MOLECULAR GENETIC RELATIONSHIPS OF THE ZOKORS (RODENTIA, MYOSPALACINAE): ANALYSIS OF D-LOOP REGION POLIMORPHISM Tsvirka Marina1, Pavlenko Marina1, Korablev Vladimir1, Pang Junfeng2

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MOLECULAR GENETIC RELATIONSHIPS OF THE ZOKORS
(RODENTIA, MYOSPALACINAE) ANALYSIS OF D-LOOP
REGION POLIMORPHISM Tsvirka Marina1, Pavlenko
Marina1, Korablev Vladimir1, Pang Junfeng2

• 1Institute of Biology and Soil Science, Far
  Eastern Branch Russian Academy of Sciences,
  Vladivostok, 690022, Russia2Kunming Institute
  of Zoology, Chinese Academy of Sciences, Kunming,
  650223, Yunnan, China

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PROBLEMS

• Among others subterranean rodents, zokors
  (Myospalacinae) are still remains poorly studied
  in viewpoint of taxonomy and evolution. Recent
  molecular phylogenetic studies have demonstrated
  that zokors are closely related to Spalacinae and
  Rhyzomyinae in the family Spalacidae (Norris et.
  al, 2004 Jansa, Weksler, 2004)
• However relationships among the species within
  Myospalacinae are not clearly understood.

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PROBLEMS
Genus Myospalax Subgenus Myospalax M. myospalax
M. aspalax M. psilurus Subgenus Eospalax M.
rothschildi M. smithii M. fontanieri ( M.
f.cansus, M. f. rufescens ,
M. f. baileyi) (Corbet, 1991)
Genus Myospalax M. myospalax M. aspalax M.
psilurus Genus Eospalax M. rothschildi M.
smithii M. fontanieri M. cansus M. rufescens
M. baileyi (Zheng, 1994 )
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Map of Zokors Distribution in the Russia
M. myospalax
M. psilurus
2n64
2n44
M. aspalax
2n62
2n62
M. armandii
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Background

• Professor N. N., Vorontsov prominent Russian
  biologist was initiator of study of fossorial
  rodents in Russia and adjacent countries

• 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.

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Electrophoretic patterns of transferrin (TF) of
zokors (Pavlenko Korablev, 2003 2005)
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3
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• 1. Tf-A M. aspalax, Transbaikalia
• 2. Tf-B M. psilurus, Primorye
• 3. Tf-C M. psilurus, Transbaikalia
• 4. Tf-C1 M. armandii
• 5. Tf-D M. myospalax, Altai region

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Map of Zokors Distribution in the China
M. fontanieri
M. cansus
M. baileyi
2n62
M. rufescens
2n62
M. smithii
M. rothschildi
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• C. Zhou and K. Zhou. The validity of different
  zokor species and the genus Eospalax inferred
  from mitochondrial gene sequences / Integrative
  Zoology 2008 3 290298.

Myospalax and Eospalax genera
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Phylogenetic relationships of zokors based on the
RAPD-PCR analysis (Tsvirka et al., 2009)
M. psilurus
100
4 species groups within genus Myospalax
54
M. smithii
74
M. aspalax M. armandii
100
M. myospalax
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Puzachenko A., Pavlenko M., Korablev V.
Craniological variability of the zokors
(Myospalacinae) with simplified first upper
molar. Abstracts of 11 International Conference
"Rodens et Spatium", 24-28 July 2008, Myshkin
(Russia). P.144 Puzachenko A., Pavlenko M.,
Korablev V. Variability of skulls in Zokors
(Rodentia,Miospalacinae) // Zool. Z. 2009. V. 88.
?1. P. 92-112.

• 3 species groups with taxonomic ranks of the
  genera or subgenera
• M. myospalax
• M. aspalax and M. armandii
• M. smithii and M. psilurus

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The aims of the present study were

• to determine the systematic position of Myospalax
  and Eospalax
• to clarify the phylogenetic relationships among
  zokor species
• to confirm or contradict the taxonomic position
  of M. armandii as a distinct species
• to confirm or disprove previous data concerning
  genetic differentiation of Manchurian zokors M.
  psilurus from Khanka Plain and Transbaikalia.

