2004

Asiatic Herpetological ResearchVol. 10, pp. 282-294

Intraspecific and Interspecific Genome Size Variation in Hynobiid
Salamanders of Russia and Kazakhstan: Determination by
Flow Cytometry
SPARTAK N. LITVINCHUK1,*, LEO J. BORKIN2 AND JURY M. ROSANOV1
1Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. Petersburg 194064, Russia;*E-mail: slitvinchuk@yahoo.com
2Zoological Institute, Russian Academy of Sciences, Universitetskaya nab. 1, St. Petersburg 199034, Russia
Abstract. - The amount of DNA per diploid nucleus in Salamandrella keyserlingii and Onychodactylus fischeri from
Russia, as well as in Ranodon sibiricus from Kazakhstan was determined by flow cytometry. Onychodactylus fischeri
had the highest genome size, Ranodon sibiricus the lowest, and Salamandrella keyserlingii was intermediate. Obvious
geographic variation in genome size was revealed for Salamandrella keyserlingii and Ranodon sibiricus. The comparison of nuclear DNA content in eleven hynobiid species supported the genus Onychodactylus as a separate lineage.
Key words. - nuclear DNA content, genome size, flow cytometry, Caudata, Hynobiidae, Onychodactylus fischeri,
Salamandrella keyserlingii, Ranodon sibiricus, Russia, Kazakhstan.

Introduction
Diploid genome size, measured in picograms (pg; 1 pg
= 10-12 g) of DNA per nucleus, varies widely in vertebrates. Living amphibians represent the largest range ofgenome size variability among terrestrial vertebrates,with a minimum of 1.7 pg in male frogs ofEleutherodactylus shrevei (Schmid et al., 2002) andmaximum of 241 pg in Necturus lewisi (Olmo, 1973).
Some extinct amphibians may have had maximum DNA
contents greater (approximately 300 pg) than the maximum known in living forms (Thoson and Muraszko,1978).
Various methods are used for the measuring ofgenome size in amphibians (for example, biochemical
analysis, Feulgen densitometry, static cell fluorometry,ultraviolet microscopy, etc.). However, modern studies
are based on a comparatively new and very precise
method of flow DNA cytometry. As a rule, standard
errors of this method (peak mean ratios) are less than
0.5% (Rosanov and Vinogradov, 1998). Among approximately 415 amphibian species examined, nuclear DNA
contents were studied by flow cytometry in about 143
species only (Gregory, 2001a).
Among some specific applications, DNA flow
cytometry has been used to examine ploidy levels(Borkin et al., 1986, 1996, 2001a; Vinogradov et al.,
1990; Sharbel et al., 1997; Litvinchuk et al., 1998,
2001), to identify morphologically similar species(Sharbel et al., 1995; MacCulloch et al., 1996;
Litvinchuk et al., 1997, 1999; Murphy et al., 1997;
Borkin et al., 2001b, 2003; Khalturin et al., 2003), andto search for hybrid individuals (Borkin et al., 1987,© 2004 by Asiatic Herpetological Research

2002; Litvinchuk et al., 2003). Many authors have noteda relationship between genome size and some biologicalparameters, such as cellular and nuclear sizes, replication time, cell-cycle length, cell division rate, metabolic
rate, longevity, morphological complexity in the brain,
etc. (Van't Hoff and Sparrow, 1963; Goin et al., 1968;
Olmo and Morescalchi, 1975; Bachmann and Nishioka,
1978; Sessions and Larson, 1987; Shakhbazov andGapchenko, 1990; Licht and Lowcock, 1991; Nevo andBeiles, 1991; Roth et al., 1994; Vinogradov, 1999;
Gregory, 2001b, 2001c; Griffith et al., 2003). However,an adaptive value of genome size is most clearly shownin its relationship with the timing of embryonic development in amphibians (Bachmann, 1972; Oeldorf et al.,
1978; Horner and Macgregor, 1983; Pagel and Jonstone,
1992; Jockusch, 1997; Chipman et al., 2001; Gregory,
2002, 2003).
This research is focused on salamanders of the family Hynobiidae, which consists of approximately 42
species. Among modern amphibians, hynobiids are recognized to be one of the primitive urodelans (e.g.,
Duellman and Trueb, 1986; Larson and Dimmick,1993). Hynobiids inhabit various mountain regions of
Palaearctic Asia, where they have quite restricted
ranges. The territory of Russia and Kazakhstan are only
inhabited by three hynobiid species. Other family members, such as the Siberian Salamander (Salamandrella
keyserlingii), is widely distributed from Northeastern
European Russia to the Kamchatka Peninsula. The
Ussuri Clawed Salamander (Onychodactylus fischeri)can be found in the southern part of Russian Far East andin the Korean Peninsula. The Semirechensk salamander(Ranodon sibiricus) inhabits the Dzhungarsky Alatau

