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 com- posed 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 form- ing 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 ex- cepting 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 field after Omura's (1967) method or in the quite rich, comprising a total of 94 anuran, six laboratory by conventional air-dry method. urodelan, and one gymnophionan species (Yang, For the hypotonic treatment, dilute KCl solution 1991). Of the first, karyotypes have been (0.4%) was applied for 30 to 40 minutes. reported only for 26 species (Li et al., 1981, Diploid chromosome numbers were determined 1990, 1991a, b; Tan and Wu, 1987; Wu and by observing more than 100 metaphase plates Zhao, 1985; Wu et al., 1989; Zhao, 1988; Zhao for each species. Ten well-spread plates were et al., 1987). photographed for each species to obtain We examined karyotypes of six anuran species chromosome measurements. Relative length from Yunnan Province, all new to our and arm ratio were then calculated for each karyological knowledge. The three species of chromosome pair. In the karyotype of each the genus Rana examined belong to two different species, chromosome pairs were numbered in the species groups, odor frogs and spine frogs, the order of decrease of the mean relative length. taxonomic treatment of which has been confus- Centromeric positions were designated on the ing. We discuss the implications of our basis of the criteria of Levan et al. (1964) as karyological findings for the systematic status of modified by Green et al. (1980). each species by comparing our results with the RESULTS published karyotypes of related Asiatic species. The one adult male of Leptobrachium cha- MATERIALS AND METHODS paensis examined had 2n = 24 bi-armed Leptobrachium chapaensis (one male) was col- chromosomes (NF = 48), consisting of six large lected in Tenchun; Rana andersonii (two males and six small pairs (Fig. 1). Pair Nos. 4 and 6 and three females) in Tenchun and Jingdong; were subtelocentric, Nos. 3, 5, 9, 10, and 11 and R. maculosa (three males and two females), submetacentric, and the remaining five pairs R. unculuana (four males and three females), metacentric (Table 1). Pair Nos. 3 and 4 did not Polypedates dugritei (two males and two differ significantly in relative length (t-test; females), and Rhacophorus rhodopus (three p>0.05), but differed completely in shape. In males and one female) in Jingdong, all in Yun- some plates, a secondary constriction was nan Province. detected on the long arm of pair No. 1. No Metaphase chromosome spreads were heteromorphic pairs were detected. prepared from bone marrow cells either in the The karyotype of Rana andersonii had 2n=26 (NF=52) chromosomes, comprising five large Accepted 8 Apr. 1993 and eight small pairs (Fig. 2A). Pair Nos. 2, 7, LIU ET AL.-KARYOLOGY OF CHINESE FROGS 23

FIG.1. Kazyotype of Leptobrachium chapaensis, family Pelobatidae. Black bar equals 10μm .

FIG.2. Karyotypes of three species in the family Ranidae, Rana andersonii (A), R. maculosa (B) and R. unculuana (C). Black bar equals 10μm . 24 Jpn. J. Herpetol. 15(1). 1993

TABLE 1. Relative length (RL) and arm ratio (AR) of the chromosome pairs in six frog species from Yunnan Province, China(N=10;Mean±SD). Abbreviations, m, sm, st and t represent metacentric, submetacentric, subtelocentric and telocentric chromosomes, respectively.

