Folia Zool. – 57(4): 337–346 (2008)
Chromosomal studies on Greek populations of four small rodent
species
George P. Mitsainas, Michail Th. Rovatsos, Irene Karamariti
and Eva B. Giagia-Athanasopoulou*
Laboratory of Zoology, Section of Animal Biology, Department of Biology, University of Patras, GR-26504 Rio,
Greece; e-mails: [email protected]; [email protected]; [email protected]; [email protected]
Received 4 August 2006; Accepted 7 October 2008
A b s t r a c t . The results of a karyological study on the dormice Myoxus glis (2n=62) and
Dryomys nitedula (2n=48), the ground squirrel Spermophilus citellus (2n=40) and the red bank
vole Myodes glareolus (2n=56) from Greece are presented. Apart from the clarification of their
diploid chromosome number, a more elaborate study of their karyotype was conducted and the Gand C- banding patterns are provided for the first time in Greek populations of these species. In
particular, heterochromatin distribution in D. nitedula seems to be more extensive than previously
thought, contrasting M. glis, in which heterochromatin seems to be absent. On the other hand,
the Y chromosome of M. glareolus was found to be a fully heterochromatic submetacentric. In
overall, the comparison of our karyological results, with those of other Eurasian populations
reinforce the belief that the karyotypes of the studied species are conservative, displaying small
degrees of variation, usually restricted to the size and morphology of the sex chromosomes.
Key words: Myoxus glis, Dryomys nitedula, Spermophilus citellus, Myodes glareolus, karyotype, heterochromatin
Introduction
Throughout the past decades, there has been an extensive effort to describe the chromosomal
constitution and variation in mammalian taxa, particularly of those distributed in Europe
(Z i m a 2000, 2004). However, the karyotypes of many Greek mammalian taxa still remain
largely unknown. Their study could contribute to the clarification of their taxonomy and
phylogenetic relationships both within and among related taxa. In this context, an effort is
being made during several years to collect and study individuals of different small mammalian
species from various localities of Greece.
Among the representatives of the family Myoxidae, the most common species in Greece
are the dormice Myoxus glis and Dryomys nitedula (O n d r i a s 1966, N i e t h a m m e r
1986, V o h r a l í k & S o f i a n i d o u 1987, 1992). Their karyotypes with 2n=62
and 2n=48, respectively, are known from their populations in several Palearctic regions
(F i l i p p u c c i et al. 1985, B e l c h e v a et al. 1988, G r a p h o d a t s k y & F o k i n
1993, C i v i t e l l i et al. 1995, P e s h e v & D e l o v 1995).
With regard to the European ground squirrel, Spermophilus citellus (Sciuridae), the
southernmost limit of its distribution lies in northern Greece. Based on the use of conventional
staining, populations of this species from northern Greece, as well as from Bulgaria, Former
Yugoslavia and European Turkey have 2n=40 (S o l d a t o v i ć et al. 1984).
The red bank vole Myodes glareolus displays a vast Palaearctic distribution
(S h e n b r o t & K r a s n o v 2005). Its karyotype is characterized by 2n=56 (K r á l 1972,
K r á l et al. 1979, Z i m a 1984) and also by variation in the centromeric position of the
Y chromosome in several European populations (K r á l et al. 1972, Ž i v k o v i ć et al.
* Corresponding author
337
1975, V o r o n t s o v et al. 1978, G a m p e r l 1982, R a d o s a v l j e v i ć et al. 1988,
V u j o š e v i ć & B l a g o j e v i ć 1997).
Since very little is known on the chromosomal constitution of the above four species
from Greece, the aim of this study was to fill this gap, using the G- and C-banding staining
techniques. The results are discussed and compared with those from previous works
concerning other European populations.
