ORIGINAL PAPER

Molecular evolution of Trichuris muris isolatedfrom different Muridae hosts in Europe

Rocio Callejón & Manuel de Rojas &

Caroline Nieberding & Pilar Foronda & Carlos Feliú &

Diego Guevara & Cristina Cutillas

Received: 7 April 2010 /Accepted: 29 April 2010 /Published online: 15 May 2010# Springer-Verlag 2010

Abstract A phylogeographic study was carried out ofTrichuris muris, nematode parasitizing Murinae rodentsfrom the Muridae family, isolated from four different hostsand from different geographical regions of Europe byamplification and sequencing of the ITS1-5.8S-ITS2 frag-ment of the ribosomal DNA. T. muris was found in theApodemus sylvaticus, Apodemus flavicollis, Mus domesticus,and Rattus rattus rodents. The molecular results confirm thepresence of DNA polymorphisms among T. muris isolatesfrom Europe. The present study shows two clear-cutgeographical and genetic lineages: one of them is wide-spread from northern Spain (Catalonia) to Denmark(Western European region), while the second is widespreadin the Eastern European region (Croatia, Rumania, andTurkey). These two genotypes can be easily distinguished

by a PCR-RFLP analysis of this sequence with the ApalIrestriction enzyme. Moreover, networks and phylogeneticreconstructions also reveal that T. muris from variousMurinae rodents did not differentiate according to the hostspecies that they parasitize. Furthermore, T. muris isolatedfrom The Canary Islands revealed a typical haplotype (H6)only present in The Canary Islands and not in continentalEurope. It is suggested that one haplotype from La GomeraIsland is the ancestor of T. muris in the Canary Islands.

Introduction

Trichuris muris is a nematode parasitizing rodent that hasbeen used as a laboratory model in many fields of research.T. muris is a direct (without intermediate host) and specificendoparasite of rodents belonging to the Muridae family.

Previous studies have reported that T. muris is foundmainly in murinae and arvicolinae rodents (Tenora 1967;Merkusheva and Bobkova 1981). Nevertheless, based onisoenzymatic techniques, Feliú et al. (2000) suggested thattrichurids-parasitizing hosts of the Arvicolinae subfamilyconstitute a separate species of Trichuris and described anew species, Trichuris arvicolae, as a parasite of theArvicolinae rodent subfamily. Cutillas et al. (2002) ampli-fied and sequenced the ITS1-5.8S-ITS2 region of theribosomal DNA (rDNA) of T. muris and T. arvicolae usingconserved primers; they reported that polymerase chainreaction (PCR) molecular techniques differentiated T. murisand T. arvicolae as two well-defined species. Furthermore,some specific recognition sites for endonucleases weredetected for this marker.

Comparative analysis of coding and noncoding regionsof ribosomal DNA has become a useful tool for the

R. Callejón :M. de Rojas :D. Guevara :C. Cutillas (*)Department of Microbiology and Parasitology,Faculty of Pharmacy, University of Seville,Profesor García González 2,41012 Seville, Spaine-mail: cutillas@us.es

C. NieberdingEvolutionary Biology Group, BDIV Research Centre,Université catholique de Louvain,Louvain-la-Neuve, Belgium

P. ForondaCanaries Tropical Disease and Public Health Institute,La Laguna University,La Laguna, Tenerife, Spain

C. FeliúDepartment of Microbiology and Parasitology,Faculty of Pharmacy, University of Barcelona,Barcelona, Spain

Parasitol Res (2010) 107:631–641DOI 10.1007/s00436-010-1908-9

construction of phylogenetic trees of many organismsincluding nematodes (Subbotin et al. 2001). The internaltranscribed spacers (ITS1 and ITS2) located in theribosomal DNA are considered appropriate molecularmarkers to resolve relationships at the species level. Studiesof phylogenetic relationships among nematodes are notonly essential to taxonomy, but also allow a more completeunderstanding of the biology of nematodes. Trichuris spp.have many features that make them good candidates forclinical use in the treatment of autoimmune diseases such asCrohn's disease (Summers et al. 2003, 2005; Reddy andFried 2007).

