The 'common vole' in Svalbard identified as Microtus epiroticus by chromosome analysis KARL F R E D G A , 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 'common vole' 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 voles from Svalbard did not belong, as earlier supposed, t o 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. B o x 7003. S-750 0 7 Uppsala. Sweden: Rolf A n k e r Ims and Harald Steen, Department of Biology, Division of Zoology, University of Oslo, P.O. B o x 1050 Blindern, N-0316 Oslo 3 , Norway; 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 L y o n 1. 69622 Villeurhanne Cedex. France; February 1990 (revised July 1990). ' In 1960 a member of the Finnish zoological expedition to Svalbard found the first vole in Svalbard at Longyearbyen. In 1964 and 1965 voles were abundant in the Longyearbyen village and district and several specimens were collected (Nyholm 1966). In 1975 voles were trapped at Hotellneset, Longyearbyen, and in 1976 at Fuglefjellet, Grumantbyen, W Adventfjorden, by Alendal (1977). The voles collected in the 1960s and 1970s were identified as Microtus aruulis (Pallas 1778) on morphological grounds. A more recent report describes voles collected at Coles Bay and Barentsburg as M . arualis (Bolshakov & Shubnikova 1988). The present study is part of a comparative ecological investigation aimed at revealing which cir- cumstances make it possible for a southern vole species to establish itself in arctic conditions. In order to find a relevant reference population it was important to find out, if possible, the origin of the ancestors. Microtus must have been brought to Svalbard by man and according to Alendal (1977), the voles might have been introduced by whale and walrus hunters from Holland in the 17th or 18th century. This conclusion was based on the identification of the specimens captured in Svalbard as Microtus arvalis. In 1972 a new Microtus species was described by Meyer et al. It was called M . subarvalis, but since this name is an absolute homonym of a fossil species, Microtus subarualis Heller 1933, the name was changed to M . epiroticus Ondrias 1966 (see Honacki et al. 1982).* It is a sibling *Recently. the namc Microtus rossiaemeridionrrlis Ognev 1924 was applied t o this species (Malygin 1983: Malygin & Yatscnko 1986). Fifteen spccimens of the 'common volc' were collcctcd in the type locality (Aninsk region. Voronej district) of the taxon M . arualis rossiaemeridionalis Ognev 1924. All fiftccn had 2n = 54 and since, according to Malygin & Yatscnko (1986). M . arualis (2n = 46) docs not cxist i n this area they conclude that thc animals described by Ogiicv also must have had 54 chromosomes. Consequcntly, the sibling spccies with 54 chromosomes should be called M . russiaemeridiorralls Ognev 1924 and not M . epiroticus Ondrias 1966. Howevcr, we arc not willing to accept their conclusion for the following reasons: I . We can not kriow which taxon Ogncv described in 1924 since the two sibling spccies of 'common voles' can not he distinguished by their external morphology. He did not study the characters that distinguish the two (chromosomes. blood proteins or sperm). 2. M . arualis 'arualis' and M . arualis 'ohvcurus' occur west and east of Voroncj, respectivelv. 1s it possiblc to excludc the present occurrence of either of these at the type locality of M . a. rossiaemeridiormlisis" Only 15 specimens were invcstigatcd. Also. the distribution of thc two sihling spccics may have changcd since 1924 ('?)when the holotypc o f ro.\iormrridionali.~ was collected. 3. Wc recommend that this question bc considcred by the Intcrnational Commission on Zoological Nomenclature. According to Article 80, Status of casc under consideration (a) (International Code of Zoological Nomenclature 1985), existing usage is to he maintained until a ruling of the Commission is published, Prcscntly, the twoauthoritative taxonomic references (Niethammcr & Krapp 1982; Honacki et al. 1982) usc the name M . epiroticus Ondrias 1966. For thcse reasons we prefer to use the name M . epiroticus in the present article. 284 K . Fredga et al. F I R . I D i d h u t i o n of M . u r u u l k (solid linc) and M . rpirulicus (\hudowcd) in Europc and western USSR (haacd o n Matygin S: Orlov 1974; K r i l CI al. 1980: Nicthammcr 8: Krapp 1982: Vorontsov el al. 1984). species of M. arualis, and is found within the central part of the vast distribution area of M . arualis (Fig. 1). T h e new species cannot be distinguished from M . arvalis by external morphology, but t h e two species have charac- teristic karyotypes: M. epiroticus has 2n = 54 and M . arvalis has 2n = 46. There is also a difference between the two species i n the size and shape of t h e spermatozoa and with regard to several proteins (Mejer et al. 1972, 1973: Sakiyan et al. 