Vol. 10, p. 283

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2004

Figure 1. Distribution of Salamandrella keyserlingii (grayish area), Ranodon sibiricus (dark square), andOnychodactylus fischeri (dark-grayish area), with localities studied (locality number as in Table 1). Dark circles designate the "Euro-Siberian" type of Salamandrella keyserlingii, white ones - "Primorsky" type.

Mountains in southeastern Kazakhstan and adjacent
China. The last two species are included in the Russianand Kazakhstan Red Data Books, respectively. Ranodon
sibiricus is also included in the IUCN Red List of
Threatened Animals under the category "vulnerable"(Borkin, 1998; Kuzmin, 1999). The goal of present
paper is to evaluate the intra- and interspecific genome
size variation in three hynobiid species of Russia andKazakhstan, with flow cytometry.

Peripheral blood cells of specimens of the Ribbed
Newt, Pleurodeles waltl, received from Prof. J. C.
Lacroix (Université Paris VI, Paris, France) were used
as a reference standard. Details of the technique havebeen published previously (Vinogradov et al., 1990;
Rosanov and Vinogradov, 1998; Borkin et al., 2001).
The relative genome size differences (RD) were calculated with use of formula: RD=(m1-m2)/(m1+m2) ×200%, where m1 and m2 are the sample means.

Materials and methods

Results

Eighty-eight metamorphosed specimens from 13 populations of three species distributed in Russia andKazakhstan were used in this study of genome size variation. Blood was taken from the clipped tail tip. The
samples of Salamandrella keyserlingii and
Onychodactylus fischeri studied are kept at the collections of the Department of Herpetology, Zoological
Institute, Russian Academy of Sciences. All specimensof Ranodon sibiricus were obtained from the Almaty
Zoo (Kazakhstan), and after preparation of blood samples, the live salamanders were sent to the Moscow Zoo.

The genome size variation in three hynobiid species varied between 54.5 and 109.0 pg (Table 1). The lowest
nuclear DNA content was recorded in Ranodon sibiricus(in average 55.4 pg), whereas the highest was in
Onychodactylus fischeri (107.7 pg). Salamandrella keyserlingii was intermediate in size (66.4 pg).
Differences between populations were studied in
two species, represented by more than one sample. In S.
keyserlingii, ten samples were allocated to two main,
distinct groups with different genome sizes (Table 1 andFig. 1). The geographic distribution of these two groups

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Asiatic Herpetological ResearchVol. 10, p. 284

Table 1. Locality of origin ("Lat" is latitude, and "Lon" is longitude), sample size, genome size (in picograms; SD is
standard deviation) with the coefficient of variation (CV; in percents) for 13 populations of three hynobiid species.
Province/Region

Locality

Salamandrella keyserlingii (Russia)
The "Euro-Siberian" group
1 Nizhny Novgorod
Pizhma2 Udmurtia
Chur
3 Ekaterinburg
Ekaterinburg
4 Tomsk
Tomsk
5 Yakutia
Khandyga
6 Kamchatka
Petropavlovsk
7 Kurile Islands
Paramushir Island
8 Sakhalin Island
Aniva
TOTAL (for the group)
The "Primorsky" type9 Primorsky
10 Primorsky

Kedrovaya Pad' Reserve
Ussuriysk Reserve
TOTAL (for the group)
TOTAL (for the species)

Onychodactylus fischeri (Russia)
11 Primorsky
Ussuriysk Reserve
Ranodon sibiricus (Kazakhstan)
12 Taldy-Kurgan
Oy-Saz River
13 Taldy-Kurgan
Borokhudzir River
TOTAL

Lat

Lon

SD

Range

CV

57°52'
57°06'
56°51'
56°31'
62°39'
53°02'
50°41'
46°43'

47°05' 5
52°59' 10
60°43' 15
84°58' 11
135°33' 2
158°38' 1
156°07' 2
142°31' 2
48

