The 'common ' in identified as epiroticus by chromosome analysis

KARL FREDGA, MAARIT JAAROLA. ROLF ANKER IMS, HARALD STEEN AND NlGEL G. YOCCOZ

Fredga, K., Jaarola, M., Ims, R. A,, Steen, FI. & Yoccoz. N. G. 1990: The '' in Svalbard identified as Microtus epiruticur by chromosome analysis. Polar Research 8, 283-2Y0. The chromosomes were studied in six individuals from a population of Microtus from Grumantbyen, Svalbard, and in six Microtur arualis (Pallas 1778) from Lauwersee, Holland. It was shown that the ' from Svalbard did not belong, as earlier supposed, to the species M. arualis (2n = 46) but to M. epiroticus (Ondrias, 1966) (2n = 54). We suggest that the Svalbard voles were introduced by man between 1920 and 1960 together with hay on Russian ships from the vicinity of Leningrad, USSR. Karl Fredga and Maarit Jaarola, Department of Genetics, Uppsala University, P.O. Box 7003. S-750 07 Uppsala. Sweden: Rolf Anker Ims and Harald Steen, Department of Biology, Division of Zoology, University of Oslo, P.O. Box 1050 Blindern, N-0316 Oslo 3, ; Nigel G. Yoccoz, Deparrment of Biology, Division of Zoology, Uniuersify of Oslo. P.O. Box I050 Blindern, N-0316 Oslo 3, Norway. or: Laboratoire de Biometrie, Unioersitk Claude Bernard Lyon 1. 69622 Villeurhanne Cedex. France; February 1990 (revised July 1990).

In 1960 a member of the Finnish zoological the name was changed to M. epiroticus Ondrias expedition to Svalbard found the first vole in 1966 (see Honacki et al. 1982).* It is a sibling Svalbard at . In 1964 and 1965 voles were abundant in the Longyearbyen village and district and several specimens were collected *Recently. the namc Microtus rossiaemeridionrrlis Ognev 1924 (Nyholm 1966). In 1975 voles were trapped at was applied to this species (Malygin 1983: Malygin & Yatscnko Hotellneset, Longyearbyen, and in 1976 at 1986). Fifteen spccimens of the 'common volc' were collcctcd in the type locality (Aninsk region. Voronej district) of the Fuglefjellet, Grumantbyen, W , taxon M. arualis rossiaemeridionalis Ognev 1924. All fiftccn by Alendal (1977). The voles collected in the had 2n = 54 and since, according to Malygin & Yatscnko 1960s and 1970s were identified as Microtus (1986). M. arualis (2n = 46) docs not cxist in this area they aruulis (Pallas 1778) on morphological grounds. conclude that thc described by Ogiicv also must have had 54 chromosomes. Consequcntly, the sibling spccies with A more recent report describes voles collected at 54 chromosomes should be called M. russiaemeridiorralls Ognev Coles Bay and Barentsburg as M. arualis 1924 and not M. epiroticus Ondrias 1966. Howevcr, we arc not (Bolshakov & Shubnikova 1988). The present willing to accept their conclusion for the following reasons: study is part of a comparative ecological I. We can not kriow which taxon Ogncv described in 1924 investigation aimed at revealing which cir- since the two sibling spccies of 'common voles' can not he distinguished by their external morphology. He did not study cumstances make it possible for a southern vole the characters that distinguish the two (chromosomes. blood species to establish itself in arctic conditions. In proteins or sperm). order to find a relevant reference population it 2. M. arualis 'arualis' and M. arualis 'ohvcurus' occur west was important to find out, if possible, the origin and east of Voroncj, respectivelv. 1s it possiblc to excludc the present occurrence of either of these at the type locality of M. of the ancestors. a. rossiaemeridiormlisis" Only 15 specimens were invcstigatcd. Microtus must have been brought to Svalbard Also. the distribution of thc two sihling spccics may have by man and according to Alendal (1977), the changcd since 1924 ('?)when the holotypc of ro.\iormrridionali.~ voles might have been introduced by whale and was collected. walrus hunters from Holland in the 17th or 18th 3. Wc recommend that this question bc considcred by the Intcrnational Commission on Zoological Nomenclature. century. This conclusion was based on the According to Article 80, Status of casc under consideration (a) identification of the specimens captured in (International Code of Zoological Nomenclature 1985), existing Svalbard as Microtus arvalis. usage is to he maintained until a ruling of the Commission is In 1972 a new Microtus species was described published, Prcscntly, the twoauthoritative taxonomic references (Niethammcr & Krapp 1982; Honacki et al. 1982) usc the name by Meyer et al. It was called M. subarvalis, but M. epiroticus Ondrias 1966. since this name is an absolute homonym of a For thcse reasons we prefer to use the name M. epiroticus fossil species, Microtus subarualis Heller 1933, in the present article. 284 K. Fredga et al.