Phylogeography is a field of research that studies theprocesses determining the geographical distribution ofgenetic lineages at the intraspecific or congeneric levelsand is useful for detecting processes such as subdivisions ofpopulations, speciation events or ecological adaptation, andmigration routes associated with past climatic changes(Avise 2000). Studies on the comparative phylogeny oftaxa strongly linked by an ecological factor, such asparasitism, have shown that the degree of phylogeneticcongruence increases with the forced character of the host–parasite relationship (Nieberding et al. 2004). Comparativephylogeographical studies in island archipielagos can reveallineage-specific differential responses to the geological andclimate partial structure and orgasnismal traits history.Island populations are invaluable for exploring the com-bined effects of spatial structure and organismal traits. Asthe surrounding sea constitutes a uniform physical barrier togene flow, various lineages may differ in their capability tocross these barriers depending on ecological or life-historycharacteristics (Heaney et al. 2005). This is predicted toaffect the rate at which distinct phylogeographic groups areformed (Papadopoulou et al. 2009).

The Canary Islands are located on a 3,000-m deepsubmarine platform off the Northwest African continent.An oceanic origin seems reasonable for the central andWestern islands, which are of the central-volcanic type(Rothe 1974). Three different species of murinae rodentsare found in the Canary Islands: Mus domesticus, Rattusnorvegicus, and Rattus rattus.

To discriminate between the alternative hypotheses ofco-speciation (host–parasite) versus geographic differentia-tion, we carried out a molecular study based on theamplification and sequencing of the ITS1-5.8S-ITS2 frag-ment of the ribosomal DNA of the Trichuris speciesisolated from Apodemus sylvaticus, Apodemus flavicollis,M. domesticus, and R. rattus sampled from differentEuropean geographical areas (Continental Europe andMediterranean and Atlantic Islands). Furthermore, westudied the existence of intraindividual and interindividualpolymorphism in T. muris isolated from the same host andbetween sequences of T. muris isolated from different hosts.

Materials and methods

Collection of samples

A total of 55 adult T. muris were collected from seven A.sylvaticus (Muridae: Murinae), five A. flavicollis (Muridae:Murinae), 19 M. domesticus ( Muridae: Murinae), and twoR. rattus (Muridae: Murinae) from different localities fromEurope: Turkey, Spain (Sant Martí, Calafell, La Riera,Montseny, Mallorca, La Gomera, Tenerife, La Palma, andHierro), Eastern Pyrenean Mountains (EPM) (France),Denmark, Croatia, and Rumania (Fig. 1, Table 1). Further-more, five Trichuris sp. from Rombomys sp. (Muridae:Gerbillinae) captured in Kazakhstan and three T. arvicolae(Cricetidae: Arvicolinae) from Myodes glareolus wereanalyzed and used as outgroups for phylogenetic purposes.Worms were washed extensively in 0.9% saline solutionand stored in 70% alcohol until required for PCR andsequencing. The identification of species of Trichuris foundin the caecum of these rodent hosts was made according toFeliú et al. (2000).

Sequence data

Genomic DNA from individual worms was extracted usingthe QIAamp Tissue Kit (Qiagen) according to the manu-facturer's protocol. Genomic DNAwas detected using 0.8%agarose gel electrophoresis and ethidium bromide. TherDNA region was amplified by PCR using a Perkin Elmerthermocycler and the following PCR mix: 10 μl 10×PCRbuffer, 2 μl 10 mM dNTP mixture (0.2 mM each), 3 μl50 mM MgCl2, 5 μl primer mix (0.5 mM each), 5 μltemplate DNA, 0.5 μl Taq DNA polymerase (2.5 units),and autoclaved distilled water to 100 μl. The followingconditions were applied: 94°C for 3 min, 35 cycles at 94°Cfor 1 min, 55°C for 1 min, 72°C for 1 min, followed by10 min at 72°C. DNA sequences of the forward primerNC5 (5′-GTAGGTGAACCTGCGGAAGGATCATT-3′)and reverse primer NC2 (5′-TTAGTTTCTTTTCCTCCGCT-3′) corresponded to the conserved 3′-5′ ends of theITS1-5.8S-ITS2 flanking the 18S and 28S gene regions(Gasser et al. 1996). For each set of PCR reactions andextraction of the DNA, samples without DNA (negative)and a known (positive) control DNA samples were alsoincluded.

