Äîêóìåíò âçÿò èç êýøà ïîèñêîâîé ìàøèíû. Àäðåñ îðèãèíàëüíîãî äîêóìåíòà : http://vertebrata.bio.msu.ru/RJG595_2006Eng.pdf Äàòà èçìåíåíèÿ: Mon Feb 8 19:41:58 2010 Äàòà èíäåêñèðîâàíèÿ: Mon Oct 1 19:27:43 2012 Êîäèðîâêà:

ISSN 1022-7954, Russian Journal of Genetics, 2006, Vol. 42, No. 6, pp. 595­604. © Pleiades Publishing, Inc., 2006. Original Russian Text © A.A. Bannikova, N.S. Bulatova, D.A. Kramerov, 2006, published in Genetika, 2006, Vol. 42, No. 6, pp. 737­747.

MOLECULAR GENETICS

Molecular Variability in the Commom Shrew Sorex araneus L. from European Russia and Siberia Inferred from the Length Polymorphism of DNA Regions Flanked by Short Interspersed Elements (Inter-SINE PCR) and the Relationships between the Moscow and Seliger Chromosome Races
A. A. Bannikova1, N. S. Bulatova2, and D. A. Kramerov3
2

Moscow State University, Moscow, 119992 Russia Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, 119991 Russia 3 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia; e-mail: hylomys@mail.ru
Received November 23, 2005

1

Abstract--Genetic exchange among chromosomal races of the common shrew Sorex araneus and the problem of reproductive barriers have been extensively studied by means of such molecular markers as mtDNA, microsatellites, and allozymes. In the present study, the interpopulation and interracial polymorphism in the common shrew was derived, using fingerprints generated by amplified DNA regions flanked by short interspersed repeats (SINEs)--interSINE PCR (IS­PCR). We used primers, complementary to consensus sequences of two short retroposons: mammalian element MIR and the SOR element from the genome of Sorex araneus. Genetic differentiation among eleven populations of the common shrew from eight chromosome races was estimated. The NJ and MP analyses, as well as multidimensional scaling showed that all samples examined grouped into two main clusters, corresponding to European Russia and Siberia. The bootstrap support of the European Russia cluster in the NJ and MP analyses was respectively 76 and 61%. The bootstrap index for the Siberian cluster was 100% in both analyses; the Tomsk race, included into this cluster, was separated with the bootstrap support of NJ/MP 92/95%. DOI: 10.1134/S1022795406060020

INTRODUCTION The common shrew Sorex araneus L., 1758 exhibits a striking range of interspecific karyotype variability, associated with Robertsonian polymorphism [1, 2]. The species proper is represented by a mosaic of distinct chromosome races, differing by the arm composition in metacentric chromosomes [3, 4]. To date, 70 chromosome races have been described across the vast range of Sorex araneus, extending from northern Europe to the Baikal Lake [5, 6]; of these, 14 races occur in European Russia and 7 races, in Siberia. The common shrew karyotype with a relatively low for mammals diploid number has formed during the evolution of the genus Sorex by centric fusions, which seem to progress also at the intraspecific level [7, 8]. The same number 2n = 20, minimum for the species, is attained by fusion of ten original acrocentrics in various pairwise combinations. The assignment to a chromosome race is established by the order of pairwise arm combinations in diagnostic metacentrics and free acrocentrics, which are denoted by Latin letters from "g" to "r" in the standard nomenclature of Sorex araneus chromosomes [9].

Chromosome polymorphism of the common shrew, genetic exchange among chromosome races, and the problem of reproductive barriers are currently extensively studied by means of such molecular genetic markers as mtDNA, microsatellites, and allozymes. However, mitochondrial markers, which indicate very recent time of formation of most chromosome races, do not allow to differentiate them by haplotypes, whose vast diversity form a starlike phylogeny [4, 10­12]. Allozymes are characterized by low inter- and intraracial variation [13]. By contrast, microsatellites are characterized by extremely high mutation rates, which results in biased estimates of gene exchange among races [14­16]. In view of the above, the molecular evolution of this species remains rather vague. No reliable molecular markers for chromosome races have been found, the only safe approach to race diagnostics still being karyotype analysis. This fact is of importance, since it suggests possible independence of molecular and chromosome evolution of the common shrew. It is also noteworthy that investigations of molecular variability, which are carried out along with studies of chromosome polymorphism in the common shrew,

595


596 2 4 1 5â 8â 6 7 15 9 12

BANNIKOVA et al.

