Folia Zool. – 57(4): 337–346 (2008)

Chromosomal studies on Greek populations of four small species

George P. Mitsainas, Michail Th. Rovatsos, Irene Karamariti and Eva B. Giagia-Athanasopoulou*

Laboratory of Zoology, Section of 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 G- and 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 (Zima 2000, 2004). However, the karyotypes of many Greek mammalian taxa still remain largely unknown. Their study could contribute to the clarification of their 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 (Ondrias 1966, Niethammer 1986, Vohralík & Sofianidou 1987, 1992). Their karyotypes with 2n=62 and 2n=48, respectively, are known from their populations in several Palearctic regions (Filippucci et al. 1985, Belcheva et al. 1988, Graphodatsky & Fokin 1993, Civitelli et al. 1995, Peshev & Delov 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 (Soldatović et al. 1984). The red bank vole Myodes glareolus displays a vast Palaearctic distribution (Shenbrot & Krasnov 2005). Its karyotype is characterized by 2n=56 (Král 1972, Král et al. 1979, Zima 1984) and also by variation in the centromeric position of the Y chromosome in several European populations (Král et al. 1972, Živković et al. * Corresponding author

337 1975, Vorontsov et al. 1978, Gamperl 1982, Radosavljević et al. 1988, Vujošević & Blagojević 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 (Hsu & Patton 1969). For chromosome identification, the G–banding technique (Seabright 1971) was implemented. In order to study the distribution of the heterochromatin, Sumner’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 and their karyological characteristics.

No. of individuals Taxon Locality 2n FNa FN Total m f Dryomys nitedula Kastritsi 6 3 3 48 92 96 -”- Lagadikia 3 1 2 48 92 96 Myodes glareolus Pisoderi 3 2 1 56 56 58 -”- Platza 4 1 3 56 56 58 Myoxus glis Charakas 2 1 1 62 120 124 -”- Kastritsi 8 2 6 62 120 124 Spermophilus citellus Seli 1 - 1 40 76 80 -”- Thermi 3 2 1 40 76 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 , 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 (Zima 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 (Dulić et al. 1971, Zima 1987, Belcheva et al. 1988, Civitelli et al. 1995, Peshev & Delov 1995), with the exception of the submetacentric form in Spanish (Diaz de la Guardia et al. 1980) and Russian populations (Graphodatsky & Fokin 1993). The large sized variant in our sample has also been reported from European Turkey (Civitelli et al. 1995). On the other hand, the small, probably acrocentric Y variant of our sample has also been recorded at least from Italy (Civitelli et al. 1995), Bulgaria (Belcheva 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 (Zima & Král 1984, Zima 1987). Finally, the single pair with secondary constrictions, shown as the 25th in Fig. 2, following Graphodatsky & Fokin (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 (Belcheva et al. 1988, Civitelli et al. 1995), leading some authors to conclude that heterochromatin absence is a chromosome feature in some dormice species (Graphodatsky & Fokin 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 (Filippucci et al. 1985, Civitelli et al. 1995, Peshev & Delov 1995), with little interpopulation variation (Zima 1987, Doğramaci & Kefelioğlu 1990, Zima 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 (Filippucci et al. 1985). Also, the secondary constriction in a small sized pair (the 21st in Fig. 3, following Civitelli 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 (Zima & Král 1984, Filippucci et al. 1985, Peshev & Delov 1995, Zima et al. 1995), however in our case it was clearly submetacentric, as was also reported by Graphodatsky & Fokin (1993) and was visible in some karyotypes presented by Civitelli 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 (Filippucci 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 (Živković et al. 1968, Savić et al. 1971, Belcheva & Peshev 1979, Soldatović et al. 1984, Özkurt 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 medium- sized submetacentric X chromosome of our sample has been previously reported with conventional staining from Greece (Soldatović et al. 1984), as well as from other regions (Živković et al. 1968, Savić et al. 1971, Belcheva & Peshev 1979, Soldatović et al. 1984, Zima & Král 1984) and seems to be the common form. However, other X variants have been described, i.e. large-sized submetacentrics (Özkurt et al. 2002) or medium-sized acrocentrics (Belcheva & Peshev 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 (Soldatović et al. 1984). Finally, the heterochromatin distribution in our sample from Greece, based on C-banding, seems to generally agree with that of Zima (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 (Král 1972, Živković et al. 1975, Vorontsov et al. 1978, Král et al. 1979, Gamperl 1982, Zima et al. 1997), however a few cases describing an acrocentric variant have been reported from southern Italy (Král et al. 1972) and former Yugoslavia (Živković et al. 1975). In the latter area, interpopulation variation has been reported (Radosavljević et al. 1988, Vujošević & Blagojević 1997), representing a gradual transition

344 from the acrocentric form in the NW to the metacentric-submetacentric form in the S-SE (Vujošević & Blagojević 1997). The submetacentric variants in our sample from northern Greece seem to support this transition. Northern Greece constitutes the south- easternmost 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 (Vorontsov et al. 1978, Vorontsov et al. 1980). With regard to C-banding, the distribution of heterochromatin in our sample is in accordance with previous descriptions (Gamperl 1982, Gamperl et al. 1982), including the appearance of a large autosomal pair with just faint centromeric heterochromatic bands (Gamperl 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 (Matthey 1973, Modi 1987).

Acknowledgements

We are grateful to J. Zima and two anonymous referees, whose detailed comments and suggestions led to the thorough improvement of our manuscript. Also, we wish to thank C. Stamatopoulos and A. Papagianopoulos for providing part of the studied material, as well as N. Turland for checking the English language of the manuscript.

LITERATURE

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 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 . 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. . 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