1. Genetics differentiation and systematic of peripheral populations of East Asian endemic rodent Myospalax psilurus (Rodentia, Spalacidae) M.V. Tsvirka, M.V. Pavlenko, V.P. Korablev Institute of Biology and Soil Science FEB RAS Vladivostok, 690022, Russia 2013

2. endemic in the Eastern Asia taxonomy and evolution are not well studied 6-10 recognized species one (Myospalax) or two (Myospalax and Eospalax) genera Fan, Shi, 1982; Musser, Carleton, 2005 PROBLEMS subfamily Myospalacinae:

3. L. Martynova, N. Vorontsov, 1975. Population cytogenetic of Zokors Chromosomes L. Martynova, 1976. Chromosomal differentiation of three species of zokors. L. Martynova, I. Fomicheva, N. Vorontsov, 1977. Electrophoretic study of blood protein of zokors. PROBLEMS M. myospalax M. aspalax M. armandii M. psilurus

4. PROBLEMS

5. - to clarify the genetic differences among two peripheral populations of the Manchurian zokors - to resolve the systematic position of M. psilurus from Khanka Plain and M. epsilanus from Transbaikalia MAIN GOAL, METHODS, MATERIALS - 45 specimens of the Manchurian zokors were studied from 12 localities : - 27 from Khanka Plain -18 from Transbaikalia by allozyme, karyological and molecular-genetic methods :RAPD- analysis, sequencing of two mt DNA fragments CR (518 bp) and cyt b (1141 bp)

6. Study area and localities of Manchurian zokor M. psilurus MAIN GOAL, METHODS, MATERIALS

7. RESULTS Myospalax epsilanus, Transbaikalia, 2n=64 (9 M-SM, 13-14 ST, 8-9 A) Myospalax psilurus, Primorye, 2n=64 (9-12 M-SM, 10-13 ST, 8-A) 1. Tf-A - Myospalax aspalax 2. Tf-B - Myospalax psilurus, Primorye 3. Tf-C - Myospalax epsilanus, Transbaikalia 4. Tf-C1 - Myospalax armandii 5. Tf-D - Myospalax myospalax

8. RESULTS 1-3 Myospalax epsilanus, Transbaikalia 4-5 Myospalax psilurus, Primorye RAPD-marker fragments and position of studied populations on the UPGMA (a) and MST (b) trees

9. Phylogenetic trees of studied Zokor species Myospalax epsilanus Transbaikalia, Chita territory 2N=64, Tf-C Myospalax psilurus Primorye territory 2N=64 Tf-B Myospalax aspalax Transbaikalia, Chita territory 2N=62, Tf-A Myospalax armandii Transbaikalia, Chita territory, 2n=62-66, Tf-C1 Myospalax smithii, China, 2n=62 RESULTS NJ cyt b 1141 bp NJ RAPD 463 fragments

10. MST phylogenetic trees of studied Zokor species RESULTS Myospalax psilurus Primorye territory Myospalax epsilanus Transbaikalia MST RAPD 463 fragments MST CR 518 bp

11. NHtHt HsHs D st G st NmD/SEx.(р) M. aspalax – M. armandii 360.14 ±0.034 0.1 ±0.02 0.03 ±0.01 0.251.480.08/0.920.00 M. epsilanus– M. psilurus 450.20 ±0.04 0.17 ±0.02 0.03 ±0.01 0.162.620.08/0.920.00 N – number of specimens, genetic diversity Dst, calculated on the basis of the total within sample genetic diversity Ht and mean sample genetic diversity Hs, coefficients of genetic differentiation Gst, number of migrants per generation Nm, genetic distances D, and genetic similarity S, and performed exact test for population differentiation Interspecific relationships within the genus Myospalax (PAPD-PCR data)

12. M. smithii M. rufescens M. myospalax M. aspalax M. armandii M. epsilanus M. psillurus M. smithii00.01740.23580.17230.18730.32760.3098 M. rufescens600.23390.16940.18380.16040.1549 M. myospalax737700.17670.15740.17850.1567 M. aspalax63605700.06730.10530.0744 M. armandii6665382300.10120.0819 M. epsilanus69 60353200.0595 M. psilurus58 512729310 Estimate value of Kimura two-parametric distance matrix (above diagonal) and transitions and transversions (Ts+Tv) (below diagonal) for 7 taxa of Myospalax Interspecific relationships within the genus Myospalax (CR- mt DNA)

13. The study was partly supported by the RFBR (12-04-00795-a) Dr. Marina V. Pavlenko Dr. Vladimir P. Korablev Thank you for your attention

14. The Zokor Myospalax is the endemic group of subterranean rodent in the East Asia. More then 10 species are whole in this group, and only 4 of them there are in Russian Far East. Manchurian zokor Myospalax psilurus (Milne-Edwards, 1874) geographically distributed from Transbaikalia to Central China. In Russia there ate two peripheral populations. The first are distributed in east part of Transbaikalia, and second one occupies Khanka plain (south-west part of Primorsky Krai). The systematic ranks of these forms are not clear. In the literature, M. epsilanus (Thomas, 1912) is regarded as a subspecies or as a synonym of M. psilurus (Ognev, 1947, Gromov, Erbaeva, 1995; Zhang et al., 1997, Wilson, Reeder, 2005), or as a separate species (Lawrence, 1991; Pavlinov, Rossolimo, 1998; Pavlenko, Korablev, 1999). The complex of the genetic methods were used to study the differentiation in the "M. psilurus - M. epsilanus" group, and to clarify the systematic position of two peripheral population from the Russian Far East and Transbaikalia. The diploid number of chromosomes are 2n = 64 in all of studied zokors. There are also the significant differences in the structure of the karyotype between populations from the Transbaikalia region (9-12 M-SM, 10-13 ST, 8-A: "epsilanus") and the Russian Far East (9 M-SM, 13-14 ST, 8-9 A: "psilurus"). Furthermore, these two populations have two different variants of the electrophoretic blood protein (transferrin), each of them is a monomorphic for studied samples within the geographical region and marks the peripheral populations. Random Amplification of Polymorphic DNA PCR (RAPD PCR) obtained the specific markers and genetic evidence of differentiation between two peripheral populations. By sequencing of two mitochondrial DNA fragments (mt DNA), a 514 base-pair fragment of the control region and a 1141 base- pair of the cytochrome b, we found the high lewel genetic differentiation between populations from Primorye and Transbaikalia. The all phylogenetic trees (NJ, MP, ML) are shown two groups of zokors from different geographical regions with high values of support. The genetic distances between these two groups are high, that comparable with those for the pair of closely related species Myospalax aspalax and M. armandii, which are regarded as a separate species taxa.Polymorphic Thus, the results confirm the hypothesis of the genetic differentiation of two populations M. psilurus from different geographical regions previously proposed on protein markers (Pavlenko, Korablev, 2003) and support a distinct taxonomic position the each of peripheral populations. Under the proposed hypothesis, populations of zokors from the territory of Primorsky Krai and Transbaikal region belong to different species - M. psilurus (Milne-Edwards, 1874) and M. epsilanus (Thomas, 1912), or semispecies within the superspecies M. psilurus.