Seliger

races Serov and Sok [6]. These, in turn, are consecutively adjoined from the east by races Novosibirsk, Tomsk, and other five Siberian chromosome races [23]. Of the latter, we have examined in the present study a karyotyped sample from the Tomsk race and a sample from the right shore of the Yenisei River, putatively assigned to the Strelka race on the basis of its geographical location. MATERIALS AND METHODS Shrews were collected by live traps and cones in different localities of European Russia and Siberia. The total size of the examined sample was 56 animals belonging to eight chromosome races; 48 animals were karyotyped. Karyological analysis. The chromosome formula of each of the karyotyped animals was established by the standard procedure, based on the metaphase chromosome G-banding. Chromosome race assignment was performed according to the recommendations of the International Sorex araneus Cytogenetics Committee (ISACC) [9, 24]. The data on preliminary karyological diagnostics of the animals are in part presented in previous reports [25­27] and obtained for the first time for samples from new localities. The geographic characteristics of the material, sample sizes for different populations, and chromosome formulas for the races used in molecular analysis are presented in Table 1 and Fig. 1. DNA isolation. DNA was isolated from ethanolfixed tissues (muscle, testicles, kidneys, and liver) by means of phenol­chloroform extraction, after treating the tissue homogenate with pronase or proteinase K [28]. Inter-SINE­PCR conditions. In this study, we used the SINE family MIR (mammalian interspersed repeats), which is represented by 105 copies probably in all mammal genomes [29­31] and the short, 179-bp retroposon SOR, specific for the family Soricidae [32]. The conditions and specific features of the interSINE­PCR method were described in detail in earlier publications [17, 21]. In the present study, inter-MIR­ PCR was carried out with two pairs of primers in combination Mil17/Mir17 and Omil17/Omir17, the sequence of which was presented earlier [30]. Primers were labeled with [32P]-ATP (1MBk) using polynucleotide kinase [28]. PCR was run in 20 µl of a reaction mixture containing 67 mM buffer TrisHCl, pH 8.6; 16.6 mM (NH4)2SO4; 2.5 mM MgCl2; 0.2 mM of each dNTP; 4 pmol of each primer; 1 U Taq polymerase (Sileks); and 25 ng of DNA. The conditions of MIR-specific amplification were as described earlier in [30]: denaturing at 94°C for 30 s; annealing at 56°C for 45 s; elongation at 72°C for 2 min. The number of cycles was 27. Preliminary denaturing was for 3 min at 94°C; final elongation, for 5 min at 72°C. In SOR-specific PCR, the annealing temperature was 65°C. The PCR was run in thermal cycler MJ Research (United States). The amplification products were denatured and
Vol. 42 No. 6 2006

13 â 14 Moscow

Fig. 1. Geographic positions of samples of the Moscow and Seliger samples in the contact zone. Sampling localities of races Moscow and Seliger are shown by respectively solid and open dots; circles, the half-solid dots mark localities where both races were found. Localities are designated by numbers; sites of occurrence of mixed karyotypes are shown by crosses.

mostly concern populations from Western Europe, whereas a significant part of the species range, covering Eastern Europe and Siberia, remains scarcely studied by molecular genetic methods. The aim of the present study was examining molecular variability and chromosome races of S. araneus from European Russia and Siberia, inferred from the data on length polymorphism of DNA regions flanked by short interspersed repeats (SINEs). We have repeatedly used this method of fragment multilocus nuclear DNA analysis--interSINE PCR (IS-PCR)--for studying interspecific taxonomic structure in insectivores and bats [17, 18], as well as for examining interspecies relationships in systematically complex taxa [19, 20]. Successful attempts to differentiate chromosome races of the common shrew [21] motivated the further development of primers and optimization of the PCR conditions for these intraspecific forms. In the present work, we focused also on the relationships between two chromosome races, Moscow and Seliger, whose karyotypes are thought to be the most distant in the European part of the range. The Moscow chromosome race, occupying the central position in the European Russia, has the diagnostic arm combination gm, hi, kr, no, pq. The range of this race is conventionally limited by a segment of the upper Volga River in the north and the Oka River in the south [22]. The Moscow race contacts with the Seliger race in the west, the Neroosa race in the south, and the Manturovo race in the north. The latter is geographically close to the Ural

RUSSIAN JOURNAL OF GENETICS


Table 1. Material examined in the study; n is the number o common shrew samples, analyzed by IS­PCR Chromosome formula go, hi, kr, mn, pq g, hn, ik, mq, o, pr Vicinity of Seliger Lake 9 localities 15 Bryansk oblast Savichki village 3 Region Geographic locality n Sample codes Ne13-99, Ne14-00, Ne19-00, (Ne21-00) (S1-03), S1-04, (S1-06), S4-10, S5-14, S7-01, S8-06, S9-02, S12-8, S15-1, S15-2, S15-3, S15-5, S3-07 M6-05, M6-06, M7-02, M13-2, M8-05, M14-1, M14-2, M14-3, M14-4 Kru3, Kru7, Kru9, Kru11 Kon 1 Kr49 S8 F1, S13 F1 1 S5 R

