MOLECULAR GENETIC RELATIONSHIPS OF MOLECULAR GENETIC RELATIONSHIPS OF THE ZOKORS (RODENTIA, THE ZOKORS (RODENTIA,

MYOSPALACINAE): ANALYSIS OF D-LOOP MYOSPALACINAE): ANALYSIS OF D-LOOP REGION POLIMORPHISMREGION POLIMORPHISM

Tsvirka MarinaTsvirka Marina11,, Pavlenko Marina Pavlenko Marina11, , Korablev VladimirKorablev Vladimir11, Pang Junfeng, Pang Junfeng22

11Institute of Biology and Soil Science, Far Eastern Branch Institute of Biology and Soil Science, Far Eastern Branch Russian Academy of Sciences, Vladivostok, 690022, RussiaRussian Academy of Sciences, Vladivostok, 690022, Russia

22Kunming Institute of Zoology, Chinese Academy of Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, Yunnan, ChinaSciences, Kunming, 650223, Yunnan, China

PROBLEMSPROBLEMS

Among others subterranean rodents, zokors Among others subterranean rodents, zokors (Myospalacinae) are still remains poorly studied in (Myospalacinae) are still remains poorly studied in viewpoint of taxonomy and evolution. Recent molecular viewpoint of taxonomy and evolution. Recent molecular phylogenetic studies have demonstrated that zokors are phylogenetic studies have demonstrated that zokors are closely related to Spalacinae and Rhyzomyinae in the closely related to Spalacinae and Rhyzomyinae in the family Spalacidae family Spalacidae (Norris et. al, 2004; Jansa, Weksler, 2004)

However relationships among the species within However relationships among the species within Myospalacinae are not clearly understood.Myospalacinae are not clearly understood.

PROBLEMSPROBLEMS

Genus Myospalax

Subgenus MyospalaxM. myospalax M. aspalax M. psilurus

Subgenus EospalaxM. rothschildi M. smithii M. fontanieri ( M. f.cansus,

M. f. rufescens , M. f. baileyi)

(Corbet, 1991)

Genus MyospalaxM. myospalax M. aspalax M. psilurus

Genus EospalaxM. rothschildi M. smithii M. fontanieriM. cansusM. rufescens M. baileyi

(Zheng, 1994 )

Map of Zokors Distribution in the RussiaMap of Zokors Distribution in the Russia

M. myospalax

M. aspalax

M. psilurus

M. armandii

2n=44

2n=622n=62

2n=64

BackgroundBackground

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

L. Martynova, N. L. Martynova, N. Vorontsov, Vorontsov, 1975.1975. Population cytogenetic of Population cytogenetic of Zokors ChromosomesZokors Chromosomes

L. Martynova, L. Martynova, 19761976.. Chromosomal Chromosomal differentiation of three differentiation of three species of zokorsspecies of zokors..

L. Martynova, IL. Martynova, I.. Fomicheva, N.Fomicheva, N. Vorontsov, Vorontsov, 19771977.. Electrophoretic study Electrophoretic study of blood protein of zokorsof blood protein of zokors..

Electrophoretic patterns of transferrin (TF) of zokors (Pavlenko & Korablev, 2003; 2005)

1.1. Tf-A – Tf-A – M. aspalaxM. aspalax, Transbaikalia, Transbaikalia2.2. Tf-B – Tf-B – M. psilurusM. psilurus, Primorye , Primorye 3.3. Tf-C – Tf-C – M. psilurusM. psilurus, Transbaikalia, Transbaikalia4.4. Tf-C1 – Tf-C1 – M. armandiiM. armandii5.5. Tf-D – Tf-D – M. myospalaxM. myospalax, Altai region, Altai region

1 2 3 4 5

M. smithii

M. rufescens

2n=62M. rothschildi

M. fontanieri

M. baileyi

M. cansus

Map of Zokors Distribution in the ChinaMap of Zokors Distribution in the China

2n=62

CC.. Z Zhouhou and K and K.. Z Zhou.hou. The validity of different zokor The validity of different zokor species and the genus species and the genus EospalaxEospalax inferred inferred from from mitochondrial gene sequencesmitochondrial gene sequences / / Integrative Zoology Integrative Zoology 2008; 3: 290–2982008; 3: 290–298..

Myospalax and Eospalax genera

PhylogeneticPhylogenetic relationships of zokorsrelationships of zokors based on the RAPDbased on the RAPD--PCRPCR analysisanalysis ((Tsvirka et al., 2009Tsvirka et al., 2009))

M. psilurus

M. smithii

M. aspalaxM. armandii

M. myospalax

4 species groups within genus Myospalax

100

74

100

54

Puzachenko A., Pavlenko M., Korablev V.Puzachenko A., Pavlenko M., Korablev V. Craniological Craniological variability of the zokors (Myospalacinae) with variability of the zokors (Myospalacinae) with simplified first upper molar. Abstracts of 11 simplified first upper molar. Abstracts of 11 International Conference "Rodens et Spatium", 24-28 International Conference "Rodens et Spatium", 24-28 July 2008, Myshkin (Russia). P.144 July 2008, Myshkin (Russia). P.144 Puzachenko A., Pavlenko M., Korablev V.Puzachenko A., Pavlenko M., Korablev V. Variability of skulls in Zokors (Rodentia,Miospalacinae) // // ZoolZool. . ZZ. . 2009. V. 88. №1. P. 92-112.2009. 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

The aims of the The aims of the present study werepresent study were

• to determine the systematic position of to determine the systematic position of Myospalax Myospalax andand Eospalax;Eospalax;

• to clarify the phylogenetic relationships among zokor to clarify the phylogenetic relationships among zokor species;species;

• to confirm or contradict the taxonomic position ofto confirm or contradict the taxonomic position of M. M. armandii armandii as a distinct species;as a distinct species;

• to to confirm or disprove previous data concerning confirm or disprove previous data concerning genetic differentiation of Manchurian zokors genetic differentiation of Manchurian zokors M. M. psiluruspsilurus from Khanka Plain and Transbaikalia. from Khanka Plain and Transbaikalia.

