I

Asiatic Herpetological Research

February 1992

Vol. 4,££.

146-157t
Karyotypic Studies of Nine Species of Chinese Salamanders

YUHUA YANG

1

^Department of Bioengineering, Sichuan University, Chengdu 610041, China

Abstract. -Four species of hynobiid salamanders, from three genera, possess bimodal and asymmetricalkaryotypes with 2n=64 or 2n=68 chromosomes. Five species of salamandrids, from four genera, have
2n=24 and unimodal, symmetrical karyotypes with no microchromosomes or telocentric chromosomes.
Karyologically, Hynobiidae is the most primitive and the Salamandridae is the most advanced family in the
Caudata. The two families conform to different models of chromosome change.

Key words: Amphibia, Caudata, Hynobiidae, Salamandridae, China, karyotype.

TABLE 1.

Locality, date of collection, and number of individuals (male, female) for the 9 species used in

the karyotypic analyses.

Hynobiidae
Sichuan Prov., 1986
Mianning Co., Sichuan Prov., July 1986
Wushan Co., Sichuan Prov., Mar. 1986
Jinzhai Co., Anhui Prov., Apr. 1984

Batrachuperus karlschmidti
B. yenyuanensis

Liua shihi
Pachyhynobius shangchengensis

F

M

2

4
2
2

1

3

Salamandridae
Weining Co., Guizhou Prov., Jun. 1986
Jingdong Co., Yunnan Prov., Aug. 1984
He Co., Guangxi Prov., Jun. 1986
He Co., Guangxi Prov., Jun. 1986
Jingdong Co., Yunnan Prov., Aug. 1984

Tylotolriton kweichowensis
T. verrucosus

Pachytriton labiatum
Paramesotriton chinensis

Cynops cyanurus yunnanensis

cytogenetic data towards
understanding the phylogeny of Chinese

Introduction

additional

There are 33 species of salamanders
China, including two
suborders, three families and 12 genera(Zhao et al., 1988). Only a few of theirkaryotypes have previously been reported(Wang et al., 1983; Yang and Zhao, 1984;
Yang et al., 1986a, 1986b; Zhu and Wei,
1981).
Karyotypic studies among the
Caudata (Kuro-o et al., 1987; Makino,
1932;
Morescalchi, 1973, 1975;
Morescalchi et al., 1977; 1979; Schmid,
1979; Sessions et al., 1982; Seto et al.,1986) have contributed evidence for thesalamanders.

known fromphylogeny of salamanders.

The present

paper reports the karyotypes of nine species

belonging to the families Hynobiidae andSalamandridae,

therebyprovidingMethods
Preparation of Chromosomes

Animals used

in

this

study

were

collected from Sichuan, Yunnan, Guizhou,

Guangxi and Anhui Provinces, China fromMay 1983 to June 1986 (Table 1). Mitotic
chromosomes were prepared using themethods of Kezer and Sessions (1979)
with minor modifications. Animals wereintraperitoneally injected with 20 mg/ml ofcolchicine solution about 40 to 60 hours
before sacrifice using a dosage of 0.01 ml/g
body weight. Liver and intestine were
removed, washed with 1% sodium citrate,

Chromosome preparations
made by air-dry and squash
Chromosomes were also
techniques.
andsliced.

werefThis publication was previously published in
Chinese by Yang (1990).

1992 by Asiatic Herpetological Researchobtained using the methods of Princee andAsiatic Herpetological Research

February 1992

Boer (1983) and Wu (1982) with slight
modifications (Yang et al., 1986a).
Specimens were injected intraperitoneallywith PHA solution (5 mg/ml) at a dosage of
0.09 ml/g body weight, administered once
every 24 hours for three days. Twenty
four hours after the third PHA injection, thespecimens received a final injection ofcolchicine (3 mg/ml) with a dosage of 30|!g/g body weight about 14 hours before

The spleen wasthey were sacrificed.
removed, washed with 1% solution of
sodium citrate, ground, and put into ahypotonic solution (0.4% KC1). A few
drops of the final cell suspensions wereused per slide, which was then placed in a
Following hypotonic
large Petri dish.
treatment and fixation in the dish, the airdried slides were stained with 5% Giemsa

PBS (pH 6.8) for 30 minutes.

