Karyological Studies on Six Anuran Species from Yunnan Province, China

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Japanese Journal of Herpetology 15(1): 22-28.

1 Japanese Journal of Herpetology 15(1): , June 1993 Karyological Studies on Six Anuran Species from Yunnan Province, China WANZHAO LIU, DATONG YANG, AND MITSURU KURAMOTO Abstract: Karyotypes of six species of frogs from Yunnan Province, China, were described for the first time. Leptobrachium chapaensis had 2n=24 chromosomes composed of six large and six small pairs. Rana andersonii and R. maculosa had 2n=26 chromosomes comprising five large and eight small pairs. The karyotype of R, unculuana was unique among the advanced anurans in having 2n=40 chromosomes forming a graded series. Polypedates dugritei had 2n=26 chromosomes consisting of five large and eight small pairs, and Rhacophorus rhodopus had 2n=26 chromosomes with five large, one medium, and seven small pairs. The six species examined here belong to the Pelobatidae, Ranidae, and Rhacophoridae, and the karyotypes of these species excepting R. unculuana agree, in general, with the common karyotypes of the three families. Taxonomic status of each species is discussed on the basis of karyological features. Key words: Karyotype; Anura; Yunnan; China The amphibian fauna of Yunnan Province is quite rich, comprising a total of 94 anuran, six urodelan, and one gymnophionan species (Yang, 1991). Of the first, karyotypes have been reported only for 26 species (Li et al., 1981, 1990, 1991a, b; Tan and Wu, 1987; Wu and Zhao, 1985; Wu et al., 1989; Zhao, 1988; Zhao et al., 1987). We examined karyotypes of six anuran species from Yunnan Province, all new to our karyological knowledge. The three species of the genus Rana examined belong to two different species groups, odor frogs and spine frogs, the taxonomic treatment of which has been confusing. We discuss the implications of our karyological findings for the systematic status of each species by comparing our results with the published karyotypes of related Asiatic species. MATERIALS AND METHODS Leptobrachium chapaensis (one male) was collected in Tenchun; Rana andersonii (two males and three females) in Tenchun and Jingdong; and R. maculosa (three males and two females), R. unculuana (four males and three females), Polypedates dugritei (two males and two females), and Rhacophorus rhodopus (three males and one female) in Jingdong, all in Yunnan Province. Metaphase chromosome spreads were prepared from bone marrow cells either in the Accepted 8 Apr field after Omura's (1967) method or in the laboratory by conventional air-dry method. For the hypotonic treatment, dilute KCl solution (0.4%) was applied for 30 to 40 minutes. Diploid chromosome numbers were determined by observing more than 100 metaphase plates for each species. Ten well-spread plates were photographed for each species to obtain chromosome measurements. Relative length and arm ratio were then calculated for each chromosome pair. In the karyotype of each species, chromosome pairs were numbered in the order of decrease of the mean relative length. Centromeric positions were designated on the basis of the criteria of Levan et al. (1964) as modified by Green et al. (1980). RESULTS The one adult male of Leptobrachium chapaensis examined had 2n = 24 bi-armed chromosomes (NF = 48), consisting of six large and six small pairs (Fig. 1). Pair Nos. 4 and 6 were subtelocentric, Nos. 3, 5, 9, 10, and 11 submetacentric, and the remaining five pairs metacentric (Table 1). Pair Nos. 3 and 4 did not differ significantly in relative length (t-test; p>0.05), but differed completely in shape. In some plates, a secondary constriction was detected on the long arm of pair No. 1. No heteromorphic pairs were detected. The karyotype of Rana andersonii had 2n=26 (NF=52) chromosomes, comprising five large and eight small pairs (Fig. 2A). Pair Nos. 2, 7,