FIR. I Didhution of M. uruulk (solid linc) and M. rpirulicus (\hudowcd) in Europc and western USSR (haacd on Matygin S: Orlov 1974; Kril CI al. 1980: Nicthammcr 8: Krapp 1982: Vorontsov el al. 1984).

species of M. arualis, and is found within the the spermatozoa and with regard to several central part of the vast distribution area of M. proteins (Mejer et al. 1972, 1973: Sakiyan et al. arualis (Fig. 1). The new species cannot be 1984). distinguished from M. arvalis by external Since both M. arvalis and M. epiroticus morphology, but the two species have charac- potentially could have been introduced from teristic karyotypes: M. epiroticus has 2n = 54 and Russia with supply ships to some of the permanent M. arvalis has 2n = 46. There is also a difference Russian settlements in Svalbard, it was important between the two species in the size and shape of to study the chromosomes of the Svalbard voles. The ‘common vole’ in Svalbard 285

Furthermore, the comparison of the chromosomes Each locality was first examined for signs of and the mitochondria1 DNA of voles from small (grazing, runways or faeces) in Svalbard with voles from Holland and other parts grassy vegetation. Such signs of Microtus activity of northern Europe might solve the problem of are conspicuous even at low densities. At places origin and perhaps also tell something about the where signs were found multiple-capture live- number of specimens introduced. traps (type: Ugglan) were set. We here show that the Microtus, belonging to Only the area between Bjarndalen, 2 km W a viable population living in natural habitats in Longyearbyen, and Coles Bay (Fig. 2) was found the surroundings of Grumantbyen, Isfjorden, are to be inhabited by Microtus. Signs of Microtus- M. epiroticus and not M. arvalis as believed activity were, however, most pronounced in the earlier (Alendal 1977). grassy slopes under Fuglefjella and the animals seemed to prefer places on peat soil with lush vegetation of grass and herbs, interspersed with patches of boulders. Generally, these preferred Material and methods areas were on stable well-drained ground. During a two-week period in late August 1989, Approximately 100 live-traps were set for three of us visited localities in the Isfjorden area 2‘12 days in the surroundings of Grumantbyen. A where Micrntus either had been captured or ‘seen’ total of 46 individuals were caught, indicating a according to Alendal(1977). Coles Bay and some dense population. The animals were brought alive other localities were also checked (Fig. 2). to the Department of Biology, University of Oslo, where a breeding colony was established. Fifty M. urvalis were live trapped in October 1989 at Lauwersee, NE Holland. All specimens were brought alive to the Department of Biology, University of Oslo, where a breeding colony was established. Six of the voles from Svalbard and six from Holland, 4males and2femalesof eachpopulation, were karyotyped. Chromosome preparations were made from bone marrow by the direct method of Fredga (1987). For G- and C-banding the techniques of Wang & Fedoroff (1972) and Sumner (1972) were used, respectively.

Results lOkm M. epiroticus The six voles studied from Svalbard had identical autosomal karyotypes and males were XY, females XX. The chromosome number was 2n = 54 and all chromosomes but the smallest pair of autosomes were telocentric (Fig. 3.4). The X Fig. 2. Map of the Isfjorden area. Circle: Voles caught during chromosome was the largest of the complement the prcsent study. Stars: Voles reported to be present by and the Y the next largest, but close in size to Nyholni (1966). Alendal (1977) or Bolshakov & Shubnikova the largest autosome. This autosome was the only (1988). hut nor found by us examining thc loealitics in August 1989. Trianglcs: Localities with no earlier reports about vole telocentric that could be identified without G- occurrence, but examined by us in August 1989 with negative banding. The rest of the single-armed autosomes results. Squares: Localities previously reported to contain voles decreased continuously in size. A G-banded (Alendal 1977; Bolshakov & Shubnikova 1988) but not karyotype is shown in Fig. 3B. After C-staining, cxarnined by us. 1 = Grurnantbyen, 2 = Longyearbyen, 3 = all autosomes showed centromeric C-bands (Fig. ColesBay, 4 = Kapp Laila.5 = Bjondhavna.Ternpelfjellet, 6 = Alkhornet, 7 = Knpp Lime. 8 = Sassendalen. 9 = Pyramiden, 3C). The distal half of the X stained as a positive 10 = Barentshurg. 11 = Bellsund. C-block and the entire Y appeared dark after 286 K. Fredga et al.