The PCR products were checked on ethidium bromide-stained 2% Tris-Borate-EDTA (TBE) agarose gels. Bandswere eluted from the agarose by using the QIAEX II GelExtration Kit (Qiagen). The isolated DNA was cloned intoEscherichia coli DH5α using pGEM-T Easy vector system(Promega). Transformed cells were selected by overnightincubation at 37°C on LBB/Amp/X-gal/IPTG plates. Inorder to check for successful cloning and to study the

632 Parasitol Res (2010) 107:631–641

intraindividual variation, at least ten single recombinants(clones) were screened for the DNA insert and sequenced.The ten clones containing the correct insert were used toinoculate 5 ml of LBB/Amp broth and incubated, shaken at37°C for 12 h. Plasmid were purified using a Wizard PlusSV (Promega) and sequenced by MWG-Biotech (Germany)with a universal primer (M13).

The intraindividual variationwas determined by sequencingfive clones of a single individual per population of T. muris.The interindividual variation was determined by sequencingthree individuals from each locality and host. Furthermore, allthe sequences were aligned and compared with each otherusing the CLUSTALW program. Alignments were manuallyadjusted.

Restriction maps

Restriction maps of the different ITS1 and ITS2 sequenceswere determined by using the “Map” program available onGen Bank. PCR products were digested directly with 2.5units of ApalI and BsePI restriction enzymes (Roche) andincubated at 37°C overnight. Digests were separated on 2%agarose-TBE (Tris-Borate-EDTA) gels.

Phylogenetic analysis

Phylogenetic relationships were analyzed using distance,maximum likelihood (ML) and maximal parsimony (MP)methods using the MEGA 4.0 program (Tamura et al.

Tu

RoCr

MaRi Ca SM

Western region

Eastern region

PyMo

De

The Canary Islands

BaPa

LLAm

La

PCLa

VGAó Al

BBCe

Fig. 1 Geographical distribution of Trichuris muris isolates and theextension of their genetic zones. Host species: filled squares: A.sylvaticus; filled circles: A. flavicollis; filled triangles: M. domesticus;filled stars: R. rattus. Localities: Tu Turkey, Mo Montseny (Catalonia),Ma Mallorca, Cr Croatia, Ro Romania, Ri La Riera (Catalonia), CaCalafel (Catalonia), SM Sant Martí (Catalonia), De Denmark, Py

Eastern Pyrenean Mountains, LL La Laguna (Tenerife), AmAguamansa (Tenerife), Ce El Cedro (La Gomera), VG Valle GranRey (La Gomera), Aó Alajeró (La Gomera), Al Alojera (La Gomera),Ba Barlovento (La Palma), Pa El Paso (La Palma), La Laurisilva (LaPalma), PC Pista Carbonero (La Palma), BB Breña Baja (La Palma)

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Table 1 Distribution of Trichuris muris isolated from different localities, hosts, and haplotypes

Host Host/Parasitesize

Locality ITS2 ITS1 Haplotypes (Numberof sequences)

