I

Asiatic Herpetological Research

June 1995

Vol. 6, pp. 161-166

Karyotypes of Four Microhylid Frogs from
Xishuangbanna, Southern Yunnan, China

XIAO-MAO ZHENG AND GUAN-FU WUChengdu Institute of Biology, Accidentia Sinica, Chengdu, Sichuan, 610041 China-The karyotypes of Microhyla butleri, ornata and pulchra and Kaloula pulchra pulchra from
Xishuangbanna, southern Yunnan are reported. The karyotype of Microhyla butleri is reported for the firsttime. Its diploid number of chromosomes (2n=22) differs from the other species investigated in the samegenus which is 24. The results of M. ornata andpulchra are also different from those obtained by theauthors.
TheofKaloula
karyotypep. pulchra (2n=28) had a slight difference from the result obtained
previousby the previous authors. The further C-banding analysis of this species revealed that an amount ofheterochromatin is located in the centric, terminal and interstitial position of chromosomes.
Abstract:M.

Key Words: Anura, Microhylidae Microhyla, Kaloula, cytotaxonomy, China,

Introduction

The karyotypes of Microhyla ornata
from Sichuan and Fujian were reported byChen (1983) and Gao et al. (1985)
respectively. The karyotypes of Microhyla
pulchra, Kaloula pulchra pulchra from
Guangzhou were reported by He (1986). InThe diploid chromosome number of M.
ornata and M. pulchra is 24, with 18 m, 4sm and 2 m or 2 sm chromosomes, whereasthat of M. butleri is 22 with 18 m and 4 sm
chromosomes. Kaloula p. pulchra had 28
including 20 m, 6 sm and 1 sm or 1 st. In
four species, the chromosome length
decreased gradually, not forming distinct

the present study, the karyotypes of thosegroups in size. The conspicuous secondary

species from Xishuangbanna, southern
Yunnan, are reported, and they are analyzed
by means of C-banding and silver-staining

constrictions (SC) were found on the long

In addition, theNORs techniques.
karyotype of Microhyla butleri from thesame locality is reported for the first time.

arm of No. 5 of K. p. pulchra, No. 8 of M.
butleri and Nos. 8, 10 of M. pulchra,whereas the unremarkable one can be soughton the long arm of No. 11 of M. ornata in afew mitotic metaphases. No consistentheteromorphic pairs were observed in all

Materials and Methods

Microhyla ornata (4 males, 4 females),M. pulchra (5 males, 5 females), M. butleri(5 males,1female) and Kaloula p. pulchra(2 males, 1 female) were captured in
Xishuangbanna, southern Yunnan, China in
May 1991. Chromosome preparations weremade from the bone marrow cells by themethod of Wu et al. (1981). C-banding andsilver-staining

NORs were carried out

four species.

The C-banding

weresuccessfullyThe centric
positive bands were discovered on allchromosomes, especially present on smaller
ones; terminal bands were shown on Nos.
obtainedinK. p. pulchra.

2-4; interstitial bands, as well, can beobserved on both the short and long arm ofNo. 1 and the short arm of No. 4. The
resultstill
revealedthehighly

following the methods of Sumner (1972)

heterochromatic region possesses two-thirds

and Tan et al. (1986).

of the length of No. 5 (Fig. 3).
Theprominent heterochromatinization areobserved in individuals of both sexes, andthere are no difference between both sexes,
theexistenceof sexindicating

Results

The karyotypes for the four species areseparately shown in Figs. 1-3 and thechromosome measurements in Table 1. The
secondary constrictions and results of AgNORs are listed in Table 2.

© 1995 by Asiatic Herpetological Researchdifferentiation.

Asiatic Herpetological ResearchVol. 6, p. 162

April 1995

Ac \y.

r .' '&* » » »*c;w••ai<»/U>.

0'>/\AA'*J[XAX

**ix

**Xtf

A*

**

BV

*-t

1//iFIG. 1. Karyotypes of Microhyla butler i (A) and A/, omala (B).

The silver-staining NORs revealed thatDiscussion

the secondary constriction in M. pulchra(Fig. 2). M. butleri and AT. p. pulchra areKaryotypes

also treated with the silver-staining NORs

The karyotypes of M. ornata and M.
pulchra from Xishuangbanna were different
from those of the other localities (Table 2).
Firstly, all 12 chromosome pairs, except
No. 3 in two species, are metacentric, butNos. 7-9 from Xishuangbanna changed to,or close to, submetacentric.
Next, theNOR is located on No. 10, associated with

Although no perfect spread ofsilver-staining NORs are in the two species,technique.

NOR associated with SC can be sought insome cells.

April 1995

Asiatic Herpetological ResearchVol. 6, p. 163„((

oft/\0\ 8s«]Al\KIay' A^:» •# #FIG. 2. Karyotype and Ag-NORs of Microhyla pulchra.

numbers and positions of SC on thechromosomes are distinctly varied between
the same species from Xishuangbanna andthe other localities (Table 2). These results

seem to show that the karyotypic type of thespecies are gradually altered due to slowly
fitting for the various environments.

