© Comparative Cytogenetics, 2009 . Vol. 3, No. 2, P. 85-89. ISSN 1993-0771 (Print), ISSN 1993-078X (Online) AFLP diversity between the Novosibirsk and Tomsk chromosome races of the common shrew (Sorex araneus) A.V. Polyakov1, V.B. Ilyashenko2, S.S. Onischenko2, V.V. Panov3, P.M. Borodin1 1Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sci- ences, Prosp. akademika Lavrentieva, 10, Novosibirsk 630090, Russia. 2Department of Zoology and Ecology Kemerovo State University, Krasnaya ul., 6, Kemerovo 650043, Russia. 3Institute of Systematics and Ecology of Animals, Siberian Branch of Russian Academy of Sciences, ul. Frunze, 11, Novosibirsk 630091, Russia. E-mails: [email protected], [email protected] Abstract. Genetic diversity between of the Novosibirsk and Tomsk chromo- some races of the common shrew (Sorex araneus) was analyzed using 39 poly- morphic AFLP (amplifi ed fragments length polymorphism) markers. Exact and F-statistics tests for population differentiation demonstrated signifi cant interra- cial difference in allele frequencies and signifi cant subdivision between the races. The value of the genetic distance between the chromosome races observed in this study corresponds to that found between subspecies of mammals studied so far. Key words: Sorex araneus, chromosome races, AFLP, interracial diversity. INTRODUCTION been found between races within a karyotypic The common shrew Sorex araneus Lin- group (Hausser, 1984; Searle, Thorpe, 1987; naeus, 1758 inhabits a huge territory includ- Hausser et al., 1991; Meyer, Searle, 1994), sig- ing the whole north part of Europe and Asia nifi cant morphological differences have been up to Baikal Lake (Corbet, 1978) and displays detected between the shrews belonging to dif- a remarkable chromosomal polymorphism ferent karyotypic groups: WEKG and EEKG determined by local fi xation of various Rob- in Poland (Chętnicki et al., 1996) and EEKG ertsonian and whole-arm reciprocal transloca- and SKG in Russia (Pavlinov, 2004). tions (Searle, Wójcik, 1998). The species area Novosibirsk and Tomsk chromosome is subdivided into a mosaic of parapatric chro- races belong to different karyotypic groups mosome races that has been classifi ed into - EEKG and SKG correspondingly (Searle, four main karyotypic groups: the West Euro- Wójcik, 1998; Polyakov et al., 2001). The pean (WEKG), the East European (EEKG), Novosibirsk race contains diagnostic chro- the North European (NEKG) and the Siberian mosomes go,hn,ik,mp,qr; the Tomsk race - (SKG) (Searle, 1984; Wójcik, 1993; Searle, gk,hi,mn,o,p,q,r. The Novosibirsk race is dis- Wójcik, 1998; Polyakov et al., 2001). Where- tributed in the lowlands, while the Tomsk race as no clear morphological divergence has – in the highlands of the West Siberia (Král, http://pensoftonline.net/compcytogen Comparative Cytogenetics doi: 10.3897/compcytogen.v3i2.14 86 A. V. Polyakov et al. Radjabli, 1974; Aniskin, Volobouev, 1981; MATERIAL AND METHODS Polyakov et al., 1996, 2000, 2001, 2003). They come into contact on the intermediate Animals landscape and form there a very narrow hybrid The shrews were collected within the zone (Polyakov et al., 2002, 2003). known territories of two chromosome races: It was known before the use of karyologi- Tomsk race (Agendarovo Vill., Kemerovo cal data that shrews of Siberian highlands dif- Distr. 54°45'N/87°01'E) and Novosibirsk race fered morphologically from other Siberian and (Novosibirsk vic. 54°49'N/83°06'E). Karyo- Uralian shrew samples (Dolgov, 1968, 1985). type of each individual was determined by Yudin (1989), combining these earlier G-banding of standard bone marrow chromo- observations and his own analysis with pio- some preparations. neering descriptions of Siberian chromosome races by Aniskin and Volobouev (1981), fi rst AFLP procedure assumed that the morphological distinction We followed Vos et al. (1995) in the AFLP between populations of shrews could be con- procedure. It was based on the selective PCR nected with their karyotypic divergence. Em- of restriction fragments from a total digest of phasizing the pronounced morphological and genomic DNA by EcoRI and MseI endonucle- karyotypic individuality of shrews from Altai ases. Two combinations of three randomly Yudin (1989) considered them as a subspecies chosen primers complementary to EcoRI and Sorex araneus rypheus in distinction to the MseI adapters with three extra-nucleotide each nominal subspecies Sorex araneus araneus. (E-ACT/M-CCG and E-ACT/M-CGT) were Subsequent investigations confi rmed that used for the analytic amplifi cation. Amplifi ed namely the shrews of the Tomsk chromosome fragments were resolved on 6% denaturing race were quite distinct morphologically from polyacrylamide gels and silver stained (Creste the shrews of neighboring Novosibirsk race et al., 2001). Gels were then dried and scanned. (Polyakov et al., 2002) as well as from some Electrophoretic bands were scored with Gel- other EEKG races (Okulova et al., 2007). Pro Analyzer system version 3.0.00.00 In this study we estimated genetic