (A), F. cf. brevipalmata (B), F. cf. keralensis (C), F. rufescens (D) and F. sahyadris (E).

some pairs 1, 2, 4, 5, 6, 7, 12 and 13 were metacentric and the remaining five pairs were submeta- centric (Table 1). The pair no. 11 exhibited the presence of a satellite region in the short arm (Fig. 1C). As seen in the congeneric species mentioned above, the C-positive bands were associated with centromeric regions of chromosomes (Fig. 2C). No sex chromosomes were identified. 2008 Comparative Chromosome Studies of Five Species of the Genus Fejervarya 247

Fig. 3. Karyotypic comparison of Fejervarya cf. limnocharis (A), F. cf. brevipalmata (B), F. cf. keralensis (C), F. rufescens (D) and F. sahyadris (E). Arrows indicate the chromosomes with a secondary constriction.

Fejervarya rufescens In this species, the diploid chromosome number was 2n26 in both sexes (Fig. 1D). The chro- mosome complement could be divided into five large and eight small pairs. All chromosomes were bi-armed and FN52. Based on the localization of centromers, the chromosome complement con- sisted of seven pairs of metacentric (nos. 1, 6, 7, 8, 10, 12 and 13) and six pairs of submetacentric chromosomes (nos. 2, 3, 4, 5, 9 and 11) (Table 1). The centromeric regions of all the chromosomes were stained C-positively (Fig. 2D). In addition to the centromeric C-bands, non-centromeric C- bands were observed in the telomeric regions of long and short arms of pair nos. 1, 2, 4 and 5. No sex chromosomes were observed.

Fejervarya sahyadris The diploid chromosome number, 2n26, was the same as the other species of the genus ana- lyzed above (Fig. 1E). The similarity extended also to the bi-armed nature of the chromosomes. The chromosomes could be divided into two groups, five pairs of large and eight pairs of small chromo- somes. Chromosome nos. 1, 5, 6, 8, 9, 11, 12 and 13 were metacentric and nos. 2, 3, 4, 7 and 10 were submetacentric (Table 1). Analysis of C-banded karyotypes revealed that the C-positive hete- rochromatin was localized in the centromeric regions of each chromosome (Fig. 2E). In the telom- eric regions of long and short arms of pair nos. 1, 2, 3 and 5, non-centromeric C-bands were seen. No sex chromosomes were detected.

