DIFFERENTIATION AND SYSTEMATICS IN DIFFERENTIATION AND SYSTEMATICS IN THE LONG-TAILED GROUND SQUIRREL, THE LONG-TAILED GROUND SQUIRREL,

SPERMOPHILUSSPERMOPHILUS UNDULATUS UNDULATUS (SCIURIDAE, (SCIURIDAE, RODENTIA)RODENTIA)

Marina V. Tsvirka, Vladimir P. Korablev, Marina V. PavlenkoMarina V. Tsvirka, Vladimir P. Korablev, Marina V. Pavlenko

Institute of Biology and Soil Science FEB RAS,Institute of Biology and Soil Science FEB RAS,Vladivostok, 690022, RussiaVladivostok, 690022, Russia

Map of the ground squirrels Map of the ground squirrels distribution in the Palaearcticdistribution in the Palaearctic

regionregion

•Genus Genus SpermophilusSpermophilus •Subgenus Subgenus UrocitellusUrocitellus •Species Species Spermophilus undulatusSpermophilus undulatus

•Subspecies Subspecies S. u. undulatus,S. u. undulatus,S. u. intercedens,S. u. intercedens,

S. u. menzbieri,S. u. menzbieri, S. u. jacutensis,S. u. jacutensis,

S. u.S. u. stramineus,stramineus, S. u. eversmanniS. u. eversmanni

PROBLEMPROBLEM

(Gromov, Erbaeva, 1995)(Gromov, Erbaeva, 1995)

two geographical forms

(Vorontsov et al., 1980;(Vorontsov et al., 1980;Frisman, Vorontsov, 1989)Frisman, Vorontsov, 1989)

“eastern”

“western”

Map of Map of S. undulatusS. undulatus distributions distributions

eastern formeastern formwestern formwestern form

species or semispeciesspecies or semispecies?

hybrid zonehybrid zone

• Morphometric dataMorphometric data (Popkov, 1978)(Popkov, 1978)

• Morphotypical studyMorphotypical study (Linetskiy, Linetskaya 1989)(Linetskiy, Linetskaya 1989)

• Karyological dataKaryological data (Korablev, 1988)(Korablev, 1988)

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

THE AIM ISTHE AIM IS

THE MAIN TASKSTHE MAIN TASKS AREARE

• To study genetic variability and To study genetic variability and differentiation of differentiation of S. undulatusS. undulatus based on based on nuclear (RAPD) and mitochondrial (D-loop) nuclear (RAPD) and mitochondrial (D-loop) DNA.DNA.

• To assess genetic divergence and To assess genetic divergence and phylogenetic relationships among subspesies phylogenetic relationships among subspesies of of S. undulatusS. undulatus..

• To specify the taxonomic rank of To specify the taxonomic rank of eversmannieversmanni (independent species (independent species S. eversmanniS. eversmanni or or subspecies of subspecies of S. undulatusS. undulatus) based on the ) based on the data obtaineddata obtained

MATERIALSMATERIALS

Map of sampling localitiesMap of sampling localities

S. relictus, S. dauricus S. relictus, S. dauricus andand S. parryi, S. taurensis S. parryi, S. taurensis and and S. xanthoprymnusS. xanthoprymnus were chosen for comparative analyses, were chosen for comparative analyses, Tamias sibiricusTamias sibiricus was chosen as was chosen as outgroup taxon.outgroup taxon.

eastern formeastern formwestern formwestern form

METHODSMETHODS RAPD-PCR analysisRAPD-PCR analysis• 166 binary characters 166 binary characters

including the including the molecular markersmolecular markers

• Ten arbitrary Ten arbitrary decanucleotide decanucleotide primersprimers

• Calculation of Calculation of genetic genetic distances (Nei, 1972)distances (Nei, 1972)

• Reconstructions: Reconstructions: NJ NJ and UPGMA with and UPGMA with bootstrapbootstrap estimation of estimation of branching robustness branching robustness in TREECONW 1.3 b in TREECONW 1.3 b (Van de Peer(Van de Peer, , De De WacherWacher, 1994 , 1994 ))

Mitochondrial DNA analysisMitochondrial DNA analysis

•5’ end of the D-loop5’ end of the D-loop (518 (518 bp)bp)

•Calculation of genetic Calculation of genetic distances based on distances based on Kimura Kimura two-parametric (two-parametric (K2P) K2P) estimator.estimator.

•Reconstructions: Reconstructions: NJ, MP, NJ, MP, ML and UPGMA with ML and UPGMA with bootstrapbootstrap estimation of estimation of branching robustness branching robustness in in MEGA 4.0 (MEGA 4.0 (TamuraTamura et al., et al., 20072007))

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-loopSequencing of the D-loop

L15702 CAACGWAGYATAATATTYCGACC

H15917 CGGAATTCCATTTTTGGTTTACAAG

polymorphicpolymorphicprofilesprofiles

monomorphicmonomorphicprofilesprofiles

Marker loci are indicated by arrows.