Chromosome race

Neroosa

Seliger

RUSSIAN JOURNAL OF GENETICS

Moscow

gm, hi, kr, no, pq

Vicinity of Seliger Lake, 5 localities Tver' oblast Staritsy district, Krutitsy village Tver' oblast Moscow oblast Vicinity of Seliger Lake 2 localities Vicinity of Seliger Lake Krasnogorsk Konakovo 1 4

9

MOLECULAR VARIABILITY IN THE COMMOM SHREW

Vol. 42

No. 6

2006

Moscow­Seliger hybrids

Moscow­Seliger recombinant karyotype go, hi, kq, mn, pr go, hn, ip, kq, mr o, hn, ip, km, qr Sverdlovsk oblast, source of Pechora R. Kemerovo oblast, valley of Tom' R. Krasnoyarskii krai Saratov oblast Kostroma oblast

Manturovo

Manturovo, Shilovo village D'yakovskii forest Ilych, foothills, right shore

1 2 5

Mant Soc (Soc2) Pech1, Pech2, Pech3, Pech4, Pech5

Sok

Serov

Tomsk

gk, hi, mn, o, p, qr

Krapivinskii district, Adzhendarovo Right shore of Yenisei R., Lebed' village

3

Ke2201, Ke2247, Ke2430

Non-karyotyped race (Strelka?) go, hi, k, m, n, p, q, r

10

Ye1,Ye2,Ye3, Ye10, Ye20, Ye22, Ye28, Ye30, Ye31 597

Note: Samples used only in inter-MIR­ or in inter-SOR­PCR are in brackets.


598

BANNIKOVA et al.

fractionated in 6% polyacrilamide gel, containing Trisborate buffer and 8 M urea [33]. The gel was 50 cm in length, 28 cm in width, and 0.4 mm in thickness. Electrophoresis was run for 9 h at constant current strength of 73 W. For radio autography, the dried gel was exposed with X-ray film RETINA for 16­48 h. Phylogenetic analysis. Fingerprints, derived in MIR- and SOR­PCR, were transformed into a binary matrix (1, the band presence; 0, the band absence), which was analyzed by Wagner's maximum parsimony (MP) and neighbor-joining (NJ) methods implemented in the PAUP* version 4.0b10 software package [34]. Statistical reliability of the groups on NJ and MP trees was estimated by bootstrap with 1000 replications. The validity of inter-SINE­PCR-derived groups was also tested by multidimensional scaling based on Nei­Li distances (DNL) [35], using the STATISTICA package [36]. RESULTS AND DISCUSSION East­West Division of the Chromosome Races The parsimony and distance trees, derived from the results of inter-MIR- and inter-SOR­PCR analyses, were similar in their general topography, demonstrating the subdivision of the total sample into two large groups. The first of these groups was formed by races Moscow, Seliger, Manturovo, and Neroosa, distributed in the central European Russia, while the second consisted of Siberian populations of the Kemerovo oblast (race Tomsk) and the right shore of the middle Yenisei River (probably, race Strelka). The Serov race, represented by the sample from the European slopes of the northern Urals (several localities from the right shore of the upper Pechora River), in this scheme proved to be closer to the Siberian races than to the East European ones. The difference between the results of inter-SOR­ PCR and inter-MIR­PCR lied in the fact that in the former case, the specimen of the Sok race from the Saratov oblast population was grouped with the Siberian races and the Serov race (bootstrap support 52%), and in the latter case, two Sok specimens from the same population tended to the East European races (bootstrap support 69%). Since the bootstrap supports of the both groups were not high, we considered the difference between the inter-SOR­PCR and inter-MIR­PCR results nonsignificant and combined the corresponding binary matrices. In what follows, we discuss the combined inter-SINE­PCR tree (Fig. 2). The topology of the combined parsimony tree (MP, 194 parsimony informative characters out of the total number of 354) and neighbor-joining (NJ) tree coincides; it is shown in Fig. 2. A clear division into the western (European Russia) and eastern (Siberia) groups is seen, with the Pechora River populations of the Serov race tending to the Siberian group. The NJ/MP bootstrap supports for the western and the eastern groups were respectively 76/61% and 100/100%. The specimen of the Sok race, examined in both analyses (inter-