1984). Since both M . arvalis and M. epiroticus potentially could have been introduced from Russia with supply ships to some of the permanent Russian settlements in Svalbard, it was important to study the chromosomes of the Svalbard voles. The ‘common vole’ in Svalbard 285 Furthermore, the comparison of the chromosomes and the mitochondria1 DNA of voles from Svalbard with voles from Holland and other parts of northern Europe might solve the problem of origin and perhaps also tell something about the number of specimens introduced. We here show that the Microtus, belonging to a viable population living in natural habitats in the surroundings of Grumantbyen, Isfjorden, are M . epiroticus and not M . arvalis as believed earlier (Alendal 1977). Material and methods During a two-week period in late August 1989, three of us visited localities in the Isfjorden area where Micrntus either had been captured or ‘seen’ according to Alendal(1977). Coles Bay and some other localities were also checked (Fig. 2). l O k m Fig. 2. Map of the Isfjorden area. Circle: Voles caught during the prcsent study. Stars: Voles reported to be present by Nyholni (1966). Alendal (1977) o r Bolshakov & Shubnikova (1988). hut nor found by us examining thc loealitics in August 1989. Trianglcs: Localities with no earlier reports about vole occurrence, but examined by us in August 1989 with negative results. Squares: Localities previously reported to contain voles (Alendal 1977; Bolshakov & Shubnikova 1988) but not cxarnined by us. 1 = Grurnantbyen, 2 = Longyearbyen, 3 = ColesBay, 4 = Kapp Laila.5 = Bjondhavna.Ternpelfjellet, 6 = Alkhornet, 7 = Knpp L i m e . 8 = Sassendalen. 9 = Pyramiden, 10 = Barentshurg. 11 = Bellsund. Each locality was first examined for signs of small rodents (grazing, runways or faeces) in grassy vegetation. Such signs of Microtus activity are conspicuous even at low densities. At places where signs were found multiple-capture live- traps (type: Ugglan) were set. Only the area between Bjarndalen, 2 km W Longyearbyen, and Coles Bay (Fig. 2) was found to be inhabited by Microtus. Signs of Microtus- activity were, however, most pronounced in the 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 areas were on stable well-drained ground. Approximately 100 live-traps were set for 2‘12 days in the surroundings of Grumantbyen. A total of 46 individuals were caught, indicating a dense population. The animals were brought alive 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 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 chromosome was the largest of the complement and the Y the next largest, but close in size to the largest autosome. This autosome was the only telocentric that could be identified without G- banding. The rest of the single-armed autosomes decreased continuously in size. A G-banded karyotype is shown in Fig. 3B. After C-staining, all autosomes showed centromeric C-bands (Fig. 3C). The distal half of the X stained as a positive 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 t h e females studied had o n e normal and one deleted X; a little more than half of t h e heterochromatic distal part was missing (Fig. 3C). T h e karyotypes of t h e voles from Svalbard a r e in perfect agreement with those of M . epiroticus from Finland (Fredga et al. unpublished) and from different parts of the USSR (Vorontsov et al. 1984; Mejer e t al. 1985). M . arvalis T h e six voles from Holland had identical karyotypes (males XY, females XX). T h e chromosome number was2n = 46. T h e autosomes may be divided into two size groups: 5 large and 17 small pairs. All t h e large chromosomes and 13 of the small were bi-armed. Four of the small chromosomes were acrocentric. T h e X Chromosome was of intermediate size and metacentric, the Y was the smallest of the complement and acrocentric (Fig. 4A). A C-banded karyotype is shown in Fig. 4B. After C-staining, centromeric blocks of heterochromatin were present in 10 of the small pairs, h bi-armed and 4 acrocentric (Fig. 4C). T h e N F value was 84 (female). Different populations of M . arvalis may be distinguished by their karyotypes ( K r a l & Lyapunova 1975; Zima & K r i l 1984). They all have 2n = 46 and 5 large, bi-armed chromosomes, but the number of small single-armed pairs varies, as well as the number of chromosomes with centromeric C-blocks. T h e karyotype of voles from this Dutch population is similar (but not identical) t o that of voles from Osnabruck in Germany (Camper1 1982). They both have 10 pairs with C-blocks and n o acrocentric without. However, t h e number of acrocentric pairs in the Dutch population is 4 compared t o 3 in the German one. Discussion T h e present chromosome study has shown that the voles collected in 1989 at Fuglefjella, Grumantbyen, belongto the species M . epiroticus. Five individuals