66.7
67.5
66.6
66.6
67.8
66.8
67.1
66.5
66.9

0.2
0.4
0.5
0.6

66.5 - 66.9
66.7 - 68.1
65.5 - 67.7
66.0 - 68.0
67.5 - 68.0

0.3
0.6
0.8
0.9

131°32' 11 64.8
132°09' 2 64.8
13 64.8
61 66.4

0.3

43°38'
44°51'
44°30'

43°07'
43°38'n Mean0.6

66.8 - 67.4
66.3 - 66.6
65.5 - 68.1

0.4

0.2
1.0

64.4 - 65.2
64.7 - 64.8
64.4 - 65.2
64.4 - 68.1

132°09' 7 107.7

0.7

106.7 - 109.0

0.7

79°02'
79°28'

0.2
0.6
0.7

54.5 - 55.1
55.1 - 56.7
54.5 - 56.7

0.3
1.0
1.3

10 54.8
10 56.0
20 55.4

Table 2. The genome size (GS; in picograms) of hynobiids, referred by some authors.
Species
LocalityGS
Reference
Hynobius (Hynobius) dunni
H. (H.) nebulosus
H. (H.) tsuensis
H. (Satobius) retardatus
H. (Pseudosalamandra) naevis
Onychodactylus fischeri

O. japonicus
Paradactylodon gorganensis
P. mustersi
Ranodon sibiricus

Salamandrella keyserlingii

0.9

unknown
33.762
unknown
38.402
unknown
32.962
unknown
38.312
unknown
40.902
Ussuriysk Reserve 45.52-//95.081-//107.71
unknown
102-1062
Shirabad cave
34.771
unknown
43.32
Tekeli50.72-//45.5911
2 localities
55.41
Yakutia
42.52
Ekaterinburg
42.32
unknown
382
Ekaterinburg
33.23-//55.4811
10 localities
66.411

1Flow cytometry; 2Feulgen's densitometry; 3Kinetics reassociation.

Olmo, 1973
Olmo, 1973
Olmo, 1973
Olmo, 1973
Olmo, 1973
Mazin, 1978
Vinogradov, 1998
Present paper
Olmo, 1983
Stöck, 1999
Morescalchi et al., 1979
Morescalchi et al., 1979
Vinogradov, 1998
Present paper
Mazin, 1978
Morescalchi et al., 1979
Grafodatsky and Grigoriev, 1982
Vladychenskaya et al., 1988
Vinogradov, 1998
Present paper

0.4
1.5

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Table 3. Genome size (2C), range of egg diameters (mm), and time of embryonic ("Embr.") and larval ("Larv.") development (days) in some hynobiids.
Taxon

2C

Reference

Egg

Embr.

LarvaeReference

Hynobius dunni

33.8

Olmo, 1973

2.0

-

-

Thorn, 1969

H. nebulosus

38.4

Olmo, 1973

2.6-5.025-303138-2741

Thorn, 1969

H. tsuensis

33.0

Olmo, 1973

4.0-

90-1501

Thorn, 1969

H. retardatus

38.3

Olmo, 1973

2.3-3.1

21-28280-7202

H. naevis

40.9

Olmo, 1973

5.0-5.3

8-411

1201

Sasaki, 1924;
Mikamo, 1956
Thorn, 1969

Onychodactylus 107.7
fischeriPresent paper

5.0-8.03602

780-15002

Smirina et al., 1994;
Griffin and Solkin, 1995;
Kuzmin, 1995

O. japonicus

104.0

Olmo, 1983

4.5-5.4

120-1503~1000

Paradactylodon
gorganensis
P. mustersi

34.8

Stöck, 1999

-

-

>3602

Iwasawa and Kera, 1980;
Hayase and Yamane, 1982
Stöck, 1999

43.3

-

>3602

Reilly, 1983

Ranodonsibiricus

55.4

Morescalchi et al., 1979
Present paper
3.0-5.021-422230-7202

Lebedkina, 1964;
Brushko, Narbaeva, 1988;
our data

Salamandrella
66.4
Present paper
2.1-3.5
144
78-1101
keyserlingii
1Laboratory conditions (temperature unknown); 2nature conditions; 3t = 10°; 4t = 16°.