SOR­PCR and inter-MIR­PCR) could not be clearly attributed to any of the groups, thus forming the third potential branch. The multidimensional scaling provided additional information on the molecular genetic relationships of the populations studied (Fig. 3a). The western and eastern chromosome race groups were distinguished without overlapping by two-dimensional scaling; the Pechora population of the Serov race grouped with two Siberian races. Table 2 presents Nei­Li mean genetic distances (DNL) among the races examined. The greatest distance was found in the pairs of races Serov/Sok and Serov/Seliger (DNL = 0.39). The mean genetic distance between the races of European Russia (without Sok) and the Siberian races was 0.3. Molecular Structure of the Siberian Race Group In the Siberian cluster, which included the Kemerovo population of the Tomsk race and the population from the right shore of middle Yenisei River basin, the group of Tomsk is supported by a high bootstrap value (92/95%), while the Yenisei population looks polymorphic and paraphyletic (Fig. 2). Nevertheless, multidimensional scaling (Fig. 3b) showed that these two groups did not overlap in the space of the two scales. The mean DNL between them was 0.16, which is rather close to the genetic distance between the Serov race from the East European group and the Yenisei population (DNL = 0.20) or races Serov and Tomsk (DNL = 0.23). Molecular Polymorphism of the Moscow Race and the Relationships with the Chromosome Race Seliger Relationships between the Moscow and Seliger races are of particular interest, because of the restricted hybrid zone, found on their mutual border near Lake Seliger (Fig. 1). In this zone, chromosome markers of the Moscow race were detected in five out of 12 sampling localities (arbitrarily regarded as the southern part of the contact zone). Only one sample (locality 14) included animals exclusively of the Moscow race, whereas the remaining four samples consisted of both races (localities 6, 7, 8) and (or) their hybrids (localities 8, 13). Interestingly, hybrid F1 karyotypes were recorded at sampling sites that limited the distribution of the Moscow race in the zone of contact with race Seliger. The representatives of the Seliger chromosome race were found in ten sampling localities (arbitrarily regarded as the northern part of the contact zone): in three of them, together with animals of the Moscow race and in one (locality 5), one individual with hybrid karyotype Seliger + Moscow (code S5R) was recorded among animals of the Seliger race. It is also of interest that mapping of the distribution of individuals of the Moscow and Seliger races in the contact zone revealed
Vol. 42 No. 6 2006

RUSSIAN JOURNAL OF GENETICS


MOLECULAR VARIABILITY IN THE COMMOM SHREW
Manturovo Manturovo S5R S3-07 60 S1-04 S8-06 52 54 S9-02 72 S12-8 S7-01 S15-5 S15-2 S15-1 S15-3 S4-10 S5-14 Seliger

599

/

51

88 79 M7-02 M13-2 M6-06

M8-05 S8 F1 76 61 65 S13 F1 M14-1 M6-05 M14-3 M14-2 M14-4

Moscow

63 Kr 49 Konakovo 55 68
/ / /

55 100 100

83 90 Ye 23 92 95

Kru 3 Kru 7 Kru 9 Kru 11 19-00 Neroosa 13-99 14-00 D'yakovka 81 76 Ye 1 Sok Ke2201 Ke2430 Ke2247 Tomsk

/

60 58
/

100 100 63
/

100 100

100 53

Ye 20 Ye 22 Ye 2 Ye 3 Strelka ? 62 Ye 10 58 Ye 28 Ye 30 Ye 31 Pech4 Pech3 Serov Pech5 Pech1 100 94 Pech2

Fig. 2. NJ tree of genetic relationships among the chromosome races of Sorex araneus, based on Nei­Li genetic distances inferred from IS­PCR. For clusters with reliability higher than 50%, bootstrap indices are shown (% of 1000 replications): NJ, above the branches and MP, under the branches. Sample codes correspond to the collection codes in Table 1.

RUSSIAN JOURNAL OF GENETICS

Vol. 42

No. 6

2006


600 Scale 3 0.8 0.6 0.4 0.2 0 ­ 0.2 ­ 0.4 ­ 0.6 ­ 0.8
Seliger

BANNIKOVA et al. (a)
Moscow

Tomsk

Enisei Strelka?

Manturovo Serov

Neroosa

Sok

­ 1.0 ­ 1.2 ­ 1.0 ­ 0.8 ­ 0.6 ­ 0.4 ­ 0.2 0 Scale 2 1.2 0.8
Ye23 Ye28 Ye30 Ye31

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Scale 1 (b)

Enisei Strelka? Pech4 Pech1 Pech5 Pech2 Pechora Serov Pech3

0.4 0 ­ 0.4

Ye22 Ye10 Ye3 Ye20 Ye2 Ye1

Ke2201 Ke2430 Kemerovo Tomsk

­ 0.8 ­ 1.2 ­ 1.6 ­ 1.0
Ke2247

­ 0.6

­ 0.2

0.2 0.6 Scale 1

1.0

1.4

1.8

Fig. 3. Multidimensional scaling of genetic variability in Sorex araneus chromosome races for 354 IS­PCR characters in the space of two coordinates. Solid dots, Moscow race; open dots, Seliger race; half-solid dots, F1 hybrids or recombinant animals. Other symbols correspond to the samples of the remaining chromosome races, used in analysis and shown in the figure. (a) Samples including all of the races examined; (b) the Siberian group of chromosome races.