Discussion All the five species examined herein had 2n26 chromosomes which were classified into two distinct size groups, five large chromosome pairs (nos. 1–5) and eight small chromosome pairs (nos. 6–13) (Fig. 1). All the chromosomes were bi-armed, hence FN52. Karyomorphological, as well as C-banding, analysis did not show any differences between the sexes. The karyotypes are rather similar in all the five species of Fejervarya (Fig. 3), and represent the common karyotype of the family Ranidae in general (e.g. King 1990). Supapron and Baimai (2004) reported the karyotype of F. limnocharis from (as Lim- 248 S. H. Joshy and M. Kuramoto Cytologia 73(3) nonectes limnocharis). Lin and Huang (1979) and He (1986) reported the karyotype of F. multistri- ata from Taiwan and Guangdong, respectively (both as Rana limnocharis). Kuramoto (1971) re- ported karyotypes of F. cf. limnocharis (as Rana limnocharis limnocharis) from Japan and F. vittig- era (as Rana limnocharis vittigera) from Philippines. All of these Southeastern and Eastern Asiatic Fejervarya species had 2n26 chromosomes with five large and eight small pairs. According to Iskandar (personal communication), the karyological characteristics of F. limnocharis and F. can- crivora from Java, Indonesia (type locality of both species) are the same as those of the species mentioned above. Sex chromosomes have not been reported in all the Fejervarya species analyzed. In F. limnocharis from Thailand, the secondary constriction was found on the short arm of no. 8 in the vicinity of centromere (Supapron and Baimai 2004). The secondary constriction was locat- ed in the short arm of no. 6 in the Taiwan population (Lin and Huang 1979) and in the short arm of no. 7 in the Guangdong population (He 1986) of F. multistriata. In F. cf. limnocharis from Japan, the secondary constriction was located on the short arm of no. 7, and in F. vittigera on the long arm of no. 9. In view of the localization of the secondary constriction, it seems highly probable that pair no. 8 of F. limnocharis from Thailand, no. 6 of F. multistriata from Taiwan, no. 7 of F. multistriata from Guangdong, and no. 7 of F. cf. limnocharis from Japan are homologous and may have a com- mon origin. Chromosome nos. 1, 12 and 13 were consistently metacentric chromosomes and no. 3 was submetacentric in the five Indian species, and also in the five east Asiatic species mentioned above. This indicates that these chromosomes were conserved during karyotypic divergence of the genus. The remaining chromosomes, however, have been subjected to various structural changes in the course of speciation. The only species (among Indian and east Asiatic Fejervarya species) that ex- hibited a subtelocentric pair (no. 9) was F. cf. brevipalmata. The no. 6 chromosome of this species was also characterized by the presence of an achromatic region in the proximal part of the long arm. The no. 11 chromosome of F. cf. keralensis is a marker with a distal satellite region in the short arm. So, it is clear that there is inter-specific variation, not only with regard to centromeric positions but also in localization of satellite regions or secondary constrictions. In addition to the common occurrence of the 2n26 karyotype with five large and eight small pairs in Ranidae, King (1990) pointed out the common localization of a secondary constriction on one of the small chromosomes, mostly on nos. 9–11. These characteristics represent the conserva- tive nature of the karyotypes. Miura (1995), comparing the late-replication banding pattern, demon- strated chromosomal homology of the genera Rana, Hyla and Bufo, belonging to different families. In cases where the chromosome number deviates from 2n26, a possible derivation of the karyotype from the basic 2n26 karyotype can be inferred based on karyometric comparisons. Vences et al. (2000) reported the karyotypic organization of Indirana species. They derived the karyotype of Indirana cf. leptodactyla (2n24) from that of Indirana sp. (2n30) by a process in- volving three Robertsonian fusions and a pericentric inversion. However, just the reverse process that the 2n30 karyotype of Indirana sp. has been derived from the 2n24 karyotype by chromo- somal fissions seems to be more probable because the former (2n30) should be regarded as a de- rived state. According to King (1990), the most probable course of chromosome reduction reported in some species of Ranidae, that is, from 2n26 to 24, 22 or 20, involves two steps; telocentric chromosome production by inversion and subsequent chromosome fusion of the derived telo- centrics. Karyological variation between species with the same chromosome number, as in the present study, should be interpreted as a result of minute chromosomal rearrangements. C-banding and late- replication banding karyological studies on Rana rugosa revealed that the W chromosome could be reconstructed from the Z chromosome by two pericentric inversions (Nishioka et al. 1993). Based on G-banding pattern of chromosomes of Buergeria buergeri (Rhacophoridae), Hanada (2002) con- cluded that the Z chromosome has been derived from the W by a single translocation. Odierna et al. 2008 Comparative Chromosome Studies of Five Species of the Genus Fejervarya 249

(2007) detected an inverted segment on the sex chromosomes of the green toad Bufo viridis from Moldavia by late-replication banding pattern. These kind of banding pattern comparisons can be applied to studies for elucidating the inter-specific karyological differences. Future banding pattern analysis will reveal structural changes of chromosomes in the course of speciation of the genus Fe- jervarya. The C-banding analysis in many ranid species have shown the common occurrence of C- positive bands on the centromeric region. Prominent non-centromeric C-bands were reported in both ends of no. 1, telomeric region of the long arm of no. 2 and of the short arm of nos. 3 and 5 chromosomes in R. ridibunda, R. lessonae and hybridogenetic R. esculenta (Heppich 1978). The telomeric C-bands were observed in several chromosome pairs of R. japonica and R. ornativentris (Iizuka 1989, Miura 1994). Chakrabarti et al. (1983) reported non-centromeric C-bands in six chro- mosome pairs of H. tigerinus. In R. temporalis, telomeric C-bands were localized in the short arm of nos. 7 and 13, and in R. curtipes they were observed in the short arm of nos. 9 and 10. Pair nos. 10, 12 and 13 exhibited telomeric C-bands on both arms in R. malabarica (Joshy et al. 2006). The telomeric C-bands were rather common in the genera Mantella and Mantidactylus (Odierna et al. 2001). In the present study, the non-centromeric C-bands were observed in the terminal portion of long and short arms of nos. 1, 2, 4 and 5 in F. rufescens, and of nos. 1, 2, 3 and 5 in F. sahyadris. The non-centromeric C-bands seen in F. rufescens and F. sahyadris are considered as species specif- ic karyological features.

Acknowledgments We thank Rev. Father Swebert D’Silva, Principal of St. Aloysius College, for providing facili- ties. Thanks are due to Rev. Father Leo D’Souza, Director of Applied Biology Laboratory at St. Aloysius College, for his guidance. We also thank the Jesuit Educational Society, Mangalore, for the encouragement and support to our research.

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