1. 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, , Russia

2. Map of the ground squirrels distribution in the Palaearctic region

3. 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)

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

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

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

7. 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

8. 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.

9. 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)

10. 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

11. 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.

12. 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).

13. Parameters of genetic differentiation on RAPD analysisn 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).

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

15. mt DNA data 519 nucleotides;
92 variable sites, 56 parsimony informative;
Mean pairwise transition/transversion ratios was 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

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

17. Genetic distances for nuclear (RAPD) and mitochondrial (mtDNA) genomes
within “eastern”
populations
within eversmanni -
0.029
between eversmanni
and “eastern” form
0.116
between distinct species of Spermophilus
0.134

18. 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.

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