a population border, which was not determined by any external barriers [37]. Inter-SINE­PCR analysis has detected considerable molecular polymorphism within the Moscow race. In our study, this race was represented by samples from four populations, one of which inhabited the contact zone near Seliger Lake, two were from two other regions of Tver' area (Staritsa and Konakovo), and the remaining one was from the Moscow area (Krasnogorsk). To get deeper insight into the relationships among these populations, we have additionally analyzed the samples, including races Moscow and Seliger, by means of multidimensional scaling, apart from the remaining samples from European Russia and

Siberia (Fig. 4a). Figure 4a clearly shows that the sample containing Moscow and Seliger races is subdivided into three groups: (1) race Seliger (code S); (2) race Moscow from the zone of sympatry with the Seliger race (code M); and (3) allopatric populations of the Moscow race from the village of Krutitsy (Staritsa district, Tver' oblast), Konakovo (Tver' oblast), and Krasnogorsk (Moscow oblast) (codes Kru, Kon, and Kr, respectively). The former two groups are closer to one another, than to the third group. The mean genetic distances between the groups were as follows: between the Seliger race and the allopatric populations of the Moscow race, 0.27; between the allopatric populations of the Moscow race and the Moscow race from the symVol. 42 No. 6 2006

RUSSIAN JOURNAL OF GENETICS


MOLECULAR VARIABILITY IN THE COMMOM SHREW Table 2. Nei­Li genetic distances within and between the races of the common shrew inferred from inter-SINE­PCR Race Seliger Moscow Manturovo Sok Tomsk Strelka? Serov Neroosa Seliger 0.17 0.23 0.24 0.38 0.34 0.33 0.39 0.24 0.17 0.18 0.33 0.32 0.30 0.35 0.24 ­ 0.27 0.32 0.30 0.34 0.22 ­ 0.36 0.33 0.39 0.35 0.10 0.16 0.23 0.30 0.11 0.20 0.30 0.09 0.31 0.07 Moscow Manturovo Sok Tomsk Strelka? Serov

601

Neroosa

patry zone with the Seliger race, 0.22; between the Seliger race and the sympatric Moscow race, 0.15. Both F1 hybrids presented in the sample in the NJ tree (Fig. 2) and in multidimensional scaling, are within the Moscow race from the contact zone; the individual with the recombinant Seliger + Moscow karyotype falls into the Seliger cluster (Figs. 2, 4). The Seliger cluster tends to exhibit internal structuring: the animals from localities 15, 12, 9, and 7 are grouped against the samples from localities 1, 4, 5, 8 (Fig. 4). However, this trend may reflect nothing more than association with geographic distance (Fig. 1). Thus, although races Moscow and Seliger demonstrate some degree of differentiation from one another, the molecular structuring is less marked than could be expected on the basis of the karyotype composition and low hybridization. This is accounted for by the presence of the Moscow population from the contact zone. If this population is excluded, both chromosome races show far higher divergence (Fig. 4b). This "buffering" Moscow population from the contact zone may have resulted from distant hybridization, while the allopatric populations from the Staritsy district of Tver' oblast and from Moscow oblast represent the "pure" race. It is also possible that the time of formation of these traces, conversely, is too small to accumulate noticeable changes at the molecular level, being sufficient for significant karyotype rearrangements. Periods of karyotypic revolutions have been recorded in macrophylogenetic studies of some mammal groups [38], but there are no grounds to extend this possibility to lower taxonomic levels. Analysis of the relationships among chromosomal races and different populations within the Moscow race showed that the divergence of the races tended to be positively associated with the geographic distance between them. This association could not be conclusively demonstrated because of the small sample size and the absence of a sufficient number of intermediate
RUSSIAN JOURNAL OF GENETICS Vol. 42 No. 6

populations between two populations located far apart. However, the revealed trend indicates that the molecular genetic variability of the common shrew population examined results from their geographic isolation rather than their unique origin. Karyotypic and Molecular Structuring of Sorex araneus Although all of the studied common shrew samples formed groups according to the detected chromosome races, the genetic structure inferred from IS­PCR did not conform to the relationships between the races based on karyological analysis. For instance, the Moscow race belongs to the Western European karyotypic group, WEKG [39], and karyotypes Manturovo and Neroosa are though to be derived from karyotypes of this group [22]. The Seliger race is assigned to the East European karyotypic group, EEKG [6]. The Sok and Seliger races belong to the Urals family of the same group and are karyologically close to the North European group, NEKG [40] or have appeared in parallel, because of canalization of karyotypic evolution [41]. These relationships among the chromosome races, inferred from karyotype analysis, are discordant with the IS­PCR-based race grouping. In our study, only the cluster formed by the Tomsk and Strelka races (Yenisei population) fully correspond to the East European race group [23]. An extension of the set of chromosome races and an increase in sample size may lead to the formation of more distinct groups or, conversely, to obliterate all borders between them in molecular analysis. However, our experience, for example, with the Neroosa race makes this assumption doubtful. Using 24, rather than 3, samples of this race in IS­PCR [21] did not result in grouping this sample together with karyotypically close races Moscow and Manturovo.
2006