12 and 13 were submetacentric, No. 3 was in- were detected in either the male or the female termediate between submetacentric and metacen- karyotype. tric, and the remaining eight pairs were metacen- Rhacophorus rhodopus had 2n=26 (NF=52) tric (Table 1). Pair No. 10 had a secondary con- chromosomes, with five large, one medium, and striction on the long arm and No. 3 had another seven small pairs (Fig. 3B). Pair Nos. 2, 3, and constriction on the short arm. No heteromorphic 8 were submetacentric, No. 10 was intermediate pairs were observed in either the male or the between submetacentric and metacentric, and female karyotype. the remaining nine pairs metacentric (Table 1). Rana maculosa had 2n=26 (NF=52) In some metaphase plates, a faint achromatic chromosomes, with five large and eight small gap, presumably representing a secondary con- pairs (Fig. 2B). Pair No. 2 was subtelocentric, striction, was observed on the short arm of pair Nos. 3, 4, 7, 9, 11, and 13 submetacentric, and No. 1, and another was observed on the long the rest metacentric (Table 1). A secondary con- arm of No. 6. No heteromorphic pairs were striction was located on the short arm of pair observed in either the male or the female No. 1. No heteromorphic pairs were observed karyotype. in either the male or the female karyotype. The karyotype of Rana unculuana was DISCUSSION remarkably different from those of the advanced The six species examined here belong to the anurans so far reported. It had 2n=40 families Pelobatidae, Ranidae, and (NF=80) chromosomes, which showed no Rhacophoridae. Except for Rana unculuana, distinct size groups (Fig. 2C). Pair No. 3 was which has a karyotype peculiar among the ad- telocentric with a distinct short arm, Nos. 7, 9, vanced anurans, these species have karyotypes 11, and 16 subtelocentric, Nos. 2, 5, 6, 12, 14, which agree, in general, with the common 15, 17, and 18 submetacentric, and the remain- karyotypes of the three families. ing seven pairs metacentric (Table 1). No secon- The familiy Pelobatidae is generally con- dary constrictions or heteromorphic pairs were sidered as transitional between the primitive (Ar- detected in either the male or the female chaeobatrachia) and the advanced anurans karyotype. (Neobatrachia), but the karyological data Polypedates d ugritei had 2n=26 (NF=52) available to date support the view that the chromosomes, consisting of five large and eight pelobatids may belong to the Neobatrachia, the small pairs (Fig. 3A). Pair Nos. 2, 8, 9, and 10 suborder in which all the higher anurans are in- were submetacentric, No. 4 was intermediate bet- cluded (Morescalchi, 1973; Morescalchi et al., ween submetacentric and metacentric, and the 1977). Of the 30 pelobatid species so far studied other eight pairs metacentric (Table 1). Pair karyologically, one (Leptolalax ventripunctatus) No. 8 had a secondary constriction on the distal has 2n=22 chromosomes, three (Leptolalax part of the long arm. No heteromorphic pairs pelodytoides, L. alpinus and Leptobrachium LIU ET AL.-KARYOLOGY OF CHINESE FROGS 25

TABLE 1. Continued.

hendricksonr) have 2n=24 chromosomes, and Ranidae is 2n=26 with some exceptions (King, the other 26 species have 2n=26 chromosomes 1990; Kuramoto, 1989, 1990; Morescalchi, (Kuramoto and Yong, 1992; Li et al., 1990, 1973). Of the three species examined here, R. 1991a, b; Morescalchi, 1973; Morescalchi et al., andersonii and R. maculosa had 2n=26 1977; Wu, 1987; Wu and Zhao, 1987). Lep- chromosomes consisting of five large and eight tobrachium chapaensis has a 24-chromosome small pairs, as in many other speciesof the fami- karyotype, as the three species of the genera Lep- ly. Rana andersonii and 11 other south Chinese tolalax and Leptobrachium mentioned above, and Taiwan species form a distinct species but chromosomes of L. chapaensis are quite group, the "odor frogs", for which Fei et al. different from those of the other species in (1990)erected a new genus Odorrana. Of these, shape. Because of the considerable difference in only five species have been examined external morphology of the adults, the 24- karyologically, and all of them have 26- chromosome karyotypes of the two genera may chromosome karyotypes (Chen et al., 1983; Li have been derived independently. and Wang, 1985a; Li et al., 1982; Wu et al., The karyotypes of L. chapaensis and L. hen- 1989; Xu et al., 1990). Their karyotypes differ dricksoni differ from those of other species of from each other in both chromosome shape and Leptobrachium (L. hasseltii and L. nigrops; position of secondary constrictions. The Morescalchi, 1973) in having fewer karyotype of R. andersonii resembles that of R. chromosomes, presumably originating from the livida reported by Li and Wang (1985a). This loss of a pair of small elements by translocation, may reflect a close relationship of the two which is a fairly common modification pattern in species, although the two karyotypes differ in the the karyological evolution among amphibians location of secondary constrictions. (Morescalchi, 1973; King, 1990). Although the The "spine frogs" of the genus Rana are often karyotypes of L. chapaensis and L. hendricksoni classified into a distinct subgenus or a distinct are characterized by many subtelocentric and genus Paa (Dubois, 1975; Fei et al., 1990). submetacentric pairs, the two karyotypes do not About 15 Chinese species, including R. correspond with each other. Thus, the maculosa and R. unculuana examined here, karyotype of L. chapaensis seems to have belong to this group. Five other species of this differentiated substantially from those of the group have been karyotyped to date; R. yun- other members of the genus Leptobrachium. nanensis (=R. phrynoides) has 2n=64 The basic chromosome number of the family chromosomes (Chen et al., 1983; Li and Wang, 26 Jpn. J. Herpetol. 15(1). 1993