Fig. 3. Karyotypes of male Microtus epiroticur from Grumantbyen, Svalbard. A) unbanded, B) G-banded, C) C-banded. Boxed: sex chromosomes from a female with one normal and one deleted X chromosome (Xd). Arrows indicate the position of the centromeres in the sex chromosomes. Preparations from bone marrow. Bar = 10 pm. Same magnification in A. B and C. The ‘common vole’ in Svalbard 287

Fig. 4. Karyotypes of Microtus arualis from Lauwersee, Holland. A) male, unbanded, B) female, G-banded, C) male, C-banded Preparations from bone marrow. Bar = 10 pm, Same magnification in A. B and C. 288 K. Fredga et al.

C-staining. One of the females studied had one attempts were made at another seven sites (Fig. normal and one deleted X; a little more than half 2). This means that we cannot exclude the of the heterochromatic distal part was missing possibility that the voles caught by earlier (Fig. 3C). investigators at Longyearbyen and its vicinities The karyotypes of the voles from Svalbard are (Nyholm 1966; Alendal 1977), Coles Bay and in perfect agreement with those of M. epiroticus Barentsburg (Bolshakov & Shubnikova 1988) from (Fredga et al. unpublished) and really were M. arualis. Bolshakov & Shubnikova from different parts of the USSR (Vorontsov et (1988) pointed out, however, that Microtus in al. 1984; Mejer et al. 1985). Svalbard needs to be karyotyped to confirm the identification of the species. The distribution of M. aroalis and M. epiroticus M. arvalis is shown in Fig. 1. M. arvalis is present in western The six voles from Holland had identical Europe, from the Atlantic coast and eastwards karyotypes (males XY, females XX). The to about longitude 90"E in western Siberia. M. chromosome number was2n = 46. The autosomes epiroticus has a more limited distribution in the may be divided into two size groups: 5 large and approximate middle third of this vast area. In 17 small pairs. All the large chromosomes and 13 Europe M. epiroticus has its northernmost of the small were bi-armed. Four of the populations in Finland and the southernmost on small chromosomes were acrocentric. The X the Balkan peninsula. These are also the two Chromosome was of intermediate size and westernmost areas of its distribution. The type metacentric, the Y was the smallest of the locality of M. epiroticus is in the Epiros mountains complement and acrocentric (Fig. 4A). in northwestern (Kuzic et al. 1Y75). A C-banded karyotype is shown in Fig. The two species exist sympatrically in many 4B. After C-staining, centromeric blocks of areas but appear to occupy different habitats in heterochromatin were present in 10 of the small the breeding season. In the winter M. epiroticus pairs, h bi-armed and 4 acrocentric (Fig. 4C). seems to have the habit of living in association The NF value was 84 (female). with human settlements, in hay barns etc. In the Different populations of M. arvalis may be vicinity of Saratov on the east bank of the Vulga distinguished by their karyotypes (Kral & River both species were caught in two haystacks Lyapunova 1975; Zima & Kril 1984). They all in early spring (Belanin ct al. 1973, quoted by have 2n = 46 and 5 large, bi-armed chromosomes, Kril et al. 1980). In both haystacks M. epiroticus but the number of small single-armed pairs varies, was by far the most common species, the as well as the number of chromosomes with proportions of epiroticus and arvalis being 38 : 3 centromeric C-blocks. The karyotype of voles and 56:2, respectively. However, the exact from this Dutch population is similar (but not ecological requirements of either species and the identical) to that of voles from Osnabruck in differences between the species have not yet been Germany (Camper1 1982). They both have 10 fully elucidated (Kral et al. 1980). pairs with C-blocks and no acrocentric without. The close association af the Svalbard voles to However, the number of acrocentric pairs in the human buildings has been pointed out by several Dutch population is 4 compared to 3 in the authors (Nyholm 1966; Alendal 1977; Bolshakov German one. & Shubnikova 1988). The apparent association of M. epiroticus with humans may have been important during the species' colonization and Discussion establishment in Svalbard. However, it is clear that the highly viable population in the Fuglefjella The present chromosome study has shown that area occurs in natural habitats independent of the voles collected in 1989 at Fuglefjella, human settlements. Grumantbyen, belongto the species M. epiroticus. Svalbard is located 657 km north of Nordkapp, Five individuals caught in 1976 in the same area midway between Norway and the North Pole, by Alendal (1977) were identified as M. arvalis, and has never been connected by a landbridge according to guidelines from literature that did with Fennoscandia or any other part of the not distinguish arvalis from epiroticus. We were Eurasian continent. Voles cannot possibly have not able to find voles anywhere else, although survived a fortuitous transport on ice or on a The ‘common vole’ in Svalbard 289 floating log because of the long distances involved. Acknowledgements. - This study was supported financially by Thus, the voles must have been brought to the Swedish Natural