602 Scale 2 1.2
Kru9

BANNIKOVA et al. (a)
Kru11 Kru3 Kru7 Kr49 S5R M7 - 02 M6 - 06 M6 - 05 M13 - 2 M14 - 3 M14 - 2 M8 - 05 M14 - 1 M14 - 4 S13 F1 S8 F1 S15-2 S15-3 S12-8 S15-5 S15-1 S9-2 S7-01

0.8 0.4 0 ­ 0.4 ­ 0.8

Kon

S4-10 S5-14 S1-04 S8-06 S3-07

­ 1.2 ­ 1.0 ­ 0.8 ­ 0.6 ­ 0.4 ­ 0.2 Scale 3 0.8
S1-04

0

0.2 0.4 0.6 0.8 1.0 1.2 1.4 Scale 1 (b)

0.6
S4-10

0.4 0.2 0

S8-06

S5-14

Kru9

S15-5 S15-2 S3-07 S15-1 S15-3 Kon Kr49 S12-8 S7-01 S9-2

Kru11 Kru7

Kru3

­ 0.2 ­ 0.4 ­ 0.6

­ 0.8 ­ 1.0 ­ 0.8 ­ 0.6 ­ 0.4 ­ 0.2

0

0.2 0.4 Scale 1

0.6

0.8

1.0

1.2

1.4

Fig. 4. Multidimensional scaling of genetic variability of chromosome races Seliger and Moscow inferred from IS­PCR. (a) Samples including all of the races examined; (b) allopatric populations of chromosome races Moscow and Seliger.

Thus, the genetic composition of the species S. araneus, manifested in the presence of numerous chromosome races, is confirmed at the molecular level, though no association was found between the molecular and karyological race grouping. Nevertheless, IS­PCR analysis showed clustering of the samples in accordance to the detected chromosome races and westerneastern differentiation of the common shrew populations studied. However, this genetic pattern may result from geographic distance between the populations. The efficiency of the barriers, separating the chromosome races, is restricted because of the gene flow between them. Even in the case of races Moscow and Seliger, whose karyotypes are maximally distant from one another, the contact of these races in the sympatry zone

increases the gene exchange, which is expressed in the IS­PCR polymorphism of the Moscow race. Thus, the problem, posed by analysis of all so far available evidence on molecular and karyological variability of the common shrew, is as follows: to what extent the easy formation of new karyotypes in this species promotes the degree of genetic differentiation of the populations that allows them to coexist without mixing and may lead to subsequent isolation and speciation. This question seems to have no clear answer yet. ACKNOWLEDGMENTS We thank the colleagues who participated in collecting shrews for karyological and molecular analyses:
Vol. 42 No. 6 2006