There are about 20 species described in
the genus Microhyla that range over Asia

(Gao et al., 1985; Guo et al., 1987); pulchra
on several pairs; inornata (Zhao, 1988) andmixtura (Guo et al., 1991) on No. 9 andbutleri on No. 8. Moreover, this evident
differentiation is reflected on the various
diploid number in the genus. Most of them
have 2n=24 except for inornata and rubra
with 2n=26 and butleri with 2n=22 (thepresent study). Diploid number of 22, 24,
26 and 28 are known for Microhylidae.

In M. nornata and mixtura,chromosomes can be observed. Thepositions of SC on the chromosomes areUsually, most species in the same genus
have the same diploid number in anurans.
On the other hand, if we supposed the 24
was the diploid number of the genus
Microhyla, it would be possible to consider
whether butleri, inornata and rubra might bequite different between these species: ornata

separated from the genus.

only.

Up to now, seven species have been
The karyotypic

analyzed karyologically.
character proved their obvious interspecificdifferentiation.
ston Nos. 3, 9 and 11; heymonsi on No. 2

Vol. 6, p. 164

Asiatic Herpetological ResearchApril 1995

ft f& « «/»t:2^Art*KMA<>

X ;{X »****> uA/.'-*\FIG. 3. Karyotype and C-bands of Kaloula p. pulchra.

The karyotypes of K. p. pulchra from
Xishuangbanna and Guangzhou arecompared (Table 2) and the difference

C-bands and NORs

between them are not obvious. Unlike inMicrohyla, five species in Kaloula whosekaryotype are known have 2n=28, and theconspicuous SC located on No. 5 except K.
picta (Kuramoto, 1980). The interspecificdifferentiation is less clear. The differences
between these species are shown on thevariety of centromere type of a few

Although many microhylids are analyzed
karyologically, their C-banding is rarely
reported. The C-bands of K. rugifera areeven mentioned (Zeng et al., 1989) in which
the interstitial and terminal bands except
centric are observed. In K. p. pulchra, notonly interstitial and terminal but also centricbands are easily seen (Fig. 3). In the twospecies, the interstitial bands associated withcorresponding chromosome pairs.

the main SC on No. 5 are enhanced, whichindicates the genetic stability in this genus.

The C-bands of the two species present theApril 1995

Asiatic Herpetological Research

Vol.6, p. 165

TABLE 1 Chromosome measurements of four microhylid species from Xishuangbanna, southern Yunnan,China.

NoVol. 6, p. 166

Asiatic Herpetological Researchheterochromatin of microhylids are widely
spread on the centric, interstitial and terminal
positions of chromosomes. The results arevery similar to those in the higher anurans.
It suggests that the evolutionary level ofmicrohylids correspond to that of the higher
anurans from cytogenetics. The obvious
heterochromatinization of No. 5 in K.
pulchra does not show sex differentiation,and it acts as part of a special sign todistinguish it from other species.

HE, H. Y.

April 1995

1986.

Comparative studies on thekaryotypes of nine anuran species of Guangdong
Province. Acta Herpetologica Sinica, Chengdu, newser. 5(3):171-175, 2 plates.
[In Chinese withEnglish abstract].

KURAMOTO, M.

1980. Karyotypes of several
frogs from Korea, Taiwan and the Philippines.
Experientia, Basel, CHE 36:826-827, 2 plates.

SUMNER, A. T.

1972.

A simple technique forcentromeric heterochromatin.
Experimental Cell Research 75:304-306, 3 plates.

demonstrating

stable and conspicuous SC isthelocation of NORs.
In fact,always

The

silver-staining NORs shows that NORs ofK. pulchra, K. rugifera, M. pulchra,butleri,M. ornata, M. mixtura, andM
M.

TAN, A. M., Z. A. WU, E. M. ZHAO AND H. X.

OUYANG.

1986.

A handy one-step method forNORs. Acta Herpetologica Sinica,
Chengdu, new ser. 5(l):72-74, 1 plate.
[Insilver-stainingChinese].
.

heymonsi are just located in the position of
their main SC.
Tymowska (1977)
concluded these species in the genus show aclose relationship due to having the samepossessing a 36-chromosome complement.
Cytogenetics and Cell Genetics, Basel, CHENORs.

18:165-181.

From

thisthepoint,

close

relationship exist between species in thegenus Kaloula for they have the same SC on

On the contrary, those species in thegenus Microhyla reveal their higherNo. 5.

interspecific differentiation level because of
their different
association with theNOR

main SC.

1977. A comparative study of theofkaryotypes
eight Xenopus species and subspecies

TYMOWSKA, J.

WU, G. F., W. M. YANG AND E. M. ZHAO.

1981.
Studies on genus Vibrissaphora (Amphibia:A preliminary observation on
Pelobatidae). 3.
karyotype of Vibrissaphora liui (Pope). Acta

Herpetologica Sinica, Chengdu, old ser. 5:139-142.
[In Chinese with English abstract].

WU, Z. A. AND H. Y. YANG.

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