Vol. 4, p. 147

morphology of the microchromosomes
varies with differences in the preparation

techniques and the mitotic chromosomes of
the cells analyzed.
For the sake ofI
have
defined theconsistency,
chromosomes less than 3.00% relative

length (in percentage of total haploid
length, including the microchromosomes)
as microchromosomes according to the factthat the minimum value of relative lengthsof the smallest chromosomes in

Salamandridae, which is the most advanced
family without microchromosomes in
The
Caudata, is 3.00 (Table 2).
abreviations of the chromosomes are as

follows:

M-metacentric;

SMT-

submetacentric; ST-subtelocentric;
telocentric; and m-microchromosome.
2.

Meiotic chromosomes.

diakinesis were

— Bivalentsinnumbered and arranged.

An intraperitoneal injection of colchicine

The relative lengths of the bivalents weremg/ml) was given using adosage of 30 (ig/g body weight for 14-24

calculated according to the principles of
ISCN (1978). The arm ratio was not
calculated due to the absence of C-band
information. The relative chiasma numbers
also were calculated (those having joinedends were also considered to be in chiasma)

solution (3hours before the specimens were sacrificed.

The testes were removed, rinsed with 1%
sodium citrate, ground, and put into 0.4%
KC1 solution for 1.0-1.5 hours at room
temperature.

After centrifugation, thewasdiscarded

andusing the method presented by Imai and
Moriwaki (1982). For chromosomes in
metaphase II, the relative length and arm

theprecipitate was fixed in a 3:1 solution of
methanol: acetic acid for three periods of 30

ratio were measured and calculated.

minutes each. Slides were prepared by the
air-dry method and stained with 5%

terminology for centromeric position
followed Levan et al. (1964).

supernatant

The

Giemsa PBS (pH 6.8) for 1 hour.
Results
Karyotype Analysis
Hynobiidae—

Mitotic
chromosomes.
Chromosomes were numbered and the
spreads which could be used forThekaryotypes were photographed.
karyotypes were prepared on the basis ofrelative lengths and arm ratios measured
and calculated from the enlarged
photographs. The chromosomes in tailed
1.

—B.

karlschmidti
12
of
pairs
macrochromosomes and 22 pairs of
microchromosomes, which is the highest

Batrachuperus.
has(2n=68)chromosome number among species ofmacrochromosomes. No general criterion

Hynobiidae reported to date except for
Onychodactylus japonicus (2n=78; Table
3). Nos. 1-3 are metacentric and the restthesubmetacentric
amongmacrochromosomes (Fig. 1, Table 4).
This is a bimodal and asymmetricalkaryotype with a formula of 6M + 18ST +of microchromosomes

44M.

amphibians grade smoothly in size from thelargest to the smallest, so that it is

impossible to distinguish between thelargest microchromosomes and the smallest
existstodate(Morescalchi, 1973, 1975; Sessions et. al,
1982; Wang et al, 1983). In addition, theB. yenyuanensis (2n = 68), consistingVol. 4, p. 148

Asiatic Herpetological Research

TABLE 2. Mitotic chromosome data for five species of Salamandridae.
Relative length (x+S.D.)

February 1992

February 1992

Asiatic Herpetological Research

TABLE 3. Chromosome data of some species in Hynobiidae.
SpeciesVol. 4, p. 149

Asiatic Herpetological ResearchVol. 4, p. 150

February 1992

HKKllMii" ••|* M "

f4

ft* #4 41
mm

«•

)f
li

** *• *• **

•* **

••

1

_

^
&*

•»

>*

i<5

at

•

M
M

«• 4* **

••

2

H i<

*• «• h*>*r« ii it m
II II M •• •• ••• § AA mm mm

«M

•«>

•••»

•«•

mmm»

Karyotypes of four species of hynobiid salamanders.
yenyuanensis, 3) Liua shihi, 4) Pachyhynobius shangchengensis.
1.

and 12 are 12.47 and 5.41, while
their relative chiasma numbers are 7.96 and

nos.

mm

•»-«

•

FIG.

•• •»

** •• M•« •» •• *a•• *• •• •• mtt •• •• ••1

8.47 respectively.
T. verrucosus (2n = 24). The haploid
chromosome number is 12. Accordingly,
the diploid chromosome number expected

24, which is consistent with those of T.
andersoni and T. verrucosus from different
localities (Ferrier and Beetschen, 1973;
Morescalchi, 1973; Seto et al, 1986). The
relative lengths of chromosomes in
metaphase II are larger than those ofisbivalents in diakinesis (Tables 5 and 6).
The chromosomes in metaphase II are

1)•«

Batrachuperus karlschmidti,!) B.

lengths are 9.36 and 5.25 respectively(Table 6).