Ishchenko et al., 1995a,b;
Berman, 1996

Table 4. Genome size (2C), diploid number of chromosomes (2n) and number of uniarmed macrochromosomes (UM)in some hynobiids.
Taxon

2C

Reference2nUMReference

Hynobius dunni

33.8

Olmo, 1973

56

0Morescalchi et al., 1979; Setoet al., 1986

H. nebulosus

38.4

Olmo, 1973

56

0-2 Seto et al., 1986; Kuro-o et al.,1987

H. tsuensis

33.0

Olmo, 1973

56

0Seto et al., 1986

H. retardatus

38.3

Olmo, 1973

40

0

Azumi and Sasaki, 1971; Kuroo et al., 1987

H. naevis

40.9

Olmo, 1973

58

0

Kohno et al., 1987

Onychodactylus
fischeri107.7 Present paper

78

0

Iizuka and Yazawa, 1994

O. japonicus

104.0 Olmo, 1983

78

0Morescalchi et al.,Kohno et al., 1991

Paradactylodon
gorganensis

34.8

Stöck, 1999

62

0

Stöck, 1999

P. mustersi

43.3

Morescalchi et al.,
1979

62

10

Morescalchi et al., 1979

Ranodon sibiricus

55.4

Present paper

66

18

Morescalchi et al., 1979

Salamandrella
keyserlingii

66.4

Present paper

62

26

Kohno et al., 1991

1979;

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Asiatic Herpetological ResearchVol. 10, p. 286

Discussion
Number of Specimens

1) Genome size values in hynobiids: literature data

Genome Size (pg)Figure 2. Genome size distribution in Salamandrella keyserlingii.

of samples proved to not be chaotic and the "EuroSiberian" (8 samples) and "Primorsky" (2 samples)groups were recognized. The data ranges of the groups
did not overlap, and the gap between both groups wasequal to 0.3 pg (Fig. 2). The "Primorsky" samples werecharacterized by smaller genome size in comparison
with the "Euro-Siberian" ones: 64.4-65.2 pg vs. 65.568.1 pg; the means were 64.8 pg vs. 66.9 pg. The differences (RD) between means were equal to 3.2%, andwere observed in five separate comparisons. Therefore,
these groups were shaped both by genome size and geographically.
The samples of R. sibiricus were taken from twosemi-isolated populations from upper parts of
Borokhudzir and Oy-Saz rivers. Both samples demonstrated different genome sizes (Table 1). The ranges ofgenome size values in these two samples slightly overlapped. The Oy-Saz sample was characterized by smaller genome size in comparison with the Borokhudzir
sample: 54.5-55.1 pg vs. 55.1-56.7 pg; the means are
54.8 pg vs. 56.0 pg. The differences (RD) between
means were equal to 2.2%.
The coefficient of variation (CV) ranged between
0.3% and 0.9% in S. keyserlingii, between 0.3% and1.0% in R. sibiricus, and was equal to 0.7% in the sample of O. fischeri (Table 1). The overall within-species
genome size variation in S. keyserlingii and R. sibiricus
were quite similar (1.5% and 1.3%, respectively).
Among the "Euro-Siberian" samples of S. keyserlingii,
the CVs ranged between 0.3% (Nizhny Novgorod
Province) and 0.9% (Tomsk Province), whereas it was
equal to 0.4% in the "Primorsky" samples of S. keyserlingii.