Accessionnumbers

Intra.V. %

Inter.V. %

Intra.V. %

Inter.V. %

Apodemussylvaticus

3/3 Turkey 0.3 0.3 0 0.4 H28(2) FN543152

H29(1) FN543153

H30(1) FN543154

H31(1) FN543155

1/3 Mallorca 0.5 0.8 0 1.6 H28(1) FN543152

H40(2) FN543164

H41(1) FN543165

H42(1) FN543166

3/3 Montseny, Catalonia 0.3 0 0.4 0.2 H32(1) FN543156

H33(1) FN543157

H34(1) FN543158

H35(1) FN543159

H36(1) FN543160

H37(1) FN543161

H38(1) FN543162

H39(1) FN543163

Apodemusflavicollis

2/3 Turkey 1.3 0.3 1.8 0.2 H24(2) FN543148

H28(1) FN543152

H43(1) FN543167

H44(1) FN543168

2/3 Croatia 0 2.1 0 2.9 H45(1) FN543169

H46(1) FN543170

H47(1) FN543171

1/3 Romania 0 0 0 0.9 H48(1) FN543172

H49(1) FN543173

H50(1) FN543174

Mus domesticus 1/1 Denmark 0 – 0.2 – H7(3) FN543131

H60(1) FN543184

2/2 Calafell, Catalonia 0.5 0.5 0.4 0 H51(1) FN543175

H52(1) FN543176

H53(1) FN543177

H54(1) FN543178

H55(1) FN543179

H56(1) FN543180

H57(1) FN543181

2/2 La Riera, Catalonia 0 – 0 – H58(2) FN543182

1/1 Sant Martí, Catalonia 0 – 0 – H59(1) FN543183

1/3 El Cedro, La Gomera – 0.78 0 0.45 H62(1) FN543186

H63(1) FN543187

H64(1) FN543188

1/3 Alojera, La Gomera 2.08 0.78 2 0.45 H6(3) FN543130

H54(1) FN543178

H65(1) FN543189

H66(1) FN543190

H67(1) FN543191

H68(1) FN543192

634 Parasitol Res (2010) 107:631–641

Table 1 (continued)

Host Host/Parasitesize

Locality ITS2 ITS1 Haplotypes (Numberof sequences)

Accessionnumbers

Intra.V. %

Inter.V. %

Intra.V. %

Inter.V. %

H69(1) FN543193

H70(1) FN543194

1/3 Valle Gran Rey, La Gomera 0 0 0.22 0.89 H6(2) FN543130

H75(1) FN543199

H76(1) FN543200

H77(1) FN543201

2/2 Alajeró, La Gomera 2.58 0.78 1.34 0.67 H6(1) FN543130

H71(1) FN543195

H72(1) FN543196

H73(1) FN543197

H74(1) FN543198

1/3 El Paso, La Palma 1.29 1.29 0 0.89 H6(1) FN543130

H7(1) FN543131

H8(1) FN543132

H9(1) FN543133

H10(1) FN543134

H11(1) FN543135

1/3 Barlovento, La Palma 0 0 0 0.45 H6(4) FN543130

H20(1) FN543144

1/3 Pista Carbonero, La Palma 0.78 0 0.22 0 H6(4) FN543130

H17(1) FN543141

H18(1) FN543142

H19(1) FN543143

2/2 Laurisilva, La Palma 1.03 1.03 1.79 1.56 H6(3) FN543130

H12(1) FN543136

H13(1) FN543137

H14(1) FN543138

H15(1) FN543139

H16(1) FN543140

1/3 Breña Baja, La Palma 0.52 0 0.89 0 H1(1) FN543125

H2(1) FN543126

H3(1) FN543127

H4(1) FN543128

H5(1) FN543129

H6(2) FN543130

1/2 Laurisilva, El Hierro 0.26 0.26 0 0 H6(2) FN543130

H26(1) FN543150

H27(1) FN543151

1/2 Aguamansa, Tenerife 0 0.26 0 0.89 H6(3) FN543130

H21(1) FN543145

H22(1) FN543146

H23(1) FN543147

Rattus rattus 1/1 Eastern Pyrenean Mountains 0.8 – 0.4 – H54(1) FN543178

1/1 La Laguna, Tenerife 0 0.22 0 H24(1) FN543148

H25(1) FN543149

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2007). A neighbor-joining (NJ) tree (Saitou and Nei 1987)was generated from Jukes and Cantor (1969) and Kimura's2-parameter method (Kimura 1980). Support for the treetopology was examined using bootstrapping (heuristicoption) (Felsenstein 1985) over 1,000 replications. Further-more, networks were constructed using the median-joiningnetwork (Bandelt et al. 1999; network 4.5.1.0 available atwww.fluxus-engineering.com). A “mismatch distribution”of substitutional differences between pairs of haplotypeswas calculated within each of the main genetic lineages andcompared with a fit to the Poisson model using dnaspversion 5.0 (Rozas and Rozas 1997).