Materials and Methods
A total of 30 individuals, belonging to the species Myoxus glis, Dryomys nitedula,
Spermophilus citellus and Myodes glareolus were karyologically studied. The collection
localities and the number of individuals collected per species are given in Table 1 and
Fig. 1, respectively. Direct chromosome preparations were made, using a modified version
of the bone marrow method (H s u & P a t t o n 1969). For chromosome identification,
the G–banding technique (S e a b r i g h t 1971) was implemented. In order to study the
distribution of the heterochromatin, S u m n e r ’ s C–banding method (1972) was used, with
some modifications. For all chromosomal studies, a Zeiss Axioscope 2 Plus light microscope
was used, equipped with a Zeiss Axiocam MRc5 (5MP) digital camera.
Table 1. Sampling localities of the studied animals and their karyological characteristics.
Taxon
Locality
Dryomys nitedula
-”Myodes glareolus
-”Myoxus glis
-”Spermophilus citellus
-”-
Kastritsi
Lagadikia
Pisoderi
Platza
Charakas
Kastritsi
Seli
Thermi
No. of individuals
Total
m
f
6
3
3
3
1
2
3
2
1
4
1
3
2
1
1
8
2
6
1
1
3
2
1
2n
FNa
FN
48
48
56
56
62
62
40
40
92
92
56
56
120
120
76
76
96
96
58
58
124
124
80
80
Results
The karyological study of the ten individuals of Myoxus glis revealed an identical karyotype
of 2n=62 (Fig. 2). All autosomes were found to be biarmed and of gradually decreasing
size. Most of them were metacentric or submetacentric, thus FNa=120. In addition, one of
the small autosomal pairs appeared to bear a secondary constriction. The X chromosome
was large sized and metacentric, whereas the Y chromosome was approximately equal in
size to the smallest autosomal pair and most likely acrocentric. The implementation of the
C–banding staining technique failed to reveal heterochromatic bands in the chromosomes of
our specimens, even in their centromeric regions.
All nine specimens of Dryomys nitedula had the same karyotype with 2n=48. Most
autosomal pairs were metacentric or submetacentric and three pairs were subtelocentric
(6, 9 and 10), therefore FNa=92 (Fig. 3). The first pair was distinctly larger than the
rest of the complement. In this karyotype there appeared a small autosomal pair, no. 21,
with a secondary constriction, which in one case was in heterozygous condition. The X
chromosome was a medium to large sized submetacentric, whereas the Y chromosome
338
Fig. 1. Map showing the collection localities of the studied animal material. For details, see Table 1.
was dot-like. C–banding showed in most autosomes at least faint heterochromatic bands
in the centromeric regions (Fig. 3b). Some pairs showed very prominent, single or double,
darkly stained pericentromeric bands (e.g. 1, 2, 5, 7, 15). Notably, one pair (10) displayed
a heterochromatic band at the distal end of its large chromosomal arms, whereas another
(19) carried heterochromatic large arms. Furthermore, chromosome no. 21 seemed largely
heterochromatic and its satellite was relatively darkly stained. With regard to the sex
chromosomes, X demonstrated only faint heterochromatic bands at interstitial and distal
positions, whereas Y was at least partially heterochromatic.
The four studied individuals of Spermophilus citellus demonstrated a karyotype of 2n=40
chromosomes (Fig. 4). All autosomal pairs were biarmed, even though some of them had
tiny short arms, whereas two of them were distinctly metacentric (12 and 19), thus FNa=76.
The X chromosome was medium-sized and submetacentric, whereas Y was a dot-like
chromosome, the smallest of the complement. The C–banding staining technique showed
that all autosomes, as well as the X chromosome, possessed prominently stained centromeres
339
Fig. 2. Karyotype of a male dormouse, Myoxus glis with 2n=62, FN=124 (G–banding). Arrows indicate the 25th
chromosomal pair with secondary constrictions.
(Fig. 4b). Some pairs demonstrated additional heterochromatic bands at different positions of
the short or long arms (6, 8, 9 and 15). The Y chromosome appeared fully heterochromatic.
The seven individuals of Myodes glareolus had the typical karyotype of this species with
2n=56, FNa=56, possessing only one pair of very small metacentric autosomes (Fig. 5). The
X chromosome was a large acrocentric, while Y was a very small metacentric chromosome.
The C–positive bands were restricted to the centromeres of all chromosomes, with the
exception of Y, which appeared fully heterochromatic (Fig. 5b).