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

MATERIALMATERIAL

M. cansus

METHODSMETHODS

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.

Sequencing 5’ end Sequencing 5’ end of the D-loopof the D-loop

RESULTSRESULTS

•The alignment of the mt DNA sequences of 7 taxa The alignment of the mt DNA sequences of 7 taxa comprises 518 nucleotides.comprises 518 nucleotides.

•206 (40%) nucleotides were variable. 206 (40%) nucleotides were variable.

•187 (36%) nucleotides were parsimony informative.187 (36%) nucleotides were parsimony informative.

•The average ratio of transition/transvertion was 3.58.The average ratio of transition/transvertion was 3.58.

NJNJ andand UPGMAUPGMA treestrees of Zokorsof Zokors based on the D-loop based on the D-loop sequencessequences ((MEGA-4.1MEGA-4.1))

M. myospalax

M. smithiiM. cansusM. rufescens

M. aspalaxM. armandii

M. psilurus Primorye

M. psilurus Zabaykalye

M. psilurus

Interspecific relationships within the genus Interspecific relationships within the genus MyospalaxMyospalax

11 22 33 44 55 66 77 88

1. 1. M. cansusM. cansus --

22. . M. smithiiM. smithii 0.1200.120 --

33. . M. rufescensM. rufescens 0.0.123123 0.0620.062 --

44. . M. aspalaxM. aspalax 0.0.190190 0.0.167167 0.0.164164 --

55. . M. armandiiM. armandii 0.0.185185 0.0.185185 0.0.181181 0.070.0700 --

66.. M. myospalax M. myospalax 0.20.22525 0.0.226226 0. 20. 22424 0.1770.177 0.1900.190 --

77.. M. psilurus M. psilurus (П)(П) 0.10.15858 0.10.15454 0.1540.154 0.00.07676 0.00.08484 0.0.159159 --

88.. M. psilurus M. psilurus (З)(З) 0.10.16464 0.10.16464 0.10.16060 0.0.108108 0.0.104104 0.0.181181 0.0630.063 --

Estimate value of Kimura two-parametric distance matrix for 8 taxa of Myospalax

DistancesDistances RAPDRAPD D-loopD-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. armandiiM. 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

Genetic distances for nuclear (RAPD) and mitochondrialGenetic distances for nuclear (RAPD) and mitochondrial(D-loop)(D-loop) DNADNA

ConclusionsConclusions Our results didn't confirm the hypothesis of validity of the genus Eospalax Our results didn't confirm the hypothesis of validity of the genus Eospalax

suggested by Zhou and Zhou (2009). The all studied zokor species suggested by Zhou and Zhou (2009). The all studied zokor species should be divided into 3 species groups within one genus should be divided into 3 species groups within one genus Myospalax:Myospalax: "myospalax", "psilurus-armandii-aspalax" and "smithii-cansus-rufescens". "myospalax", "psilurus-armandii-aspalax" and "smithii-cansus-rufescens".

The species within the "psilurus-armandii-aspalax" (The species within the "psilurus-armandii-aspalax" (M. psilurus, M. M. psilurus, M. armandii, M. aspalaxarmandii, M. aspalax) as well as within "smithii-cansus-rufescens" () as well as within "smithii-cansus-rufescens" (M. M. smithii, M. cansus, M. rufescenssmithii, M. cansus, M. rufescens) groups were closely related among ) groups were closely related among themselves.themselves. The The M. myospalaxM. myospalax, differs from the other species in , differs from the other species in chromosomal and molecular features, is more closely related with the first chromosomal and molecular features, is more closely related with the first species group than the second one.species group than the second one.

The hypothesis of species status of The hypothesis of species status of M. aspalaxM. aspalax suggested on suggested on craniometrical analysis of zokors were confirmed (Puzachenko et al., craniometrical analysis of zokors were confirmed (Puzachenko et al., 2009). 2009).

The hypothesis about the independent species status of two geographical The hypothesis about the independent species status of two geographical populations of M. psilurus from Khanka Plain and Transbaikalia earlier populations of M. psilurus from Khanka Plain and Transbaikalia earlier suggested on t biochemical and karyological data (Pavlenko, Korablev, suggested on t biochemical and karyological data (Pavlenko, Korablev, 2003) was also confirmed on the results of mt DNA analysis .2003) was also confirmed on the results of mt DNA analysis .

The AuthorsThe AuthorsMarina Pavlenko

Vladimir Korablev Junfeng Pang

Marina Tsvirka

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

ACKNOWLEDGMENTSACKNOWLEDGMENTS