RUSSIAN JOURNAL OF GENETICS


MOLECULAR VARIABILITY IN THE COMMOM SHREW

603

E.I. Krysanov, N.A. Shchipanov, A.A. Kalinin, V.B. Il'yashenko, S.G. Dmitriev, and A.V. Bobretsov. This study was supported by the Russian Foundation for Basic Research (grant no. 05-04-49240) and INTAS (grant no. 03-51-4030). REFERENCES
1. Orlov, V.N. and Bulatova, N.Sh., Sravnitel'naya tsitogenetika i kariosistematika mlekopitayushchikh (Comparative Cytogenetics and Karyosystematics of Mammals), Moscow: Nauka, 1983. 2. Searle J.B., Three New Karyotypic Races of the Common Shrew Sorex araneus (Mammalia: Insectivora) and a Phylogeny, Syst. Zool., 1984, vol. 33, pp. 184­194. 3. Searle, J.B and Wojcik, J.M, Chromosomal Evolution: The Case of Sorex araneus, Evolution of Shrews, Wojcik, J.M. and Wolsan, M., Eds., Bialowieza: Mammal Res. Inst. Polish Acad. Sci., 1998, pp. 219­268. 4. Wojcik, J.M., Ratkiewicz, M., and Searle, J.B., Evolution of the Common Shrew Sorex araneus: Chromosomal and Molecular Aspects, Acta Theriologica, 2002, vol. 47, suppl. 1, pp. 139­167. 5. Wojcik, J.M., Borodin, P.M., Fredga, K., et al., The List of the Chromosome Races of the Common Shrew Sorex araneus (Updated 2002), Mammalia, 2003, vol. 67, no. 2, pp. 169­178. 6. Orlov, V.N., Bulatova, N.Sh., Kozlovskii, A.I., and Balakirev, A.E., Hierarchy of Intraspecific Taxa of Common Shrew Sorex araneus (Insestivora), and Taxonomic Species Structure in Mammals, Zool. Zh., 2004, vol. 83, no. 2, pp. 199­212. 7. Volobouev, V.T. and Catzeflis, F., Mechanisms of Chromosomal Evolution in Three European Species of the Sorex araneus­arcticus Group (Insectivora: Soricidae), Zeitschrift fur Zool. Systematik und Evolutionsforschung, 1989, vol. 27, pp. 252­262. 8. Zima, J., Is the Trend Toward Low 2Na Numbers Inescapable for Sorex araneus Populations?, Mem. Societe Vaudoise Sci. Natur., 1991, vol. 19, pp. 63­71. 9. Searle, J.B., Fedyk, S., Fredga, K., et al., Nomenclature for the Chromosomes of the Common Shrew (Sorex araneus), Mem. Societe Vaudoise Sci. Natur., 1991, vol. 19, pp. 13­22. 10. Ratkiewicz, M., Fedyk, S., Banaszek, A., et al., The Evolutionary History of the Two Karyotypic Groups of the Common Shrew, Sorex araneus, in Poland, Heredity, 2002, vol. 88, pp. 235­242. 11. Andersson, A.-C., Postglacial Population History of the Common Shrew (Sorex araneus) in Fennoscandia: Molecular Studies of Recolonisation, Sex-Biased Gene Flow and the Formation of Chromosome Races, Acta Universitatis Upsaliensis, Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 986, Uppsala: Uppsala Univ, 2004. 12. Bannikova, A.A., Bulatova, N.S., Lebedev, V.S., and Kramerov, D.A., Mitochondrial and Nuclear DNA Variability of the East European and Siberian Chromosome Races of the Common Shrew Sorex araneus, Evolution in the Sorex araneus Group: Cytogenetic and Molecular Aspects: 7th Meeting Intern. Sorex araneus Cytogenetics Committee (ISACC), St. Petersburg, 2005, pp. 14­15.
RUSSIAN JOURNAL OF GENETICS Vol. 42 No. 6

13. Ruedi, M., Protein Evolution in Shrews, Evolution of Shrews, Wojcik, J.M. and Wolsan, M., Eds., Bialowieza: Mammal Research Inst. Polish Acad. Sci., 1998, pp. 269­294. 14. Hedrick P.W., Highly Variable Loci and Their Interpretation in Evolution and Conservation, Evolution, 1999, vol. 53, pp. 313­318. 15. Balloux, F., Brunner, H., Lugon-Moulin, N., et al., Microsatellites Can Be Misleading: An Empirical and Simulation Study, Evolution, 2005, vol. 54, no. 4, pp. 1414­ 1422. 16. Lugon-Moulin, N., Balloux, F., and Hausser, J., Genetic Differentiation of Common Shrew Sorex araneus Populations among Different Alpine Valleys Revealed by Microsatellites, Acta Theriologica, 2000, vol. 45, no. 1, pp. 103­117. 17. Bannikova, A.A., Matveev, V.A., and Kramerov, D.A., Using Inter-Sine­RCR to Study Mammalian Phylogeny Rus. J. Genet., 2002, vol. 38, no. 6, pp. 714­724. 18. Bannikova, A.A., Lavrenchenko, L.A., and Kramerov, D.A., Phylogenetic Relationships between Afrotropical and Palaearctic Crocidura Species Inferred from Inter-SINE­PCR Data, Biochem. Syst. and Ecol., 2004, vol. 33, pp. 45­59. 19. Bannikova, A.A., Kramerov, D.A., Vasilenko, V.N., et al., DNA Polymorphism in Erinaceus Hedgehogs and Polytypicism of E. concolor (Insectivora, Erinaceidae), Zool. Zh., 2003, vol. 82, no. 1, pp. 1­11. 20. Matveev, V.A., Kruskop, S.V., and Kramerov, D.A., Revalidation of Myotis petax Hollister, 1912 and Its New Status in Connection with M. daubentonii (Kuhl, 1817) (Vespertilionidae, Chiroptera), Acta Chiropterologica, 2005, vol. 7, no. 1, pp. 23­37. 21. Bannikova, A.A., Bulatova, N.S., Krysanov, E.Y., and Kramerov, D.A., DNA Polymorphism of Sorex araneus and Its Relationships with Associated Species As Derived from Inter-SINE-PCR, Mammalia, 2003, vol. 38, no. 2, pp. 263­274. 22. Bulatova, N., Searle, J.B., Bystrakova, N., et al., The Diversity of Chromosome Races in Sorex araneus from European Russia, Acta Theriologica, 2000, vol. 45, no. Suppl. 1, pp. 33­46. 23. Polyakov, A.V., Panov, V.V., Ladygina, T.Yu., et al., Chromosomal Evolution of the Common Shrew Sorex araneus L. from the Southern Urals and Siberia in the Postglacial Period, Rus. J. Genet., 2001, vol. 37, no. 4, pp. 351­357. 24. Hausser, J., Fedyk, S., Fredga, K., et al., Definition and Nomenclature of Chromosome Races of Sorex araneus, Folia Zoologica, 1994, vol. 43, suppl. I, pp. 1­9. 25. Bulatova, N., Kalinin, A., Alexandrova, A., and Alexandrov, D., Perspectives of Studying a Border Zone between Two Chromosome Races--Moscow and Seliger--of Sorex araneus, Evolution in the Sorex araneus Group: Cytogenetic and Molecular Aspects, 6th Meeting Intern. Sorex araneus Cytogenetics Committee (ISACC), Paris, 2002, p. 18. 26. Bulatova, N., Shchipanov, N., and Searle, J., The Moscow­ Seliger "Strong" Hybrid Zone--a Model System in European Russia, Evolution in the Sorex araneus Group: Cytogenetic and Molecular Aspects: 7th Meeting Intern. Sorex
2006