2 LIU ET AL.-KARYOLOGY OF CHINESE FROGS

3 Jpn. J. Herpetol. 15(1) TABLE 1. Relative length (RL) and arm ratio (AR) of the chromosome pairs in six frog species from submetacentric, subtelocentric and telocentric chromosomes, respectively. 12 and 13 were submetacentric, No. 3 was intermediate between submetacentric and metacentric, and the remaining eight pairs were metacentric (Table 1). Pair No. 10 had a secondary constriction on the long arm and No. 3 had another constriction on the short arm. No heteromorphic pairs were observed in either the male or the female karyotype. Rana maculosa had 2n=26 (NF=52) chromosomes, with five large and eight small pairs (Fig. 2B). Pair No. 2 was subtelocentric, Nos. 3, 4, 7, 9, 11, and 13 submetacentric, and the rest metacentric (Table 1). A secondary constriction was located on the short arm of pair No. 1. No heteromorphic pairs were observed in either the male or the female karyotype. The karyotype of Rana unculuana was remarkably different from those of the advanced anurans so far reported. It had 2n=40 (NF=80) chromosomes, which showed no distinct size groups (Fig. 2C). Pair No. 3 was telocentric with a distinct short arm, Nos. 7, 9, 11, and 16 subtelocentric, Nos. 2, 5, 6, 12, 14, 15, 17, and 18 submetacentric, and the remaining seven pairs metacentric (Table 1). No secondary constrictions or heteromorphic pairs were detected in either the male or the female karyotype. Polypedates d ugritei had 2n=26 (NF=52) chromosomes, consisting of five large and eight small pairs (Fig. 3A). Pair Nos. 2, 8, 9, and 10 were submetacentric, No. 4 was intermediate between submetacentric and metacentric, and the other eight pairs metacentric (Table 1). Pair No. 8 had a secondary constriction on the distal part of the long arm. No heteromorphic pairs were detected in either the male or the female karyotype. Rhacophorus rhodopus had 2n=26 (NF=52) chromosomes, with five large, one medium, and seven small pairs (Fig. 3B). Pair Nos. 2, 3, and 8 were submetacentric, No. 10 was intermediate between submetacentric and metacentric, and the remaining nine pairs metacentric (Table 1). In some metaphase plates, a faint achromatic gap, presumably representing a secondary constriction, was observed on the short arm of pair No. 1, and another was observed on the long arm of No. 6. No heteromorphic pairs were observed in either the male or the female karyotype. DISCUSSION The six species examined here belong to the families Pelobatidae, Ranidae, and Rhacophoridae. Except for Rana unculuana, which has a karyotype peculiar among the advanced anurans, these species have karyotypes which agree, in general, with the common karyotypes of the three families. The familiy Pelobatidae is generally considered as transitional between the primitive (Archaeobatrachia) and the advanced anurans (Neobatrachia), but the karyological data available to date support the view that the pelobatids may belong to the Neobatrachia, the suborder in which all the higher anurans are included (Morescalchi, 1973; Morescalchi et al., 1977). Of the 30 pelobatid species so far studied karyologically, one (Leptolalax ventripunctatus) has 2n=22 chromosomes, three (Leptolalax pelodytoides, L. alpinus and Leptobrachium

LIU ET AL.-KARYOLOGY OF CHINESE FROGS hendricksonr) have 2n=24 chromosomes, and the other 26 species have 2n=26 chromosomes (Kuramoto and Yong, 1992; Li et al.