Presently, the nuclear DNA content has been determinedfor eleven hynobiid species. However, among them,
only four species were examined through flow cytometry (Table 2). The first data collected about genome sizeof nine hynobiid species was obtained by Italian
researchers with an application of Feulgen densitometry(Olmo, 1973, 1983; Olmo and Morescalchi, 1975;
Morescalchi et al., 1979). According to these data,
genome size in hynobiids ranged from 32.96 pg in
Hynobius tsuensis to 102-106 pg in Onychodactylus
japonicus (Table 2). According to Mazin (1978), thenuclear DNA content in two Russian species,
Onychodactylus
fischeriandSalamandrella("Hynobius") keyserlingii, measured by Feulgen densitometry, were similar to each other (45.5 and 42.5 pg,
respectively). Grafodatsky and Grigoriev (1982) estimated genome size of Salamandrella keyserlingii (38pg), perhaps, by means of Feulgen densitometry.
Vladychenskaya et al. (1988) studied kinetics of DNA
reassociation; they found that the nuclear DNA content
of Salamandrella keyserlingii was equal to 33.2 pg.
Using DNA flow cytometry, Vinogradov (1998) estimated genome size values as 95.08 pg for
Onychodactylus fischeri, 45.59 pg for Ranodon sibiricus, and 55.48 pg for Salamandrella keyserlingii.
Finally, Stöck (1999) determined that genome size of
Batrachuperus gorganensis is 34.77 pg, using DNA
flow cytometry as well.
The literature values of genome size for
Onychodactylus fischeri, Ranodon sibiricus, andSalamandrella keyserlingii expressed in absolute units
vary sometimes more than two-fold (ranges are 45.595.1 pg, 45.6-50.7 pg, and 33.2-55.5 pg, respectively).
Therefore, the comparison of data provided by various
authors should be made cautiously. The contradictions
may be explained by an application of different techniques (Feulgen densitometry, kinetics reassociation,and flow cytometry), dyes, and laboratory conditions(cell preparation methods, devices for measurements,
types of reference cell standards, etc.). It has been shownthat genome size measured with fluorochromes of different nucleotide specificity may differ markedly (e.g.,
Johnston et al., 1987; Birshtein et al., 1993; Vinogradov
and Borkin, 1993). For instance, the determination of
genome size by means of flow cytometry for cell samples of Salamandrella keyserlingii stained witholivomycin and Hoechst (GC- and AT-specific fluorochromes, respectively) provided 6.68 and 4.77 arbitrary units (Rana temporaria was taken as an internalreference; Vinogradov, 1998). To exclude the influenceVol. 10, p. 287

Asiatic Herpetological Researchof AT/GC-structure, it is necessary to use ethidium bromide or propidium iodide (Vinogradov and Borkin,1993), which were used in our research.
To convert genome size from the relative units topicograms, it is necessary to have data about genome
size of reference cells. Such data should be obtained
without using stains. Unfortunately, the data available
today, mentioned by various authors, do not correspondto each other. For instance, Vinogradov (1998) reported
a genome size of reference standard Mus musculus to be
6.5 pg. Our estimations of genome size of some mammals (Homo sapiens, Mus musculus, Rattus norvegicus)
were the closest to that mentioned by Bianchi et al.
(1983). In our work, we used the genome size of malesof Mus musculus (C57B1) as a basic reference standard
with value of 6.8 pg. Other authors preferred other reference standards, which have different base-pair-specificity of some stains widely used in flow cytometry.
Vinogradov and Borkin (1993) listed the AT- and GCpair specific DNA contents (CAT and CGC) for many
species of amphibians. For instance, CAT/CGC was
equal to 1.42 in Xenopus laevis (Mazin's reference standard), and 1.00 in Rana lessonae (= Rana "esculenta";
Olmo's and Morescalchi's standard).
The estimations of Italian authors (Olmo, 1973,
1983; Morescalchi et al., 1979) and our genome size values for Ranodon sibiricus (50.7 and 54.5-56.7 pg,
respectively) and members of genus Onychodactylus(102.0-106.0 and 106.7-109.0 pg, respectively) are quite
similar. However, data for Salamandrella keyserlingii(42.3 and 64.4-68.1 pg, respectively) are in obvious discordance. The genome size of Onychodactylus fischeri,
Ranodon sibiricus, and Salamandrella keyserlingii mentioned by Vinogradov (1998), was lower than our values, who used other stains and lower genome size estimation of basic reference standard.
Unfortunately, some authors did not supply any
information about sample sizes and localities. However,some differences in genome size might be influenced byintra-population and geographic variation as well.
2) Within-species variation
Some authors discussed the levels of intraspecific variation in genome size. We recognized two kinds of such avariation; namely, the "within-population" variation and"between-population" (or geographical) variation.
A) Within Population Variation
Among amphibians, the greatest intrapopulational variation (CV = 7.5%, the data of Licht and Lowcock, 1991
were recalculated by us) was recorded for the Western
Red-back Salamander (Plethodon vehiculum). However,the variation in other amphibian species was considerably lower (Licht and Lowcock, 1991; MacCulloch et2004al., 1996; Murphy et al., 1997; Lizana et al., 2000). The
variation within populations of three hynobiid species(CVs were 0.3-1.0%, mean was 0.67 ± 0.12%) was quite
similar to that in salamandrids (range is 0.1-1.7%, meanis 0.64 ± 0.03%, 99 populations studied), pelobatids, andother anurans studied in our laboratory at the same conditions (Litvinchuk et al., 1997, 1999, 2001a,b, 2003;
Rosanov and Vinogradov, 1998; Borkin et al., 2001b,
2003; our data).
Sexual dimorphism in genome size has been registered in some amphibian species (Schmid et al., 2002;
our data). Unfortunately, in the hynobiids examined byus, sexual differences are not expressed in external characters beyond the breeding time. Our study was based
mostly on juvenile and non-breeding adult animals, and,
therefore, we failed to reliably identify the sex without
anatomical dissections.
B) Geographical Variation
The significant geographical variation in genome size
was revealed for several amphibian species (Licht andLowcock, 1991; Murphy et al., 1997; Litvinchuk et al.,1999, 2001b). In a few cases, differences (RD) exceeded 8%. However, in the majority of species studied, suchdifferences were about 1% (Licht and Lowckock, 1991;
Borkin et al., 1997, 2000, 2001, 2003; Litvinchuk et al.,1997, 1999, 2001b, 2003; our data). In Salamandrella
keyserlingii, the maximum genome size difference (RD= 4.5%) was found between samples from the Khandyga(Yakutia Republic) and Kedrovaya Pad' Reserve(Primorsky Territory). The average differences (RD)between the "Euro-Siberian" and the "Primorsky" sample groups of the species were equal to 3.2%.
3) Interspecies differences: developmental and karyological correlations
Eleven species of the family Hynobiidae may be divided into two groups by their genome size. Two species of
the genus Onychodactylus form a group with the largest
genomes (104-108 pg). They also have the longest
embryonic and larval development periods (Table 3), as
well as the greatest number of chromosomes (Table 4).
Another group includes the remaining nine species(33-67 pg). Among them, Salamandrella keyserlingii
has the largest genome size, and the seven species fromthe genera Hynobius and Batrachuperus have smaller
sizes. Ranodon sibiricus has an intermediate genome
size. Such a distribution of genome sizes in the second
group does not seem to be associated with ovum diameter, and, perhaps, with time of embryonic and larval
development (Table 3).
The comparison of genome size values in the second group with karyological data evidenced no signifi-