Science Research Council, the Norwcgian Polar Research Institute, the ‘Fondation Franco-Norvegiennc Svalbard by man. (We also exclude the possibility pour la recherche scicntifique et techniquc ct le d&Aoppcmcnt that voles were introduced secretly to Svalbard industriel’ (FFN) and the Norwcgian Research Council for by scientists as part of an ecological experiment.) Scicnce and thc Humanities (NAVF). Both arualis and epiroticus exist in the Leningrad region (Pavlovsk, Volosovo and Volchov districts), but in the areas closest to the coast (Leningrad and the Lomonosov district) References only epiroticus was found (Mejer et al. 1972; Alendal, E. 1977: Sdrlig markmus har fatt fotfcste pi Svalbard. Malygin & Orlov 1974; Kril et al. 1980). Fauna 30, bll. However, the recent discovery of M. arualis in Belnntn, A. N., Vcnig, L. L.. Lark, N. I. 9r Sonin. K. A. southeastern Finland (Fredga et al. unpublished) 1973: Characteristics of tlic karyotypes of the common volc indicatesthat bothspeciesrnay occursyrnpatrically (Microtus drualis Pall.) froin thc Povoliie. Fzzid. popu/. ekol. iw.,Sarutoo I,6672. along the easternmost coasts of the Gulf of Bolshakov, V. N. & Shuhnikova, 8.N. 1988: Common volc - Finland. However, M. epiroricus is by far the Microtus uroalis (RodLntia, Muridae) on Spitshergcn most common species in Finland an3 adjacent archipelago. 2001.Zhurn. 67, 3OR-310 (in Russian). parts of the USSR. Frcdga, K. 1987: Chromos:ime preparations in the ficld from Hence, Fixiand, western USSR and Balkan are long after dzath. Stain Techno/. 62, 167-171. Gamperl, R. 1982: Die Chromoromcn van Microrus uroalis the only areas where M. epiroticus exists near (Rodcntia, Micro!inac). Z. Siiugelierkundc 47, 35h-363. water and harbours. We are not aware of any Hod, A. 1966: Sun1bui.d I. Sverrc Kildah!s Boktrykkeii, Oslo. transportations from the eastern Mediterranean Honacki. J. H., Kinman, K. E. & Koeppl, J. Mi. (eds.) 1982: (Yugoslavia or Greece) or from the Black Sea Murnmol Species of the Wxldc A luxonwiic and Geographic Rcference. Allen Press and The Association of Systematic (USSR, Rumania or ) to Svalbard. Co!lections, Lawrence. Kansas, U.S.A Finland has not been involved in any industrial KrBI, B., Bclanin, A. N., Ziina, J., Malygin, V. M.. Gajccnko, or agricultural activities in Svalbard. Thus, we V. A. & Orlov, V. N. 1980: Distribution of Microtus aruulis find it unlikely that the voles in Svalbard originate and M. epirolicus. Actu Sc. Net. Brno 14, 1-30, from the Balkan Peninsula or Finland but from Kral, 6. & L.yapunova, E. A. 1975: Karyotypes of 46- chromosome Microtm arudi,s (Micratidac. Rodentia). Zool. the western parts of the USSR, bordering the Listy 24, 1-11, Gulf of Finland. Malygin, V. M. 1983: Systcmurics ofih~C;rnrun uolc. Nauka, The Russians have exploited coal mines in Moscow. 208 i)p (in hssian). Grumantbyen since 1920 (Hoe1 1966) and at Malygin, V. M. & Orlov, V. N. 1974: A:-xof 4 species of voles (aupcrspccics Microtus aruulis) by karyologicai data. Coles Bay since 1938 (Bolshakov & Shubnikova Zool. Zhurn. 53, 610-622 (in Russian). 1988). Horses were used for transportation and Malygin, V. M. 9, Yatsenko, V. N. 1986: Taxonomic hay must have been brought there for their winter nomcnclaturc of sibling species of the common volc survival. We conclude that the voies were (Rodenria, Cricctidae). Zd. Zhurn. 35, 579-591 (in introduced together with hay by Russian ships Russian). Mejcr, M. N., Moroz, J. M., Orlov, V. N. B Scholl, E. D. from Leningrad (or nearby harbours) in the 1973: Zwillingsartcn der Feldmaus, Mzcrotus nrudb (Pallah). period 1920-1960, and thus originate from the Mitt. Zool. MLLS.Berlin 49, 387-402. vicinity of Leningrad. Prof. Charles Elton visited Mcjcr, M. N., Orlov, V. N. & Scholl, E. D. 1372: On thc Svalbard in 1921, 1923 and 1924 as a member of nomenclature of 46- and 54-chromosome voles of ?he type Microrrrs orua1i.s (Pall.) (Rodentia, ). Zoo/. Zkurn. the Oxford expeditions to Svalbard. ‘I feel certain 51, 157-161 (in Russian). they [the voles] were not introduced by 1924 - Mejer, M. N., Radjabli, S. J.. Bulatova, N. S. & Golenischcv, the last time I went there! I would have been F. N. 1985: Karyotypical peculiarities and probable relations told’ (Charles Elton, in a letter to K.F.). of common volesof the group ‘uroulis’(Rodentia, Cricetidac, Further studies may tell whether Microrus). 2001.Zhurn. 64, 417-428 (in Russian). of DNA us Niethammer, J. & Krdpp, F. 1982: Microtw aroalis (Pallas, voles were introduced to Svalbard more than 1779) - Feldmaus. Pp. 284-318 in Niethammer, J. & Krdpp, once, and may also support the hypothesis about F. (eds.): Hundbuch der Saugetiere Europas 2(1). Rodentiu: their origin put forward here. We cannot exclude 11. Akademische Verlagsgesellschaft. Wiesbaden. the possibility that M. arvalis also has been Nyholm. E. S. 1966: Observations on some birds and mammals of . Ann. 2001. Fenn. 3, 173175. introduced. But so far, the only vole species that Ride, W. D. L., Sabrosky, C. W., Bernardi, G. & Melville, has been proven to exist in Svalbard is M. R. V. (eds.) 1985: International Code of Zoologicul epiroticus. Nomenclature (3rdedition). InternationalTrust for Zoological 290 K. Fredga et al.