604

BANNIKOVA et al. araneus Cytogenetics Committee (ISACC), St. Petersburg, 2005, p. 20. Sheftel, B.I. and Krysanov, E.Yu., Chromosome Polymorphism of Race Neroosa (Sorex araneus) on the Territory with Radioactive Pollution After Chernobyl Accident, Evolution in the Sorex araneus Group: Cytogenetic and Molecular Aspects: 6th Meeting Intern. Sorex araneus Cytogenetics Committee (ISACC), Paris, 2002, pp. 29­30. Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Lab., 1989. Smit, A. and Riggs, A., MIRs are Classic, tRNA-Derived SINEs that Amplified before the Mammalian Radiation, Nucl. Acids Res., 1995, vol. 23, no. 1, pp. 98­102. Jurka, J., Zietkiewicz, E., and Labuda, D., Ubiquitous Mammalian-Wide Interspersed Repeats (MIRs) are Molecular Fossils from the Mesozoic Era, Nucl. Acids Res., 1995, vol. 23, no. 1, pp. 170­175. Gilbert, N. and Labuda, D., Evolutionary Inventions and Continuity of CORE-SINEs in Mammals, J. Mol. Biol., 2000, vol. 298, pp. 365­377. Borodulina, O.R. and Kramerov, D.A., Short Interspersed Elements (SINEs) from Insectivores: Two Classes of Mammalian SINEs Distinguished by A-Rich Tail Structure, Mammalian Genome, 2001, vol. 12, pp. 779­786. Slatko, B.E and Albright, L.M, DNA Sequencing, in Short Protocols in Molecular Biology, Ausubel, F.M., et al., Eds., New York: Wiley, 1992, pp. 48­53. 34. Swofford, D.L., PAUP: Phylogenetic Analysis using Parsimony (and Other Methods), Version 4, Sunderland: Sinauer, 1998. 35. Nei, M. and Li, W.-H., Mathematical Model for Studying Genetic Variation in Terms of Restriction Endonucleases, Proc. Natl. Acad. Sci. USA, 1979, vol. 76, pp. 5269­5273. 36. Statsoft: Statistica for Windows, Tulsa: StatSoft, 1995, pp. 74 104­74 442. 37. Shchipanov, N.A., Population as a Unit of a Species Existence: Small Mammals, Zool. Zh., 2003, vol. 82, no. 4, pp. 450­469. 38. Trifonov, V.A., Perel'man, P.L., Romanenko, S.V., et al., Phylogenomics of Mammals: Cytogernetic Aspects, Biol. Membr., 2005, vol. 22, no. 3, pp. 212­226. 39. Orlov, V., Bulatova, N., Kozlovsky, A., et al., Karyotypic Variation of the Common Shrew (Sorex araneus) in European Russia: Preliminary Results, Hereditas, 1996, vol. 125, pp. 117­121. 40. Polyakov, A.V., Zima, J., Searle, J.B., et al., Chromosome Races of the Common Shrew Sorex araneus in the Ural Mountains: A Link Between Siberia and Scandinavia?, Acta Theriologica, 2000, vol. 45, suppl. 1, pp. 19­26. 41. Orlov, V.N. and Kozlovsky, A.I., On the Role of the Glacial Period in the Development of Chromosomal Polymorphism in Common Shrew Sorex araneus L. (Insestivora, Mammalia), Dokl. Akad. Nauk, 2002, vol. 386, pp. 423­426.

27.

28. 29. 30.

31. 32.

33.

RUSSIAN JOURNAL OF GENETICS

Vol. 42

No. 6

2006