2004

Asiatic Herpetological Researchcant relations between the nuclear DNA content anddiploid chromosome numbers (Table 4). Nevertheless,we found the positive correlation (r = 0.9998) between
genome size and uniarmed macrochromosome numbers.
4) Intergeneric relationships
Based on recent studies (Fei and Ye, 2000a; Fu et al.,2001), approximately eight or ten genera of the hynobiids could be recognized. The most speciose genus,
Hynobius, consists of about 24 species, which may bearranged into three subgenera (Matsui et al., 1992;
Mizuno et al., 1995; Borkin, 1999), namely
Pseudosalamandra (7 species from Japan and Taiwan),
Satobius (1 species from Hokkaido Island), andHynobius (11 species from Japan, and, perhaps, 5species from Korea and China). The family also includes
the genera Liua (1 species), Onychodactylus (2 species),
Pachyhynobius (1 species), Protohynobius (1 species),
Pseudohynobius (2 species), Ranodon (1 species),
Salamandrella (1 species, which penetrates to eastern
Europe), and Batrachuperus (sensu lato) (about 9
species). The taxonomic position of Ranodon, Liua, andPseudohynobius was discussed (Fei and Ye, 2000b;
Kuzmin and Thiesmeier, 2001). Based on mitochondrial
DNA study, T. Papenfuss (Pers. comm. in Fu et al.,2001) showed that the genus Batrachuperus is paraphyletic, and consists of two groups with distinct geographic distributions. The Eastern (Chinese) groupbelongs to Batrachuperus (sensu stricto) and consists ofabout six species (Fei and Ye, 2000b; Fu et al., 2001;
Song et al., 2001). The western (Iran and Afganistan)group includes two or three species (Stöck, 1999), andmay be allocated to Paradactylodon.
Various authors suggested several configurations ofevolutionary relationships among hynobiids. For
instance, based on reproductive biology characters,
Thorn (1969) proposed to recognize two families:
Ranodontidae, with the genus Ranodon only, andHynobiidae, with remaining genera. Using a set of morphological and biological data, Zhao and Hu (1984) separated two "natural" groups among Chinese species: theHynobius group with predominantly terrestrial species(Hynobius and Salamandrella), and the Ranodon groupwith predominantly aquatic inhabitants (Ranodon,
Onychodactylus, Batrachuperus, and Liua). Later, Zhao
and Zhang (1985) assigned the genus Pachyhynobius toa third group. Combining morphological characters andmitochondrial DNA data, Larson and Dimmick (1993)
found the closest relationships between the genera
Salamandrella and Hynobius, thus confirming the traditional acceptance of the similarity of these taxa.
However, the genus Onychodactylus was more closely
related to that lineage, whereas Batrachuperus (the east-