Nomenclature in association with British Museum (Natural Sumner, A. T. 1972: A simple technique for demonstrating History), London, and Univ. of California Press, Berkeley centromeric heterochromatin. Exp. Cell Res. 75, 304306. and Los Angeles. Vorontsov, N. N., Lyapunova, E. A., Belyanin, A. N., Kral, Ruzic, A,, Petrov, B. & Zivkovic, S. 1975: On the species B., Frisman, L. V., Ivnitsky, S. B. & Yanina, I. Y. 1984: independence of the 54-chromosome vole Microfurs epiroticus Comparative genetic methods of diagnostics and estimation Ondrias 1966 (Mammalia, Rodentia), its distribution, ecology of the degree of divergence for the sibling species of common and importance as a pest in the west part of Balkan peninsula. voles Microtus aroalis and M. epiroticw. Zool. Zhurn. 63, 1. Sci. Agri. Res. Beograd 28, 153-160. 1555-1566 (in Russian). Sakiyan, S. M., Kulbakina, N. A,, Serov, 0. L., Mejer, M. N. Wang, H. C. & Fedoroff, S. 1972: Banding in human & Zharkikh, A. 1984: Assessment of the degree of genetic chromosomes treated with trypsin. Nature New Bid. 235. divergcnce of the twin species of the common vole Microfurs 52-53. urvalis and Microtus subaroulis (Rodentia). Genetika 20, Zima, J. & Kril, B. 1984: Karyotypcs of European Mammals 1365- 1372. 11. Acfu Sc. Nar. Brtio 18, 1-62.