—

P. labiatum (2n = 24).
Pachytriton.
in diakinetic cells
12bivalents
areThere(n=12).
Accordingly, the diploid
chromosome number should be 24 (Fig.
2), in accord with that of P. brevies (Zhuand Wei, 1981).

numbers of nos.

The relative chiasma
1-6

bivalents

vary

basically with their relative lengths, while
the relative chiasma numbers of nos. 7-12
are more constant (Table 6).

Cynops.

— C. cyanurus yunnanensis

metacentric except nos. 6, 8 and 12 ofsubmetacentric chromosomes and there are

There are 12 bivalents indiakinetic cells and 12 chromosomes inno microchromosomes. The karyotype isunimodal and symmetric. 2). The relative
chiasma numbers are not proportional torelative lengths, for example, the relative

metaphase II cells (Fig. 2). Consequently,
the 2n should be 24. The chromosomes in(2n=24).

metaphase II are metacentric except no. 8(SM), without microchromosomes (Fig.

February 1992

Asiatic Herpetological Research

TABLE 4. Macrochromosome data for four species of Hynobiidae.
Relative length (x±S.D.)

Vol. 4, p. 151

Asiatic Herpetological ResearchVol. 4, p. 152

February 1992

^X«w 00 }»*JU&AX* W0*» |*WIf(tiiiiutiu

21^Knkka3?•«*•• »!«••

FIG. 2. Karyotypes of five species of salamandrid salamanders. 1-3) Tylototriton kweichowensis, mitotic
chromosomes (1), chromosomes in metaphase II (2), and meiotic bivalents (3). 4, 5) T. verrucosus,
chromosomes in metaphase II (4), and meiotic bivalents (5). 6) Meiotic bivalents of Pachytriton labiatum.
7, 8) Cynops cyanurus yunnanensis, chromosomes in metaphase II (7), and meiotic bivalents (8). 9)
Meiotic bivalents of Paramesotriton chinensis.

shown in table 6. The relationship ofchiasma numbers and the lengths of thebivalents is not a straight line, for example,
the relative lengths of the nos. 2 and 12 are

11.89 and 3.55 respectively, while theirrelativechiasma numbers are the same,

8.10.

Discussion

and microchromosomes) and asymmetrical(with metacentric and telocentricchromosomes) karyotype is considered to
be primitive (Morescalchi, 1975) inreference to the karyotypic evolution in
Caudata.
Among some species ofthediploid numbers are 40-66
Hynobiidae,

and their karyotypes are bimodal andasymmetrical except for Hynobius
retardatus [=Satobius retardatus (Adler andIt

has been suggested from karyotypic

data that Hynobiidae is the most primitive
group of salamanders, while Salamandridaeisthemost advanced group in Caudata(Morescalchi, 1973, 1975; Morescalchi etal., 1979). The same conclusion is derived

from morphological comparisons (Zhaoand Hu, 1984). There are some differencesin the evolutionary ways of the two groups.
Karyotypic Evolution in Hynobiidae

The bimodal (with macrochromosomes

Zhao, 1990)], (Table 3). The karyotypesof the four species (in three genera) I
studied here are all bimodal andasymmetrical, with 2n=64 or 68
chromosomes. Batrachuperus is one of 2
hynobiid genera in China that are aquaticand different fromThe diploid
number of B. karlschmidti and B.
yenyuanensis is 68, the second highest
number among species of Caudata studied
so far (Table 3). Onychodactylus is morederived and allied with Ranodon andfor their entire lives

other genera morphologically.

February 1992

Asiatic Herpetological Research

TABLE 5. Metaphase II chromosome data for 3 species of Salamandridae.

Vol.4, p. 153

Vol. 4, p. 154

Asiatic Herpetological Research

TABLE 6. Bivalent data for 5 species of Salamandridae.
Relative length (x+S.D.)