FIG.3. Kazyotypes of two species in the family Rhacophoridae, Polypedates dugritei (A) and Rhacophorus rhodopus (B). Black baz equals 10μm.

1985b; Liu and Zan, 1984; Wu and Zhao, 1984), plete absence of telocentric pairs in R. un- and the other four species have 2n=26 culuana suggests that the ancestral form of R. chromosomes as most ranids (Chen et al., 1983; unculuana was a polyploid and that the unique Tan and Wu, 1987). The karyotype of R. karyotypes of R. unculuana and R. yunnanensis maculosa here examined resembles those of R. have evolved independently. Rana unculuana boulengeri, R. shini, and R. spinosa in the shape is distinct from the other spine frogs, including of most chromosome pairs, but is distinct from R. yunnanensis, in external morphology, and so these three in having one subtelocentric pair and Fei et al. (1990), while recognizing Paa as an secondary constrictions at different sites. The independent genus, erected a new subgenus karyotype of R. maculosa is characterized by the Unculuana for this species. Our karyological presence of a subtelocentric pair, which has not evidence supports the subgeneric separation of been reported for the other spine frogs. this species from the other spine frogs, though The karyotype of another spine frog, R. un- the generic status of Paa is not certain. culuana, is strikingly distinct among the advanc- All Asiatic species of the family ed anurans (Neobatrachia) in having 2n=40 Rhacophoridae examined so far have 2n=26 chromosomes and, apart from the polyploid chromosomes with five large and eight small species, the highest NF value (NF=80). It is pairs as in most members of the Ranidae very difficult to interpret the origin of the (Kuramoto, 1977, 1990; Tan, 1987; Tan et al., karyotype of this species. If it has derived from 1989). The karyotype of Polypedates dugritei a 26-chromosome karyotype, it should have in- agrees well with this common karyotype. The volved a considerable amount of structural rear- karyotype of Rhacophorus rhodopus is distinct rangement of chromosomes through Robertso- in the genus Rhacophorus in having one pair of nian processes and intra- and interchromosomal medium size chromosomes, which may have translocations. Another species of the spine been produced through translocations. Because frogs in Yunnan Province, R. yunnanensis, also the karyotype of this species from another locali- has a peculiar 64-chromosome karyotype, but it ty lacks this feature (Wu and Zeng, 1992) and the consists of all telocentric chromosomes without external morphology, especially color pattern of short arms (Liu and Zan, 1984; Wu and Zhao, specimens from Jingdong, Yunnan Province, 1984), and accordingly its NF value (NF=64) is differs from those of the other populations (Hu less than that of R. unculuana. 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要旨中国雲南省のカエル6種の核型苅万兆・楊大同・倉本満雲南省のカエル6種の核型を初めて報告した. rhodopusは大形5対,中形1対,小形7対の Leptobrachium chapaensisは大形6対,小形6 2n=26の染色体をもっていた.R. unculuana 対よりなる2n=24の染色体,Rana andersonii を除き,これらの種の核型はそれぞれの科の基とR.maculosaは大形5対,小形8対よりなる本的な核型にほぼ合致していた.核型の比較に 2n=26の染色体をもっていた.Rana un- 基づいて,それぞれの種の分類上の問題点を論の核型は高等なカエル類culuana の核型としてじた. は特殊なもので,2n=40の染色体をもち,それらを大きさによって区分することはできなか (中国雲南省昆明市中国科学院昆明動物研った.Polypedates dugriteiは大形5対,小形究所(苅・楊)・811-41福岡県宗像市赤間福 8対の2n=26の染色体,Rhacophorus 岡教育大学生物学教室(倉本))