DIFFERENTIATION AND SYSTEMATICS IN THE LONG-TAILED GROUND SQUIRREL, SPERMOPHILUS UNDULATUS (SCIURIDAE, RODENTIA) Marina V. Tsvirka, Vladimir P. Korablev, Marina V. Pavlenko Institute of Biology and Soil Science FEB RAS, Vladivostok, 690022, Russia

Map of the ground squirrels distribution in the Palaearctic region

two geographical forms PROBLEM Genus Spermophilus Subgenus Urocitellus Species Spermophilus undulatus Subspecies S. u. undulatus, S. u. intercedens, S. u. menzbieri, S. u. jacutensis, S. u. stramineus, S. u. eversmanni two geographical forms “eastern” “western” (Vorontsov et al., 1980; Frisman, Vorontsov, 1989) (Gromov, Erbaeva, 1995)

? Map of S. undulatus distributions species or semispecies western form eastern form hybrid zone ? species or semispecies

Morphometric data (Popkov, 1978) Morphotypical study (Linetskiy, Linetskaya 1989) Karyological data (Korablev, 1988)

THE AIM IS to better understand species subdivision in S. undulatus , and ultimately speciation, in Spermophilus.

THE MAIN TASKS ARE To study genetic variability and differentiation of S. undulatus based on nuclear (RAPD) and mitochondrial (D-loop) DNA. To assess genetic divergence and phylogenetic relationships among subspesies of S. undulatus. To specify the taxonomic rank of eversmanni (independent species S. eversmanni or subspecies of S. undulatus) based on the data obtained

Map of sampling localities MATERIALS Map of sampling localities western form eastern form S. relictus, S. dauricus and S. parryi, S. taurensis and S. xanthoprymnus were chosen for comparative analyses, Tamias sibiricus was chosen as outgroup taxon.

Mitochondrial DNA analysis METHODS RAPD-PCR analysis 166 binary characters including the molecular markers Ten arbitrary decanucleotide primers Calculation of genetic distances (Nei, 1972) Reconstructions: NJ and UPGMA with bootstrap estimation of branching robustness in TREECONW 1.3 b (Van de Peer, De Wacher, 1994 ) Mitochondrial DNA analysis 5’ end of the D-loop (518 bp) Calculation of genetic distances based on Kimura two-parametric (K2P) estimator. Reconstructions: NJ, MP, ML and UPGMA with bootstrap estimation of branching robustness in MEGA 4.0 (Tamura et al., 2007)

Sequencing of the D-loop L15702 CAACGWAGYATAATATTYCGACC H15917 Primer Sequence (5' - 3') RAPD OPA-02 TGCCGAGCTG OPC-02 GTGAGGCGTC OPC-05 GATGACCGCC OPC-09 CTCACCGTCC OPC-12 TCGGCGATAG OPC-20 ACTTCGCCAC OPD-05 TGAGCGGACA OPE-20 AACGGTGACC POC-10 TGTCTGGCTG OPC-16 CACACTCCAG Sequencing of the D-loop L15702 CAACGWAGYATAATATTYCGACC H15917 CGGAATTCCATTTTTGGTTTACAAG polymorphic profiles monomorphic profiles

RESULTS Electrophoregrams of S. undulatus obtained using primers OPD-05 and OPE-20 “eastern” form common for all of subspecies eversmanni menzbiery Marker loci are indicated by arrows.

Genetic variability among S. undulatus subspecies RAPD analysis Genetic variability among S. undulatus subspecies N Np A / P intercedens menzbieri jacutensis eversmanni 166 126 0.42/0.15 0.48/0.23 0.45/0.27 0.38/0.17 N – total number of loci, Np – number of polymorphic loci, A – allele frequencies, Р – percentage of polymorphic loci (as the proportion of loci polymorphic within each population tested).

Parameters of genetic differentiation on RAPD analysis   n DN p (Ex) intercedens / menzbieri 10 0.7 1.00 menzbieri / jacutensis 12 0.8 intercedens / jacutensis 0.6 eversmanni / “eastern” form 19 0.40 0.00 S. parryi / S. relictus 6 0.41 S. dauricus / S. relictus 0.42 S. dauricus / S. parryi 0.46 n- number of samples, DN - genetic distances (Nei, 1972), p (Ex) - probability (exact test for interpopulation differentiation (Raymond and Rousset, 1995).

NJ and UPGMA trees based on RAPD data “eastern” form eversmanni UPGMA “eastern” form eversmanni

mt DNA data 519 nucleotides; 92 variable sites, 56 parsimony informative; Mean pairwise transition/transversion ratios was 1.284. 1 2 3 4 5 6 1. S. u. jacutensis - 2. S. u. interscedens 0.043 3. S. u. menzbieri 0.0 5 0.22 4. S. u. eversmanni 0. 113 0.107 0.115 5. S. xanthoprymnus 0.159 0.165 0.142 6. S. taurensis 0.175 0.160 0.166 0.162 0.134 Estimates of mean divergence between main lineages of Spermophilus given as genetic distances

NJ and UPGMA trees based on KP2 distances calculated from mtDNA data

Genetic distances for nuclear (RAPD) and mitochondrial (mtDNA) genomes within “eastern” populations 0.026-0.06 0.022-0.05 within eversmanni - 0.029 between eversmanni and “eastern” form 0.39-0.41 0.116 between distinct species of Spermophilus 0.41-0.46 0.134

CONCLUSIONS Both nuclear and mitochondrial DNA analyses showed that the subspecies of “eastern” form were close to each other and far from subspecies of “western” form. 2. Our molecular genetic data showed an important level of genetic divergence between the animals from Altai (S. u. eversmanni) and Far East of Russia (S. u. intercedens, S. u. menzbieri, S. u. jacutensis), similar to those observed between different Spermophilus species (S. relictus, S. dauricus, S. parryi, S. xanthoprymnus, S. taurensis). 3. The results confirm a distinct taxonomic position of eversmanni. It should be considered as a species taxon within Urocitellus group.

THANKS FOR YOUR ATTENTION ACKNOWLEDGMENTS The study was supported by the RFBR (grant nos. 06-04-39015) and FEBRAS (grant nos. 09-III-В-138). THANKS FOR YOUR ATTENTION