“eastern” form

eversmanni

menzbiery

common for allof subspecies

RESULTSRESULTS Electrophoregrams of Electrophoregrams of S. undulatusS. undulatus

obtained using primers OPD-05 and OPE-obtained using primers OPD-05 and OPE-2020

NN – – total number of loci, , NpNp – – number of polymorphic locnumber of polymorphic locii, , A A –– allele allele

frequenciesfrequencies, , РР – – percentage of polymorphicpercentage of polymorphic loci (as the proportion of loci (as the proportion of loci polymorphic withinloci polymorphic within each population testedeach population tested).).

Genetic variability among Genetic variability among S. undulatusS. undulatus subspeciessubspecies

NN NpNp A / PA / P

intercedenintercedenss

menzbiermenzbierii

jacutensijacutensiss

eversmaneversmannini

166166 126126 0.40.42/0.152/0.15 0.48/0.230.48/0.23 0.45/0.20.45/0.277

0.38/0.170.38/0.17

RAPD analysisRAPD analysis

Parameters of genetic differentiation Parameters of genetic differentiation on RAPD analysison RAPD analysis

n- number of samples, DN - genetic distances (Nei, 1972), p (Ex) - probability (exact test for interpopulation differentiation (Raymond and Rousset, 1995).

   nn DDNN pp (Ex)(Ex)

intercedens / menzbieriintercedens / menzbieri 1010 0.70.7 1.001.00

menzbieri / jacutensismenzbieri / jacutensis 1212 0.80.8 1.001.00

intercedens / jacutensisintercedens / jacutensis 1010 0.60.6 1.001.00

eversmanni / “eastern”eversmanni / “eastern”formform

1919 0.40.400 0.000.00

S. parryi / S. relictusS. parryi / S. relictus 66 0.410.41 0.000.00

SS. . dauricus / S. relictusdauricus / S. relictus 66 0.0.4242 00.00.00

SS. . dauricus / S. parryidauricus / S. parryi 66 0.460.46 0.000.00

NJ and UPGMA NJ and UPGMA treestrees based on based on

RAPD dataRAPD data““eastern” eastern”

formform

eversmannieversmanni

““eastern” eastern” formform

eversmannieversmanni

UPGMAUPGMA

11 22 33 44 55 66

1. 1. S. u. jacutensisS. u. jacutensis --

2. 2. S. u. S. u. interscedensinterscedens

0.0430.043 --

3. 3. S. u. menzbieriS. u. menzbieri 0.0 50.0 5 0.220.22 --

4. 4. S. u. S. u. eversmannieversmanni 0. 0. 113113

0.1070.107 0.1150.115 --

5. 5. S. S. xanthoprymnusxanthoprymnus

0.1590.159 0.1590.159 0.1650.165 0.1420.142 --

6. 6. S. taurensisS. taurensis 0.1750.175 0.1600.160 0.1660.166 0.1620.162 0.130.1344

--

mt DNA datamt DNA data

Estimates of mean divergence between main lineages of Estimates of mean divergence between main lineages of SpermophilusSpermophilus given as geneticgiven as genetic distancesdistances

• 519 519 nucleotidesnucleotides; • 92 variable sites92 variable sites,, 56 parsimony informative; 56 parsimony informative; • Mean pairwise transition/transversion ratios was 1.284. Mean pairwise transition/transversion ratios was 1.284.

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

mtDNA datamtDNA data

Distances Distances RAPD mtDNA

within “eastern”populations

0.026-0.06 0.022-0.05

within eversmanni - 0.029

between eversmanniand “eastern” form

0.39-0.41 0.116

between distinct species of

Spermophilus

0.41-0.46 0.134

Genetic distances for nuclear Genetic distances for nuclear (RAPD) and mitochondrial (RAPD) and mitochondrial

(mtDNA) genomes(mtDNA) genomes

1.1. Both nuclear and mitochondrial DNA analyses Both nuclear and mitochondrial DNA analyses showed that the subspecies of “eastern” form were showed that the subspecies of “eastern” form were close to each other and far from subspecies of close to each other and far from subspecies of “western” form.“western” form.

2. Our molecular genetic data showed2. Our molecular genetic data showed an important an important level of genetic divergence between the animals level of genetic divergence between the animals from Altai (from Altai (S. u. eversmanniS. u. eversmanni) and Far East of Russia ) and Far East of Russia ((S. u. intercedens, S. u. menzbieri, S. u. jacutensisS. u. intercedens, S. u. menzbieri, S. u. jacutensis), ), similar to those observed between different similar to those observed between different Spermophilus Spermophilus species (species (S. relictus, S. dauricus, S. S. relictus, S. dauricus, S. parryi, S. xanthoprymnus, S. taurensisparryi, S. xanthoprymnus, S. taurensis).).

3. The results confirm a distinct taxonomic position of 3. The results confirm a distinct taxonomic position of eversmanni. eversmanni. It should be considered as a species It should be considered as a species taxon within taxon within Urocitellus Urocitellus group.group.

CONCLUSIONSCONCLUSIONS

The study was supported by the RFBR (grant nos. 06-The study was supported by the RFBR (grant nos. 06-04-39015) and FEBRAS (grant nos. 09-III-04-39015) and FEBRAS (grant nos. 09-III-ВВ-138).-138).

ACKNOWLEDGMENTSACKNOWLEDGMENTS