Results

A single PCR product (about 1,040 base pairs) wasamplified from the genomic DNA of T. muris isolated fromdifferent localities and hosts. The sequences of T. muriswere of 1040 base pairs (bp), corresponding with 448 bp ofthe ITS1; 161 bp of the 5.8S; 384 bp of the ITS2, and 47 bpof the 5′ end of the 28S rDNA gene.

All the sequences (haplotypes) of T. muris isolated fromdifferent hosts and localities were submitted to the GenBankwith the accession numbers showed in Table 1. Furthermore,intraindividual and interindividual variations were observedin the ITS1 and ITS2 sequences of different individualsisolated from different hosts and regions (Table 1).

Different repetitive nucleotide sequences were found inthe ITS2 sequences of T. muris isolated from differentlocalities and hosts. Thus, in T. muris, Poly (AGC) andPoly (GGT) were observed at positions 79 and 176,respectively. Poly (TGC), Poly (TC), Poly (TG), and Poly(GT) were observed at positions 82, 237, 247, and 253,respectively, in all the ITS1 sequences of T. muris fromdifferent hosts and regions analyzed.

Phylogenetic studies of T. muris isolates

The alignment of all sequences obtained from the fourspecies of hosts (A. sylvaticus, A. flavicollis, M. domesticus,

and R. rattus) (Muridae: Murinae) isolated from differentEuropean regions (continental and islands) (Fig. 1) and thesubsequent phylogenetic trees, revealed that T. murisisolated from different hosts did not differentiate accordingto the host species that they parasitized. Rather, 78haplotypes were observed for the 114 sequences analyzed(Table 1).

The results of our phylogenetic analysis support thefindings of previous studies which showed that T. arvicolaeisolated from voles (Cricetidae: Arvicolinae) is clearlyseparated from T. muris isolated from Murinae rodents.Furthermore, Trichuris sp sequences from Rombomys(Muridae: Gerbillinae) were different to those from T. murisand T. arvicolae (Fig. 2).

The phylogenetic trees (NJ, MP, and MLH), constructedfor T. muris isolated from continental European regions,showed 35 haplotypes distributed in two clear-cut clusterscorresponding to two different geographic regions(Fig. 2a.1, a.2). Although each geographical region (Westernand Eastern European regions), showed highly similar ITS1-5.8S-ITS2 rDNA sequences between different isolates ofT. muris (99.8% to 100% homology), nevertheless, whensequences of this rDNA fragment of T. muris isolated fromthe Western region (Catalonia, France and Denmark) werecompared with those from the Eastern region (Croatia,Turkey and Rumania) they displayed slightly different(98.7% to 98.9% homology). Furthermore, two Çhaplotypesfrom Turkey (H43) and Croatia (H47) were clustered withthose from the Western continental region (Figs. 3, 4).

Based on the ITS1-5.8S-ITS2 fragment sequences, arestriction map was carried out; two endonucleases locatedat positions 315 (ApaLI) and 313 (BsePI) clearly differen-tiated both geographical regions—Western and EasternEuropean regions (not shown).

The networks and phylogenetic trees (NJ, MP, andMLH) constructed for T. muris isolated from the CanaryIslands (Fig. 2b.1, b.2) revealed the existence of 42haplotypes. A typical haplotype (H6) observed in TheCanary Islands and not in Continental Europe was the mostfrequent haplotype (showed by 25 taxa). Furthermore,haplotype H76 (La Gomera) appeared separated from the

Table 1 (continued)

Host Host/Parasitesize

Locality ITS2 ITS1 Haplotypes (Numberof sequences)

Accessionnumbers

Intra.V. %

Inter.V. %

Intra.V. %

Inter.V. %

Outgroups

Rombomys sp. 1/5 Kazahkstan – – – – H78(1) FN543202

Myodes glareolus 1/1 Montseny, Catalonia – – – – H61(1) FN543185

Trichuris sp. isolated from Rombomys sp. and Trichuris arvicolae from Myodes glareolus has been used as outgroups in the phylogenetics studies