Discussion
This is the first time that the G–banding pattern, as well as the heterochromatin distribution
with the use of C–banding is reported for Greek populations of the studied species. With regard
to Myoxus glis, our sample exhibited in overall the typical, conservative karyotype of this
species, similar to what has been described in previous works for other European populations.
The small differences between published karyotypes for this species may be the result of the
subjective arrangement of the chromosomes (Z i m a et al. 1995), which is particularly true
for karyotypes with high diploid chromosome numbers. Like in our case, the X chromosome
in European populations is commonly reported as metacentric (D u l i ć et al. 1971, Z i m a
1987, B e l c h e v a et al. 1988, C i v i t e l l i et al. 1995, P e s h e v & D e l o v 1995), with
the exception of the submetacentric form in Spanish (D i a z d e l a G u a r d i a et al. 1980)
and Russian populations (G r a p h o d a t s k y & F o k i n 1993). The large sized variant
in our sample has also been reported from European Turkey (C i v i t e l l i et al. 1995).
On the other hand, the small, probably acrocentric Y variant of our sample has also been
recorded at least from Italy (C i v i t e l l i et al. 1995), Bulgaria (B e l c h e v a et al. 1988)
340
Fig. 3. a) G–banded and b) C–banded karyotype of a male dormouse, Dryomys nitedula with 2n=48, FN=96.
Arrows indicate the 21st chromosomal pair with a heterozygous secondary constriction.
341
Fig. 4. a) G-banded karyotype of a female and b) C-banded karyotype of a male ground squirrel, Spermophilus
citellus with 2n=40, FN=80.
and former Czechoslovakia (Z i m a & K r á l 1984, Z i m a 1987). Finally, the single pair
with secondary constrictions, shown as the 25th in Fig. 2, following G r a p h o d a t s k y &
F o k i n (1993), has been mentioned in previous works, indicating that it is a common feature
342
Fig. 5. G–banded and b) C–banded karyotype of a male red bank vole, Myodes glareolus with 2n=56, FN=58.
of this species’ karyotype. With regard to the heterochromatin distribution and in agreement
with our efforts, such bands are very rarely demonstrated. In fact, only a few relevant reports
exist for M. glis (B e l c h e v a et al. 1988, C i v i t e l l i et al. 1995), leading some authors
to conclude that heterochromatin absence is a chromosome feature in some dormice species
(G r a p h o d a t s k y & F o k i n 1993).
343
Our results on Dryomys nitedula and those of previous works support the view that this
species is characterized by a rather conservative karyotype of 2n=48 (F i l i p p u c c i et al.
1985, C i v i t e l l i et al. 1995, P e s h e v & D e l o v 1995), with little interpopulation
variation (Z i m a 1987, D o ğ r a m a c i & K e f e l i o ğ l u 1990, Z i m a et al. 1995).
In all studies so far, the first autosomal pair was remarkably larger than the rest, and it
seems that among Myoxidae species this is a unique karyotypic feature of D. nitedula
(F i l i p p u c c i et al. 1985). Also, the secondary constriction in a small sized pair (the
21st in Fig. 3, following C i v i t e l l i et al. (1995)) has been frequently described in the
karyotype of this species. The X chromosome is commonly recorded as a medium to large
sized metacentric (Z i m a & K r á l 1984, F i l i p p u c c i et al. 1985, P e s h e v &
D e l o v 1995, Z i m a et al. 1995), however in our case it was clearly submetacentric,
as was also reported by G r a p h o d a t s k y & F o k i n (1993) and was visible in some
karyotypes presented by C i v i t e l l i et al. (1995). On the other hand, all studies agree with
the very small-sized, occasionally dot-like, appearance of the Y chromosome. With regard
to the C–banding pattern, this is the first time, to the best of our knowledge, that numerous
heterochromatic bands at several chromosomal positions (centromeric, interstitial etc.) are
observed for this species. In a previous work (F i l i p p u c c i et al. 1985), the metacentric
X chromosome was found with two prominent C–bands at pericentromeric position,
whereas in our case the submetacentric X displayed only faint heterochromatic regions,
indicating that the X chromosome in this species may vary both in centromeric position and
heterochromatin content.