4 LIU ET AL.-KARYOLOGY OF CHINESE FROGS hendricksonr) have 2n=24 chromosomes, and the other 26 species have 2n=26 chromosomes (Kuramoto and Yong, 1992; Li et al., 1990, 1991a, b; Morescalchi, 1973; Morescalchi et al., 1977; Wu, 1987; Wu and Zhao, 1987). Leptobrachium chapaensis has a 24-chromosome karyotype, as the three species of the genera Leptolalax and Leptobrachium mentioned above, but chromosomes of L. chapaensis are quite different from those of the other species in shape. Because of the considerable difference in external morphology of the adults, the 24- chromosome karyotypes of the two genera may have been derived independently. The karyotypes of L. chapaensis and L. hendricksoni differ from those of other species of Leptobrachium (L. hasseltii and L. nigrops; Morescalchi, 1973) in having fewer chromosomes, presumably originating from the loss of a pair of small elements by translocation, which is a fairly common modification pattern in the karyological evolution among amphibians (Morescalchi, 1973; King, 1990). Although the karyotypes of L. chapaensis and L. hendricksoni are characterized by many subtelocentric and submetacentric pairs, the two karyotypes do not correspond with each other. Thus, the karyotype of L. chapaensis seems to have differentiated substantially from those of the other members of the genus Leptobrachium. The basic chromosome number of the family Ranidae is 2n=26 with some exceptions (King, 1990; Kuramoto, 1989, 1990; Morescalchi, 1973). Of the three species examined here, R. andersonii and R. maculosa had 2n=26 chromosomes consisting of five large and eight small pairs, as in many other species of the family. Rana andersonii and 11 other south Chinese and Taiwan species form a distinct species group, the "odor frogs", for which Fei et al. (1990) erected a new genus Odorrana. Of these, only five species have been examined karyologically, and all of them have 26- chromosome karyotypes (Chen et al., 1983; Li and Wang, 1985a; Li et al., 1982; Wu et al., 1989; Xu et al., 1990). Their karyotypes differ from each other in both chromosome shape and position of secondary constrictions. The karyotype of R. andersonii resembles that of R. livida reported by Li and Wang (1985a). This may reflect a close relationship of the two species, although the two karyotypes differ in the location of secondary constrictions. The "spine frogs" of the genus Rana are often classified into a distinct subgenus or a distinct genus Paa (Dubois, 1975; Fei et al., 1990). About 15 Chinese species, including R. maculosa and R. unculuana examined here, belong to this group. Five other species of this group have been karyotyped to date; R. yunnanensis (=R. phrynoides) has 2n=64 chromosomes (Chen et al., 1983; Li and Wang,

Jpn. J. Herpetol. 15(1). 1993 1985b; Liu and Zan, 1984; Wu and Zhao, 1984), and the other four species have 2n=26 chromosomes as most ranids (Chen et al., 1983; Tan and Wu, 1987). The karyotype of R.

5 Jpn. J. Herpetol. 15(1) b; Liu and Zan, 1984; Wu and Zhao, 1984), and the other four species have 2n=26 chromosomes as most ranids (Chen et al., 1983; Tan and Wu, 1987). The karyotype of R. maculosa here examined resembles those of R. boulengeri, R. shini, and R. spinosa in the shape of most chromosome pairs, but is distinct from these three in having one subtelocentric pair and secondary constrictions at different sites. The karyotype of R. maculosa is characterized by the presence of a subtelocentric pair, which has not been reported for the other spine frogs. The karyotype of another spine frog, R. unculuana, is strikingly distinct among the advanced anurans (Neobatrachia) in having 2n=40 chromosomes and, apart from the polyploid species, the highest NF value (NF=80). It is very difficult to interpret the origin of the karyotype of this species. If it has derived from a 26-chromosome karyotype, it should have involved a considerable amount of structural rearrangement of chromosomes through Robertsonian processes and intra- and interchromosomal translocations. Another species of the spine frogs in Yunnan Province, R. yunnanensis, also has a peculiar 64-chromosome karyotype, but it consists of all telocentric chromosomes without short arms (Liu and Zan, 1984; Wu and Zhao, 1984), and accordingly its NF value (NF=64) is less than that of R. unculuana. The almost complete absence of telocentric pairs in R. unculuana suggests that the ancestral form of R. unculuana was a polyploid and that the unique karyotypes of R. unculuana and R. yunnanensis have evolved independently. Rana unculuana is distinct from the other spine frogs, including R. yunnanensis, in external morphology, and so Fei et al. (1990), while recognizing Paa as an independent genus, erected a new subgenus Unculuana for this species. Our karyological evidence supports the subgeneric separation of this species from the other spine frogs, though the generic status of Paa is not certain. All Asiatic species of the family Rhacophoridae examined so far have 2n=26 chromosomes with five large and eight small pairs as in most members of the Ranidae (Kuramoto, 1977, 1990; Tan, 1987; Tan et al., 1989). The karyotype of Polypedates dugritei agrees well with this common karyotype. The karyotype of Rhacophorus rhodopus is distinct in the genus Rhacophorus in having one pair of medium size chromosomes, which may have been produced through translocations. Because the karyotype of this species from another locality lacks this feature (Wu and Zeng, 1992) and the external morphology, especially color pattern of specimens from Jingdong, Yunnan Province, differs from those of the other populations (Hu et al., 1987; Liu and Hu, 1961), cryptic species