Intra. V. intraindividual variation, Inter. V. interindividual variation

636 Parasitol Res (2010) 107:631–641

a. Continental Europe and Balearic Islands

b. Canary Islands

Gerbillinae

Murinae

Arvicolinae

Muridae

Gerbillinae

Murinae

Arvicolinae

H59

H7

H57 H39 H56

H54

H52H51

H35

H53 H58

H55

H32

H50

H47

H38

H78

H37

H33

H42

H28

H46

H30

H44 H40

H61

H78

H25

H72

H24

H74

H4 H23

H13 H19

H22

H6

H8

H70

H15

H12

H11

H62

H20 H66

H21

H78

H61

a.1

a.2

b.1

b.2 H51

H53

H52

H55

H32

H54

H37

H39

H56

H33

H58

H35

H34

H7

H41

H60

H43

H36

H57

H42

H38

H47

H40

H46

H50

H49

H45

H48

H30

H44

H24

H31

H28

H29

H59

H61

H78

99

95

65

67

65

96

62

70

100

66

68

0.05

H15

H70

H12

H8

H23

H65

H74

H25

H24

H72

H19

H10

H77

H6

H13

H20

H71

H17

H18

H27

H3

H5

H9

H22

H75

H67

H54

H68

H73

H64

H62

H63

H1

H11

H2

H14

H26

H7

H4

H16

H66

H21

H69

H76

H61

H78

63

98

95

95

74

100

61

87

72

0.05

Muridae

H6

H76

WCE

ECE

Fig. 2 Phylogenetics analyses of Trichuris muris based on the ITS1-5.8S-ITS2 fragment of ribosomal DNA. Median-joining (MJ) net-works and Neighbor-joining (NJ) trees were constructed using thehaplotypes. Sizes of circles are proportional to frequency of thehaplotypes. Black diamonds symbolize median vectors that represent

hypothetical missing or unsampled ancestral haplotypes. Numbersindicated on the branches correspond to bootstrap support above 60%obtained in the NJ analyses. The geographical origin for eachhaplotype is shown in Table 1. WCE Western Continental Europe,ECE Eastern Continental Europe

Parasitol Res (2010) 107:631–641 637

rest of the haplotypes (bootstrap value 100%; seeFig. 2b.2). It is noteworthy to pay attention to differenthaplotypes from La Gomera (H24, H72, H74,) and Tenerife(H25) that were clustered with those of eastern continentalEurope while the other 37 haplotypes were clustered withthose from western continental European regions (Fig. 4).

The Balearic Islands showed four different haplotypes:two of them only present in Mallorca and clustered withthose from the western continental European region andCanary Islands, while the others two were clustered withhaplotypes from the eastern region (Figs. 3, 4).

The comparative analysis of the ITS1-5.8S-ITS2 sequencesof all T. muris haplotypes and T. muris isolates from Franceobtained from the GenBank revealed these isolates clusteredwith the western continental region.

In conclusion, T. muris isolates from the EuropeanEastern regions form a well-defined cluster (bootstrap value100%), including five haplotypes from the Canary Islands andtwo from Mallorca, whereas the isolates from the Cataloniaregion (La Riera, Montseny, and Calafell; Spain) appear to bequite similar and clustered with Denmark, France, theBalearics, and the Canary Islands (Western Europe).

Discussion

The results of the analysis of the ITS1-5.8S-ITS2 sequenceof the ribosomal DNA confirm the presence of DNApolymorphisms among T. muris isolates from Europe. Theexistence of intraindividual and intraspecific variations in T.muris agrees with our previous results (Cutillas et al. 2002).Polymorphism has been cited by different authors in theITS rDNA in some species of strongylid nematodes(Campbell et al. 1995; Stevenson et al. 1995), and severalauthors suggest that they are the consequence of mutationalexchange during DNA replication, the extent of which

Distribution of Western haplotypes at different countries

Montseny

Calafell

La Riera

Sant Martí

Pirineos

Mallorca

Turkey

Croatia

Rumania

La Gomera

La Palma

El Hierro

Tenerife

Distribution of Eastern haplotypes at different countries

Montseny

Calafell

La Riera

Sant Martí

Pirineos

Mallorca

Turkey

Croatia

Rumania

La Gomera

La Palma

El Hierro

Tenerife

0

2

4

6

8

10

12

14

16

18

20

Montseny Calafell La Riera Sant Martí Pirineos Mallorca Turkey Croatia Rumania La Gomera La Palma El Hierro Tenerife