Comparison of our data for Spermophilus citellus with those of previous studies
in populations from Greece, the former Yugoslavia, Bulgaria and European Turkey
(Ž i v k o v i ć et al. 1968, S a v i ć et al. 1971, B e l c h e v a & P e s h e v 1979,
S o l d a t o v i ć et al. 1984, Ö z k u r t et al. 2002), demonstrate that all Balkan
populations have the same diploid chromosome number, 2n=40, with some differences in
chromosome size and in the interpretation of the chromosome morphology. The mediumsized submetacentric X chromosome of our sample has been previously reported with
conventional staining from Greece (S o l d a t o v i ć et al. 1984), as well as from other
regions (Ž i v k o v i ć et al. 1968, S a v i ć et al. 1971, B e l c h e v a & P e s h e v 1979,
S o l d a t o v i ć et al. 1984, Z i m a & K r á l 1984) and seems to be the common form.
However, other X variants have been described, i.e. large-sized submetacentrics (Ö z k u r t
et al. 2002) or medium-sized acrocentrics (B e l c h e v a & P e s h e v 1979). On the
contrary, the Y chromosome commonly appears as dot like, with the exception of one study
that reported a significantly larger, metacentric variant from Bulgaria and European Turkey
(S o l d a t o v i ć et al. 1984). Finally, the heterochromatin distribution in our sample from
Greece, based on C-banding, seems to generally agree with that of Z i m a (1984), but in our
case the Y chromosome is entirely heterochromatic, instead of negatively stained.
The red bank vole Myodes glareolus exhibits a fairly conservative karyotype of 2n=56,
FN=58, also demonstrated in our sample. The only notable polymorphism in this species
seems to be in the Y chromosome morphology. Specifically, the Y chromosome appears
metacentric-submetacentric in most examined populations from Europe and Asia (K r á l
1972, Ž i v k o v i ć et al. 1975, V o r o n t s o v et al. 1978, K r á l et al. 1979, G a m p e r l
1982, Z i m a et al. 1997), however a few cases describing an acrocentric variant have been
reported from southern Italy (K r á l et al. 1972) and former Yugoslavia (Ž i v k o v i ć et al.
1975). In the latter area, interpopulation variation has been reported (R a d o s a v l j e v i ć
et al. 1988, V u j o š e v i ć & B l a g o j e v i ć 1997), representing a gradual transition
344
from the acrocentric form in the NW to the metacentric-submetacentric form in the S-SE
(V u j o š e v i ć & B l a g o j e v i ć 1997). The submetacentric variants in our sample
from northern Greece seem to support this transition. Northern Greece constitutes the southeasternmost distribution border of M. glareolus in Europe, and the respective populations
are considered peripheral rather than central within its distribution range. Therefore, the
prevalence of the submetacentric Y variant in Greece is rather interesting, since it has
been postulated elsewhere that it is the acrocentric variant that characterizes the peripheral
populations of the species (V o r o n t s o v et al. 1978, V o r o n t s o v et al. 1980). With
regard to C-banding, the distribution of heterochromatin in our sample is in accordance
with previous descriptions (G a m p e r l 1982, G a m p e r l et al. 1982), including
the appearance of a large autosomal pair with just faint centromeric heterochromatic
bands (G a m p e r l 1982). In this species, the overall morphology and heterochromatin
distribution of the karyotype indicates that it is rather primitive within Arvicolinae,
since it shows great similarity to the postulated ancestral karyotype of this group: high
diploid chromosome number (2n=56) with acrocentric chromosomes and restriction
of heterochromatin to the centromere of all chromosomes, apart from a possibly fully
heterochromatic Y chromosome (M a t t h e y 1973, M o d i 1987).
Acknowledgements
We are grateful to J. Z i m a and two anonymous referees, whose detailed comments and suggestions led
to the thorough improvement of our manuscript. Also, we wish to thank C. S t a m a t o p o u l o s and
A. P a p a g i a n o p o u l o s for providing part of the studied material, as well as N. T u r l a n d for checking
the English language of the manuscript.