diversity Data scoring and statistical analysis between samples from populations of the No- Only precisely and evenly expressed elec- vosibirsk and the Tomsk chromosome races of trophoretic bands were chosen for analysis. the common shrew using polymorphic AFLP Length of visible fragments of DNA on gels (amplifi ed fragments length polymorphism) ranged between 60 and 800 nucleotides, how- markers. This method can generate large num- ever the area of fragments longer than 500 bers of molecular markers without any previ- nucleotides was not clear enough. For this ous knowledge of the genetic constitution of reason they were excluded from the analysis. the genotypes under investigation (Vos et al., We regarded each band as a locus with two al- 1995). Because of its universal applicability, ternative alleles: present (1) or absent (0). The reproducibility and a high power of discrimi- identifi cation of 39 polymorphic bands (=loci) nation this approach has been used success- led to the construction of a 47 individuals x 39 fully in numerous phylogenetic and ecological loci data matrix, which was analyzed for di- studies (see review of Bonin at al., 2007). This versity within and between races. The genetic is the fi rst time this method is used to analyze data was analyzed using the Tools for Popula- populations of the common shrew. tion Genetic analysis (TFPGA) program ver- Comparative Cytogenetics Comp. Cytogenet., 2009 3(2) Chromosome races of Sorex araneus 87 Table 1. Summary statistics of the sample sizes, to obtain variance estimates and confi dence number and % (in parentheses) of polymorphic loci and intervals. Nei’s (1973) index of gene diversity for the Novosi- birsk and the Tomsk chromosome races. RESULTS A summary of the genetic diversity esti- Race Sample Number of Gene mated from the AFLP data is given in Table size polymorphic diversity 1. Amount of polymorphic loci and the levels loci of gene diversity within two races were about Novosibirsk 24 31(79) 0.30±0.18 Tomsk 23 34(87) 0.37±0.17 similar with slight priority within the Tomsk race sample. Exact test however demonstrated the high signifi cance of the difference in bands 2 sion 1.3 (Miller, 1997) for dominant markers frequencies between the races: χ =189.6 (P< in diploid organism, assuming Hardy–Wein- 0.001). Two-level hierarchy F- statistics also berg equilibrium. Both band-based and allele revealed the signifi cant differences among the frequency-based approaches were applied to intra and interracial substructures. Bootstrap- extract the statistical information from AFLP ping (1000 replications) confi dence intervals data (Bonin at al., 2007). Exact test for pop- of within and between races jackknifi ng vari- ances θ estimates (θ =0.2237 ± 0.0366 and ulation differentiation (Raymond, Rousset, P θ =0.0315 ± 0.0441) did not overlap at the 1995) was applied to determine signifi cant R differences in the bands frequencies between 95% confi dence level. races. Genetic distance D (Nei, Li, 1979) Two estimators of genetic distance between NL races gave the cognate data: D = 0.0713 and was calculated from the bands frequencies N D = 0.0983. using the phylogenetic package PHYLIP 3.6 NL (RESTDIST and NEIGHBOR subroutines) DISCUSSION (Felsenstein, 2005). Summarizing the series of observations Unbiased genetic distance DN (Nei, 1978) and gene diversity (Nei, 1973) were calculated Nei (1975) designed the scale of relatedness on the base of allele frequencies. Frequency between the genetic distance and taxonomic of the null allele was calculated as a square categories. By this scale the genetic distance root of the frequency of null homozygotes between local populations of a species ranges (Weir, 1990). from 0 to 0.02 and from 0.02 to 0.2 between To evaluate the substructure of the stud- subspecies. Genetic differentiation between ied samples Weir and Cockerham’s (1984) the Novosibirsk and Tomsk races, estimated methods of calculating Wright’s F-statistics by DN and DNL was higher than that between were applied to the data. Two-level hierarchy local populations. Nearly the same values of (within and between races) was expressed us- genetic distance were published recently for ing the terminology of Weir (1990) where θ four sibling species of the blind mole rat Spa- lax ehrenbergi: S. galili, S. golani, S. carmeli, correspond to unbiased Wright’s FST. For hi- erarchical data set two θ values were calcu- and S. judaei (Polyakov et al., 2007). The lat- ter result was also obtained with the use of lated: θP measured differentiation within races AFLP method and is thus completely com- and θR estimated the differentiation between the races. Jackknifi ng and bootstrapping over patible with our data. Like the shrew chro- loci (using 1000 replications) were generated mosome races Novosibirsk and Tomsk these Comparative Comp. Cytogenet., 2009 3(2) Cytogenetics 88 A. V. Polyakov et al. four species are distinguished by chromosome Bannikova A.A., Bulatova N.S., Krysanov E.Y., complements and form parapatric areas (Nevo Kramerov D.A.