6 LIU E T AL.-KARYOLOGY OF CHINESE FROGS may be involved in R. rhodopus. ACKNOWLEDGMENTS. -We thank Wenjing Dong, Jingdong County, Yunnan Province, for help in the field and Wenxian Zuo for help in preparing specimens. LITERATURE CITED LI, S. -S., L. FEI, AND C. -Y. YE. 1991b. Cytogenetic study on three Oreolalax pelobatoides from Yunnan. Acta Zool. Sinica 37 (2): (in Chinese, with English abstract) LI, S. -S., Y. -X. WANG, C. -Y. LI, R. -F. WANG, AND G. -Z. LN A comparative investigation of the karyotypes from four amphibian species. Zool. Res. 2 (1): 17-24, pl. 4 (in Chinese, with English abstract) LI, S. -S., Y. -X. WANG, C. -Y. LI, R. -F. WANG, AND G. -Z. LIV An investigation for the karyotypic and C-banding pattern on the two anuran amphibia. Acta Genet. Sinica 9: , pl. 1. (in Chinese, with English abstract) LIU, C. -C. AND S. -Q. HU Tailless Amphibians of China. Science Press, Beijing. 364 p. (in Chinese) LIU, W. -G. AND R. -G, ZAN A special CHEN, W. -Y., Z.-S. WANG, X. -Z. WANG, Y. -H. YANG, AND Q. -L. SONG A comparative study of the karyotypes from six species of frogs in Sichuan. Zool. Res. 4(1) : 83-88, pl. 2. (in Chinese, with English abstract) DUBOIS, A Un nouveau sous-genre (Paa) et trois nouvelles especes du genre Rana. Remarques sur la phylgenie des Ranides (Amphibians, Anoures). Bull. Natn. Hist. Nat., Ser. 3, 324 (Zool. 231): karyotype in the genus Rana-an investigation of FEI, L., C. -Y. YE, AND Y. -Z. HUANG Key to the karyotype, C-banding and Ag-NORs of Rana Chinese Amphibia. Scientific and Technological phrynoides Boulenger. Acta Genet. Sinica 11: 61- Press, Chunqing. 364 p. (in Chinese) 64, pl. 1. (in Chinese, with English abstract) GREEN, D. M., J. P. BOGART, E. H. ANTHONY, AND MORESCALCHI, A Amphibia. In: A. B. D. L. GENNER An interactive, microcomputer-based Chiarelli and E. Capanna (eds.), Cytotaxonomy and karyotype analysis system for Vertebrate Evolution. p Academic Press, phylogenetic cytotaxonomy. Comput. Biol. Med. New York. 10: HU, S. -Q. (Ed.) Amphibia-Reptilia of Xizang. Science Press, Beijing. 153 p. (in Chinese) MORESCALCHI, A., E. OLMO, AND V. STINGO Trends of karyological evolution in pelobatid frogs. Experientia 33(12) : KING, M Animal Cytogenetics 4. Chordata 2. OMURA, T A method for chromosome preparations Amphibia. Gebruder Borntraeger, Berlin. 241 p. from amphibian bone marrow cells without in KURAMOTO, M A comparative study of vitro culture and centrifugation. Zool. Mag. karyotypes in the treefrogs (family Rhacophoridae) from Japan and Taiwan. Caryologia 30(3) : 333- (Tokyo) 76 (7): (in Japanese, with English abstract) 342. TAN, A. -M A rare case of karyotype in KURAMOTO, M Karyological studies on some anura-a preliminary study on the karyotype of Philippine frogs. In: M. Matsui, T. Hikida and Philautus doriae (Boulenger) with different diploid R. C. Goris (eds.), Current Herpetology in East numbers of 26 and 16. Chinese Herpetol. Res. 1987: Asia. p Herpetol. Soc. Japan, Kyoto , pl. 1. KURAMOTO, M A list of chromosome numbers TAN, A. -M. AND G. -F. WU Preliminary studies of anuran amphibians. Bull. Fukuoka Univ. Educ., on the karyotypes of three "spine-frogs" and the Part III, 39: karyotypic evolution of the subgenus Paa (Anura: KURAMOTO, M. AND H. -S. YONG Karyotypes of several frog species from peninsular Malaysia. Herpetologica 48 (4): LEVAN, A. K., K. FREDGA, AND A. A. SANDBERG. Ranidae, Rana). Acta Herpetol. Sinica 6(4) : 35-38, pl. 1. (in Chinese, with English abstract) TAN, A. -M., G. -F. WU, AND E. -M. ZHAO The karyotype of the treefrog Rhacophorus reinwardtii Nomenclature for centromeric position on (Boie) from Xishuangbanna Daizu autonomous chromosomes. Hereditas 52 (2): prefecture, Yunnan province, China. Chinese LI, B. -H. AND Z. -D. WANG. 1985a. Karyotypes of Herpetol. Res. 2 (2): Rana livida. Acta Herpetol. Sinica 4 (1): 56. (in WU, G. -F Cytotaxonomical studies on Chinese Chinese, with English abstract) pelobatids. III. The analysis of the karyotypes of LI, B. -H. AND Z. -D. WANG. 1985b. Karyotypes of Megophrys lateralis and Atympanophrys shapingensis. Rana spinosa. Acta Herpetol. Sinica 4 (1): Acta Herpetol. Sinica 6 (3): 45-48, pl. 2. (in (in Chinese, with English abstract) LI, S. -5., L. FEI, AND C. -Y. YE Studies of Chinese, with English abstract) WU, G. -F. AND X. -M. ZENG Studies on karyotypes, Ag-NORs and C-banding on five mountain pelobatid toads from China. Acta. Zool. Sinica 36 (3): (in Chinese, with English abstract) LI, S. -S., L. FEI, AND C. -Y. YE. 1991a. Cytogenetic studies on two Leptolalax pelobatids (Pelobatidae, Anura) from China. Acta Genet. Sinica 18 (6): 495- karyotypes of nine rhacophorid species from China (abstract). Asian Herpetological Meeting. p. 72. WU, G. -F. AND E. -M. ZHAO A rare karyotype of anurans, the karyotype of Rana phrynoides. Acta Herpetol. Sinica 3 (1): 29-32, pl. 1. (in Chinese, with English abstract) 499, pl. 1. (in Chinese, with English abstract) WU, G. -F. AND E. -M. ZHAO Preliminary