L I T E R AT U R E
Belcheva R.G. & Peshev D.T. 1979: Intersubspecific sex chromosome difference in Citellus citellus L., (Rodentia,
Sciuridae). Experientia 35: 595–596.
Belcheva R.G., Topashka-Ancheva M.N. & Atanassov N.I. 1988: Karyological studies of five species of mammals
from Bulgaria’s fauna. Comp. rend. bulg. Acad. Sci. 42(2): 125–128.
Civitelli M.V., Filippucci M.G., Kurtonur C. & Özkan B. 1995: Chromosome analysis of three species of
Myoxidae. Hystrix 6(1–2): 117–126.
Diaz de la Guardia R., Girela M.R. & Ladron de Guevara R.G. 1980: Los cromosomas del Glis glis pyrenaicus.
Bol. Soc. Espagnola Hist. Nat. (Biol.) 78: 165–168.
Doğramaci S. & Kefelioğlu H. 1990: (The karyology of Dryomys nitedula (Mammalia: Rodentia) in Turkey).
Turk. J. Zool. 14: 316–328 (in Turkish with English summary).
Dulić B., Savić I. & Soldatović B. 1971: The chromosomes of two rodent species, Dolomys bogdanovi (V. and E.
Martino 1922) and Glis glis (Linnaeus 1766) (Mammalia, Rodentia). Caryologia 24(3): 299–305.
Filippucci M.G., Civitelli M.V. & Capanna B. 1985: Le caryotype du létorin Dryomys nitedula (Pallas) (Rodentia,
Muridae) Mammalia 49(3): 365–368.
Gamperl R. 1982: Chromosomal evolution in the genus Clethrionomys. Genetica 57: 193–197.
Gamperl R., Ehmann C. & Bachmann K. 1982: Genome size and heterochromatin variation in rodents. Genetica
58: 199–212.
Graphodatsky A.S. & Fokin I.M. 1993: (Comparative cytogenetics of Gliridae (Rodentia)). Zool. Zh. 72(11):
104–113 (in Russian with English summary).
Hsu T.C. & Patton J.L. 1969: Bone marrow preparations for chromosome studies. In: Benirschke K. (ed.),
Comparative mammalian cytogenetics. Springer, Berlin: 454–460.
Král B. 1972: Chromosome characteristics of Muridae and Microtidae from Czechoslovakia. Acta Sc. Nat. Brno
6(12): 1–78.
345
Král B., von Lehmann E. & Zejda J. 1972: Die Hybriden zweier Unterarten der Rötelmaus (Clethrionomys
glareolus Schreb.). Zool. Listy 21: 43–61.
Král B., Zima J., Herzig-Straschil B. & Štěrba O. 1979: Karyotypes of certain small mammals from Austria. Folia
Zool. 28(1): 5–11.
Matthey R. 1973: The chromosome formulae of eutherian mammals. In: Chiarelli A.B. & Capanna B. (eds),
Cytotaxonomy and vertebrate evolution. Academic Press, London and New York: 531–616.
Modi W.S. 1987: C-banding analyses and the evolution of heterochromatin among arvicolid rodents. J. Mammal.
68: 704–714.
Niethammer J. 1986: Über griechische Nager im Museum A. Koenig in Bonn. Ann. Naturhist. Mus. Wien 88/89
B: 245–256.
Ondrias J.C. 1966: The taxonomy and geographical distribution of the rodents of Greece. Säug. Mitt.
14(Sonderheft): 1–136.
Özkurt S., Yiğit N. & Çolak E. 2002: Karyotype variation in Turkish populations of Spermophilus (Mammalia,
Rodentia). Mamm. Biol. 67: 117–119.
Peshev D.T. & Delov V. 1995: Chromosome study of three species of dormice from Bulgaria. Hystrix 6(1–2):
151–153.
Radosavljević J., Vujošević M. & Živković S. 1988: Intrapopulation variability of the Y chromosome of the bank
vole (Clethrionomys glareolus, Schreber 1780). Arh. biol. nauka 40: 3P–4P.