Western haplotype

Eastern haplotype

H6 (Canary Islands)

Fig. 3 Diagrammatic distribution of the different haplotypes by different localities

Fig. 4 Phylogenetics analyses of Trichuris muris based on the ITS1-5.8S-ITS2 fragment of ribosomal DNA. Median-joining (MJ) net-works and Neighbor-joining (NJ) trees were constructed using thehaplotypes. Sizes of circles are proportional to frequency of thehaplotypes. Black diamonds symbolize median vectors that representhypothetical missing or unsampled ancestral haplotypes. Numbersindicated on the branches correspond to bootstrap support above 60%obtained in the NJ analyses. CI Canary Island, WCE WesternContinental Europe, ECE Eastern Continental Europe. The geographicalorigin for each haplotype is shown in Table 1

�

638 Parasitol Res (2010) 107:631–641

H18

H71

H20

H66

H41

H13

H77

H10

H9

H19

H1

H11

H2

H14

H27

H5

H3

H17

H59

H16

H6

H67

H43

H36

H57

H22

H75

H60

H64

H62

H63

H7

H4

H69

H76

H26

H21

H23

H8

H47

H38

H12

H15

H70

H42

H34

H35

H73

H54

H55

H52

H51

H53

H32

H33

H58

H68

H37

H39

H56

H65

H74

H40

H30

H72

H44

H24

H25

H29

H31

H28

H48

H45

H49

H46

H50

H61

H78

61

65

82

66

66

65

62

50

76

85

96

83

41

57

54

15

35

35

59

41

63

100

38

66

26

10

49

59

50

55

15

42

0

28

1

1

0

0

0.05

Arvicolinae Gerbillinae

ECE

Muridae

Murinae

WCE + CI

CI

WCE

CI

CI

CI

CI WCE

WCE

CI

Continental Europe and Balearic and Canary Islands

H46

H50

H72

H30

H11

H74

H6

H42

H13 H39 H18

H1

H62

H69

H32 H55

H4

H56

H43 H68 H19

H35 H58

H8

H23

H70

H38 H15 H12

H36

H57

H47

H78

H61

H50

CI

WCE

ECE

Mallorca

WCE

CI + WCE

WCE

Mallorca

ECE + Mallorca

CI

Mallorca ECE CI ECE

CI

CI WCE + CI

WCE

ECE

CI

ECE

Parasitol Res (2010) 107:631–641 639

appears to differ depending on the taxonomic group(Wesson and Collins 1992; Gasser et al. 1996).

The presence of repetitive nucleotide sequences has beenreported for trichurid nematodes (Cutillas et al. 2002) andalso by different authors in the eukaryote genome as Poly(G) or Poly (TG) (Hamada et al. 1982). Thus, micro-satellites have been found in Trichinella pseudospiralis((TGC)n) (Zarlenga and Dame 1992). Furthermore, thevariability in the length of the microsatellite repeatobserved in different clones and individuals of T. murishas also been reported by Zarlenga et al. (1996) as a typicalphenomenon observed in closely related species, and theyargued that variability would occur in the length of themicrosatellite repeat only, and not in its location nor in itspresence within the genome.

The biogeographical study carried out in continentalEurope shows the existence of two clear-cut geographicaland genetic lineages: one of them is widespread fromnorthern Spain (Catalonia) to Denmark (Western Europeanregion) while the second one is widespread for the EasternEuropean region (Croatia, Rumania and Turkey). T. murishave been found in A. sylvaticus, A. flavicollis, M.domesticus, and R. rattus. Moreover, the phylogeneticreconstructions also reveal that T. muris individuals in thevarious Muridae rodent hosts are not differentiated accord-ing to the host species that they parasitize. Thus, T. murisshows no host specificity since it does not have lineagesthat segregate among host species, rather, T. muris lineagesdifferentiated in allopatric geographic areas. Furthermore,these two genotypes can be easily distinguished amongthem by a PCR-RFLP analysis of this sequence with theApalI restriction enzyme.