Vol. 10, p. 288

ern group) proved to be more distant. Recently, Fei andYe (2000a) have erected a new subfamily for the newly
discovered Protohynobius puxiongensis, whereas allother hynobiids were allocated to another subfamily.
Genome size data are not in agreement with allthese suggestions, which did not recognize separate
position of the genus Onychodactylus. Apart from thelargest genome size, distincton of the genus was also
supported by morphological and karyological data
(Morescalchi et al., 1979; Olmo, 1983; Kohno et al.,
1991; Iizuka, Yazawa, 1994; Kuro-o et al., 2000;
Litvinchuk and Borkin, 2003).
5) Taxonomic considerations
Based on the intraspecific analysis, Bassarukin andBorkin (1984), and Borkin (1994) outlined the peculiarities of Salamandrella keyserlingii from the southernpart of the Russian Far East. Moreover, these authors
suggested that local populations would be considered asa geographic race of the species (the species type territory is the Kultuk Village, Baikal Lake, Irkutsk
Province, Russia; restricted by Borkin, 1994). Indeed,the "Euro-Siberian" and "Primorsky" groups of populations are different in allozymes (Litvinchuk et al.,2001a), in some morphological characters (Ostashko,
1981; Bassarukin and Borkin, 1984; Borkin, 1994;
Litvinchuk and Borkin, 2003), in breeding sites(Kuzmin, 1990), and in the shape of egg sacs and timeof larval development (Korotkov, 1977; Bassarukin andBorkin, 1984; Sapozhnikov, 1990; Vorobyeva et al.,1999). Therefore, geographic differences in genome size
revealed by us are in concert with differences in othercharacters. Based on a concordance of various characters, including genome size, we would recognize a distinct status of the "Primorsky" samples, at least, of a subspecific rank. Formerly, Nikolsky (1906) and Dybowski(1928) have coined the names Salamandrella keyserlingii var. tridactyla and Salamandrella keyserlingii var.
kalinowskiana, respectively, for animals from theRussian Far East. Obviously, the first name has the priority. Therefore, populations from Russian Far East
(Primorsky Territory) should be named Salamandrella
keyserlingii tridactyla Nikolsky, 1906.
The range of Ranodon sibiricus is quite limited.
Nevertheless, it consists of several semi-isolated areas(Brushko et al., 1988; Kuzmin et al., 1998; our data).
The populations from the upper parts of Borokhudzir
and Oy-Saz Rivers are separated from each other by amountain range. Therefore, it is not surprising that the
genome size difference (RD) between them is relatively
large (2.2%). However, geographical variation in thespecies is poorly studied, and so the taxonomic value ofthat difference is still unclear.

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Acknowledgments
We thank A. G. Borissovsky (Izhevsk), V. G. Ishchenko(Ekaterinburg), V. N. Kuranova (Tomsk), J. C. Lacroax(Paris), I. V. Maslova (Spassk-Dal'ny), N. Panfilova(Almaty), M. V. Pestov (Nizhniy Novgorod), G. P.
Sapozhnikov (Vilnus), V. T. Sedalishchev (Yakutsk), E.
I. Vorobyeva (Moscow), Y. Zhuravlyev (Almaty), andthe staff of Almaty and Moscow Zoo terraria for help us
in the field or providing animals. We also thank
Theodore Papenfuss (Berkeley) for valuable comments
and English corrections of the earlier version of thepaper. The research was funded by the INTAS (grant no97-11909), the Russian Foundation for Basic Research(the grant No. 02-04-49631), and the Federal State
Program "Integration" (the grant No. E-0121).

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Herpetologica Sinica 4:36-40. (In Chinese).

Vol. 10, p. 294