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MATERIAL

• 54 specimens of 7 species from 30 different
  localities in Russia and China.
• 1 specimen of Spalax judaei from GenBank was
  used as outgroup.

M. cansus
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METHODS
Sequencing 5 end of the D-loop

• Phylogenetic reconstructions using MEGA 4 (Tamura
  et al., 2007) Neighbor-Joining (NJ), Maximum
  Parsimony (MP), Minimum Evolution (ME),
  unweighted pair-group method with arithmetic
  averaging (UPGMA)
• Calculation of genetic distances based on Kimura
  two-parametric (K2P) estimator.

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RESULTS

• The alignment of the mt DNA sequences of 7 taxa
  comprises 518 nucleotides.
• 206 (40) nucleotides were variable.
• 187 (36) nucleotides were parsimony informative.
• The average ratio of transition/transvertion was
  3.58.

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NJ and UPGMA trees of Zokors based on the D-loop
sequences (MEGA-4.1)
M. psilurus Primorye
M. psilurus
M. psilurus Zabaykalye
M. aspalax M. armandii
M. smithii M. cansus M. rufescens
M. myospalax
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Interspecific relationships within the genus
Myospalax
1 2 3 4 5 6 7 8
1. M. cansus -
2. M. smithii 0.120 -
3. M. rufescens 0.123 0.062 -
4. M. aspalax 0.190 0.167 0.164 -
5. M. armandii 0.185 0.185 0.181 0.070 -
6. M. myospalax 0.225 0.226 0. 224 0.177 0.190 -
7. M. psilurus (?) 0.158 0.154 0.154 0.076 0.084 0.159 -
8. M. psilurus (?) 0.164 0.164 0.160 0.108 0.104 0.181 0.063 -
Estimate value of Kimura two-parametric distance
matrix for 8 taxa of Myospalax
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Genetic distances for nuclear (RAPD) and
mitochondrial (D-loop) DNA
Distances RAPD D-loop
within species 0.05 0.003-0.028
within forms of M. psilurus 0.04-0.05 0.003
between species (M. aspalax - M. armandii M. smithii - M. rufescens) 0.08 0.062- 0.07
between two forms of M. psilurus 0.08 0.063
between distant species (Myospalax Eospalax) 0.25-0.35 0.154-0.226
between genera (Myospalax Spalax) 0.31 0.238-0.304
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Conclusions

• Our results didn't confirm the hypothesis of
  validity of the genus Eospalax suggested by Zhou
  and Zhou (2009). The all studied zokor species
  should be divided into 3 species groups within
  one genus Myospalax "myospalax",
  "psilurus-armandii-aspalax" and
  "smithii-cansus-rufescens".
• The species within the "psilurus-armandii-aspalax"
  (M. psilurus, M. armandii, M. aspalax) as well
  as within "smithii-cansus-rufescens" (M. smithii,
  M. cansus, M. rufescens) groups were closely
  related among themselves. The M. myospalax,
  differs from the other species in chromosomal and
  molecular features, is more closely related with
  the first species group than the second one.
• The hypothesis of species status of M. aspalax
  suggested on craniometrical analysis of zokors
  were confirmed (Puzachenko et al., 2009).
• The hypothesis about the independent species
  status of two geographical populations of M.
  psilurus from Khanka Plain and Transbaikalia
  earlier suggested on t biochemical and
  karyological data (Pavlenko, Korablev, 2003) was
  also confirmed on the results of mt DNA analysis
  .

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The Authors
Marina Pavlenko
Marina Tsvirka
Vladimir Korablev
Junfeng Pang
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ACKNOWLEDGMENTS
The study was supported by the RFBR (grant nos.
06-04-39015) and FEBRAS (grant nos. s
09-III-?-138).
THANKS FOR YOUR ATTENTION