Wu et al. (2007) studied the genetic relationships amongT. pseudospiralis isolates from Australia, Nearctic, andPalearctic regions. They demonstrated that the Palearcticpopulation (seven isolates from six different hosts from fourEuropean and two Asian countries) consisted of a uniformgenotype suggesting that there is a frequent gene flowamong the isolates. Furthermore, the two isolates from theNearctic region were 100% identical between them andquite different (in four gene loci) from isolates from thePalearctic region. These different genotypes could be easilydistinguished among them by a PCR-RFLP analysis of theCOI gene with the RsaI restriction enzyme. Nevertheless,Nagano et al. (1999) cited the existence of a geographicpolymorphism in Trichinella britovi (isolated from Europeand Japan) and a lack of gene flow between populations ofthe Western and Eastern parts of the distribution range ofthis species.

Nieberdeing et al. (2008) studied the phylogeography ofparasites of the genus Heligmosomoides sampled fromdifferent species of murid rodents of the genus Apodemus.Based on mitochondrial and rDNA data, these authors

showed that all lineages were differentiated according to ageographic pattern and independently from the sampledhost species. Similar results were found by Nieberding et al.(2004) for Heligmosomoides polygyrus parasitizing A.sylvaticus. They found that the parasite and the host partlydisplayed the same phylogeographic history. Indeed, theparasite and host continental European populations bothdifferentiated into three main geographical and geneticlineages: one of them was widespread for Western andcentral Europe, while the others were located in continentalItaly and Sicily, respectively. They showed that partialcongruent phylogeographic patterns in Western Europe, incontinental Italy and Sicily could be observed betweenspecies linked by a strong ecological trait, such as specificand direct-cycle endoparasitism.

In host–parasite interactions, co-speciation, i.e., the jointspeciation between host and its parasite, can arise if the twoorganisms share a common evolutionary history, so that theparasite follows the speciation events of its host. T. muris isan endoparasite of rodents with a direct life cycle, thus, allthe ecological traits affecting the host also affect T. muris.According to the hosts, the Murinae are the largestsubfamily of mammals. Steppan et al. (2005) carried out amultigene phylogeny ofMurinae, revealing distinct geograph-ical lineages and suggesting that the subfamily originated insoutheast Asia and that rapid diversification associated withrange expansion led to one or more coincident colonizationsof Africa and central and northern Asia. Murines expandingout of Southeast Asia probably passed through Western Asiaon the way to Africa. Thus, we suggest that T. muris couldhave evolved from the eastern region of Asia to the easternregion of Europe and subsequently to the Western region ofEurope. These two geographical lineages (Western andEastern continental Europe) could have arisen through theexistence of geographical barriers.

The biogeographical analyses of T. muris from theCanary Islands revealed one typical haplotype (H6)observed in 25 taxa. Furthermore, considering the NJ tree,a monophyletic origin of T. muris in these islands issuggested on the basis that oceanic islands have a typicaldevelopmental life cycle and one commonly supportedpattern involves taxa showing a pattern of dispersion fromolder to younger islands within an archipelago, withspeciation occurring on newly colonized islands (Funkand Wagner 1995). Thus, we suggest that the H76haplotype (La Gomera) could be the ancestor of T. murisin the Canary Islands, and the existence of four haplotypesfrom La Gomera and one from Tenerife which appearedclustered with those of Eastern continental Europe could beexplained according to the older origin of these islands andthe outcoming of hosts by ship from the Oriental zone thatseems to be the geographical origin of the host, and asubsequent dispersal to the other islands.

640 Parasitol Res (2010) 107:631–641

Acknowledgements We wish to thank Dr. Voitto Haukisalmi andDr. Heikki Henttonen from the Finnish Forest Research Institute,Vantaa (Finland) who provided samples from different Europeanregions. We also wish to thank Mr. Geoffrey Giddings for the criticalreading of the manuscript. The research has been funded by threegrants from the Ministry of Science and Technology (CGL2004-00630/BOS, CGL 2006-04937/BOS, CGL2008-01459/BOS).

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