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
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