7 Jpn. J. Herpetol. 15(1) studies on karyotypes of the genus Amolops of the Hengduan mountains. Acta Herpetol. Sinica 4 (4): , pl. 3. (in Chinese, with English abstract) WU, G. -F. AND E. -M. ZHAO Cytotaxonomical studies on Chinese pelobatids II. The karyotype of Vibrissaphora ailaonica, with a discussion on the synonymy of V. liui and V. yaoshanensis. Acta Herpetol. Sinica 6 (3): 42-44, pl. 1. (in Chinese, with English abstract) WU, G. -F., A. -M. TAN, AND X. -M. ZHEN The karyotypes of six species of Rana. In: M. Matsui, T. Hikida and R. C. Goris (eds.), Current Herpetology in East Asia. p Herpetol. Soc. Japan, Kyoto. YANG, D. -T. (Ed.) The Amphibia-Fauna of Yunnan. China Forestry House, Beijing. 259 p. (in Chinese) XU, N., G. WEI, AND D. -J. LI An investigation of the karyotype, C-band and Ag-NORs pattern on Rana schmackeri. Hereditas (Beijing) 12 (3): 22-24, pl. 1. (in Chinese) ZHAO, E. -M Microhyla inornata Boulenger, 1890, found in mainland China, with its karyotypic report. Acta Herpetol. Sinica 7 (2): (in Chinese, with English abstract) ZHAO, E. -M., A. -M. TAN, AND G. -F. WU Karyotypes of Chinese species of Occidozyga (Family Ranidae), with discussion on the taxonomic status of O. laevis martensi. Chinese Herpetol. Res. 1987: 7-11, pl. 1. Kunming Institute of Zoology, Academia Sinica, Kunming, Yunnan , CHINA (WL and DY) and Department of Biology, Fukuoka University of Education, Munakata, Fukuoka, JAPAN (MK)