February 1992

Asiatic Herpetological Research

February 1992

Vol. 4, p. 155

Pericentric inversions do not change thediploid number, but could increase the

Tylototriton are all subtelocentric, while thekaryotypes of C. orientalis and P. brevipes

number of metacentric chromosomes andhave no subtelocentric chromosomes, only

the stability of karyotypes.

andsubmetacentricmetacentrictheIn
chromosomes.
addition,
chromosome differences in morphology
were seen among 3 species in Tylototriton,

B. karlschmidti and B. yenyuanensis
of
and
23
22
pairs
possess
microchromosomes respectively and lackmacrochromosomes.

telocentricContrastingly, P. shangchengensis and L.

shihii have 21 and 20 pairs of
microchromosomes and 5 and 9 pairs of
It istelocentric macrochromosomes.
concluded that the karyotypic evolution of
the 4 species above has involved
Robertsonian centric fusion as well as

4 pairs of submetacentric chromosomes inT. andersoni, 3 pairs in T. kweichowensis
and 2 pairs in T. verrucosus. 2. The dataof chromosomes in meiotic metaphase II(Table 5): C. cyanurus yunnanensis has
1submetacentric
of
pair
only
chromosomes, while there are 4 pairs inboth T. verrucosus and T. kweichowensis.
Numbers 6, 7, 8, and 12 areinversion.
However, the4
could not beofthespeciesphylogeny
established based on the present data. It is

submetacentric chromosomes in T.
verrucosus and nos. 6, 8, 11, and 12 in T.

information fromchromosome banding and biochemistry inorder to define the structures and functionsof microchromosomes and telocentricchromosomes.

in diakinesis (Table 6):of no. 1 bivalents of T. verrucosus and T.
kweichowensis are similar, 12.49 and12.47 respectively, but 13.27, 12.62 and11.92 in P. chinensis, P. labiatum and C

Karyotypic Evolution in Salamandridae

relative chiasma numbers of no. 1 bivalents

The 5 salamandrid species studied here
have 2n=24 chromosomes, lackreveal the intergeneric and intrageneric
It is noteworthy that thevariations.
andintraspecific variations ofinterspecificmicrochromosomes, and possess unimodaland symmetrical karyotypes (Fig. 2, Tables
2 and 5) as consistent (Ferrier andindividual bivalents on relative chiasma
number in 2 species of Tylototriton are
more distinctive than those in other genera

Beetschen, 1973; Seto et al., 1986; Zhu
Morescalchi (1975)
and Wei, 1981).
suggested that all species studied possess
similar karyotypes that differ very little
The
even at the intergeneric level.
differences between these karyotypes
predominantly concern the absolute size ofchromosomes and quantity of DNA.

and species. The relative chiasma numbers

Accordingly, the karyotypic diversity
among the species has chiefly resulted frompericentric inversions that result inin mitotic metaphase, more submetacentric

pericentric

necessary

to

have

kweichowensis.

3.

The data of bivalents
the relative lengths

cyanurus yunnanensis individually.

all

The

are apparently not directly proportional to
the relative lengths.

Tylototriton has karyotypically been
considered to be the most primitive genus
inSalamandridae, based on the fact that

there are more subtelocentric chromosomes

individual
betweendifferences
chromosomes by changing the telocentricchromosomes in meiotic metaphase II, andmore variations of relative chiasma
The same
numbers in diakinesis.
based onwasreachedconclusion

chromosomes into metacentric ones, ormorphological comparisons (Zhao and Hu,changing the metacentric chromosomes into
submetacentric, subtelocentric and1984). However, as shown in table 2, T.
andersoni has 2 more submetacentric

The difference,in mitotic metaphase: as shown in table 2,chromosomes pairs (nos. 10-11) than T.
kweichowensis and 1 more submetacentricchromosomes pair (no. 10) than T.
The relative length ofverrucosus.
1
is largest and theno.
chromosome

the no. 12 chromosomes of 3 species inrelative length of no. 12 is the shortest in T.

telocentric chromosomes.

which occured not only at the intergenericlevel, but also at the intrageneric level, areas follows:1.

The data of chromosomes

Asiatic Herpetological ResearchVol. 4, p. 156

andersoni among the 3 species above.
These data could provide cytogenetic

February 1992

MORESCALCHI, A. 1973. Pp. 233-259. In A. B.
Chirelli and E. Capama (eds.), Cytotaxonomy

evidence for reestablishing Echinototriton.

and vertebrate evolution. Academic Press, New

The mitotic chromosome number andYork.

morphology of C. orientalis are the same as
those of P. brevipes, but the color, size anddistribution of C-bands are different fromeach other. Consequently, the authors
proposed that the differences ofheterochromaticcomponents

MORESCALCHI, A.

Chromosome

1975.

evolution in the caudata Amphibia.

387.

In

T.

et

Dobzhansky

Evolutionary Biology, vol.
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genera could be the block of interspecificfertilization (Zhu and Wei, 1981).

MORESCALCHI, A., G. ODIERNA, AND E. OLMO.
1977.

Karyological relationships between thecryptobranchid
33:1579-1581.

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