Savić I., Milošević M. & Živković S. 1971: Chromosomes of ground squirrel (Citellus citellus Linnaeus, 1766)
from Yugoslavia. Arh. biol. nauka 23(1–2): 35–37.
Seabright M. 1971: A rapid banding technique for human chromosomes. Lancet 11: 971–972.
Shenbrot G.I. & Krasnov B.R. 2005: Atlas of the geographic distribution of the arvicoline rodents of the world
(Rodentia, Muridae: Arvicolinae). Pensoft Publishers, Sofia.
Soldatović B., Zimonjić D., Savić I. & Giagia E. 1984: Comparative cytogenetic analysis of the populations of
european ground squirrel (Citellus citellus L.) on the Balkan Peninsula. Bulletin T. LXXXVI de l’Académie Serbe
des Sciences et des Arts – Classe des Sciences naturelles et mathématiques – Sciences naturelles 25: 47–56.
Sumner A.T. 1972: A simple technique for demonstrating centromeric heterochromatin. Exp. Cell Res. 75:
304–306.
Vohralík V. & Sofianidou T. 1987: Small mammals (Insectivora, Rodentia) of Macedonia, Greece. Acta Univ.
Carolinae-Biol. 1985: 319–354.
Vohralík V. & Sofianidou T. 1992: Small mammals (Insectivora, Rodentia) of Thrace, Greece. Acta Univ.
Carolinae-Biol. 1992: 341–369.
Vorontsov N.N., Lyapunova E.A., Ivanitskaya E.Y., Nadler C.F., Král B., Kozlovsky A.I. & Hoffman R.S. 1978:
(Variability of sex chromosomes in mammals. I. Geographical variability of morphology of Y-chromosome in voles
of genera Clethrionomys (Rodentia, Microtinae)). Genetika 14(8): 1432–1446 (in Russian with English summary).
Vorontsov N.N., Lyapunova E.A., Borissov Y.M. & Dovgal V.E. 1980: Variability of sex chromosomes in
mammals. Genetica 52–53: 361–372.
Vujošević M. & Blagojević J. 1997: Y chromosome polymorphism in the bank vole Clethrionomys glareolus
(Rodentia, Mammalia). Z. Säugetierkd. 62: 53–57.
Zima J. 1984: Chromosomes of certain small mammals from southern Bohemia and the Šumava Mts. (ČSSR).
Folia Zool. 33(2): 133–141.
Zima J. 1987: Karyotypes of certain rodents from Czechoslovakia (Sciuridae, Gliridae, Cricetidae). Folia Zool.
36: 337–343.
Zima J. 2000: Chromosomal evolution in small mammals (Insectivora, Chiroptera, Rodentia). Hystrix 11(2): 5–15.
Zima J. 2004: Karyotype variation in mammals of the Balkan Peninsula. In: Griffiths H.I., Kryštufek B. & Reed
K.M. (eds), Balkan Biodiversity: Pattern and process in the European hotspot. Kluwer Academic Publishers,
Dordrecht: 109–133.
Zima J. & Král B. 1984: Karyotypes of European mammals II. Acta Sc. Nat. Brno 18(8): 1–62.
Zima J., Macholán M. & Filippucci M.G. 1995: Chromosomal variation and systematics of Myoxids. Hystrix
6(1–2): 63–76.
Zima J., Macholán M., Kryštufek B. & Petkovski S. 1997: Karyotypes of certain small mammals (Insectivora,
Rodentia) from Macedonia. Scopolia 38: 1–15.
Živković S., Petrov B., Soldatović B. & Savić I. 1975: Two morphologically different Y chromosomes in the bank
vole (Clethrionomys glareolus Schreb.) from Serbia (Yugoslavia). Acta Vet. 25: 241–246.
Živković S., Soldatović B., Milošević M. & Savić I. 1968: Analyse des Chromosomen der drei Populationen von
Ziesel (Citellus citellus) aus Serbien. Zool. Anz. 181(3–4): 181–185.
346