caught in 1976 in the same area by Alendal (1977) were identified as M. arvalis, according t o guidelines from literature that did not distinguish arvalis from epiroticus. We were not able to find voles anywhere else, although attempts were made a t another seven sites (Fig. 2). This means that we cannot exclude the possibility that the voles caught by earlier investigators at Longyearbyen and its vicinities (Nyholm 1966; Alendal 1977), Coles Bay and Barentsburg (Bolshakov & Shubnikova 1988) really were M . arualis. Bolshakov & Shubnikova (1988) pointed out, however, that Microtus in Svalbard needs t o be karyotyped t o confirm the identification of the species. T h e distribution of M . aroalis and M . epiroticus is shown in Fig. 1. M . arvalis is present in western Europe, from the Atlantic coast and eastwards to about longitude 90"E in western Siberia. M . epiroticus has a more limited distribution in the approximate middle third of this vast area. In Europe M . epiroticus has its northernmost populations in Finland and the southernmost on the Balkan peninsula. These are also the two westernmost areas of its distribution. The type locality of M . epiroticus is in the Epiros mountains in northwestern Greece (Kuzic e t al. 1Y75). T h e two species exist sympatrically in many areas but appear t o occupy different habitats in the breeding season. In the winter M . epiroticus seems t o have the habit of living in association with human settlements, in hay barns etc. In the vicinity of Saratov on the east bank of the Vulga River both species were caught in two haystacks in early spring (Belanin c t al. 1973, quoted by K r i l et al. 1980). In both haystacks M . epiroticus was by far the most common species, the proportions of epiroticus and arvalis being 38 : 3 and 5 6 : 2 , respectively. However, the exact ecological requirements of either species and the differences between t h e species have not yet been fully elucidated (Kral e t al. 1980). T h e close association af t h e Svalbard voles to human buildings has been pointed o u t by several authors (Nyholm 1966; Alendal 1977; Bolshakov & Shubnikova 1988). T h e apparent association of M . epiroticus with humans may have been important during the species' colonization and establishment in Svalbard. However, it is clear that the highly viable population in the Fuglefjella area occurs in natural habitats independent of human settlements. Svalbard is located 657 km north of Nordkapp, midway between Norway and the North Pole, and has never been connected by a landbridge with Fennoscandia o r any other part of the Eurasian continent. Voles cannot possibly have survived a fortuitous transport on ice or on a The ‘common vole’ in Svalbard 289 floating log because of the long distances involved. Thus, the voles must have been brought t o Svalbard by man. ( W e also exclude t h e possibility that voles were introduced secretly t o Svalbard by scientists as part of an ecological experiment.) Both arualis and epiroticus exist in the Leningrad region (Pavlovsk, Volosovo and Volchov districts), but in t h e areas closest to the coast (Leningrad and the Lomonosov district) only epiroticus was found (Mejer et al. 1972; Malygin & Orlov 1974; K r i l e t al. 1980). However, the recent discovery of M . arualis in southeastern Finland (Fredga et al. unpublished) indicatesthat bothspeciesrnay occursyrnpatrically along t h e easternmost coasts of the Gulf of Finland. However, M. epiroricus is by far the most common species in Finland a n 3 adjacent parts of t h e USSR. Hence, Fixiand, western USSR and Balkan are the only areas where M . epiroticus exists near water and harbours. W e a r e not aware of any transportations from the eastern Mediterranean (Yugoslavia o r Greece) or from the Black Sea (USSR, Rumania or Bulgaria) to Svalbard. Finland has not been involved in any industrial o r agricultural activities in Svalbard. Thus, we find it unlikely that the voles in Svalbard originate from the Balkan Peninsula or Finland but from the western parts of the USSR, bordering the Gulf of Finland. T h e Russians have exploited coal mines in Grumantbyen since 1920 (Hoe1 1966) and at Coles Bay since 1938 (Bolshakov & Shubnikova 1988). Horses were used for transportation and hay must have been brought there for their winter survival. W e conclude that the voies were introduced together with hay by Russian ships from Leningrad (or nearby harbours) in the period 1920-1960, and thus originate from the vicinity of Leningrad. Prof. Charles Elton visited Svalbard in 1921, 1923 and 1924 as a member of the Oxford expeditions to Svalbard. ‘I feel certain they [the voles] were not introduced by 1924 - the last time I went there! I would have been told’ (Charles Elton, in a letter to K.F.). 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