Genetic clustering of Trypanosoma cruzi I lineage evidenced by intergenic miniexon gene sequencing

Infection, Genetics and Evolution 7 (2007) 587–593 www.elsevier.com/locate/meegid

Genetic clustering of Trypanosoma cruzi I lineage evidenced by intergenic miniexon gene sequencing§ Olivia O’Connor a, Marie-France Bosseno a, Christian Barnabe´ a, Emmanuel J.P. Douzery b, Simone Fre´de´rique Brenie`re a,* a

Institut de Recherche pour le De´veloppement (IRD), De´partement Socie´te´s et Sante´, UR 008 et 165, Montpellier, France Universite´ Montpellier 2 Sciences et Technologie, De´partement Biologie Evolution Environnement, Montpellier, France

b

Received 22 February 2007; received in revised form 27 April 2007; accepted 1 May 2007 Available online 6 May 2007

Abstract American trypanosomiasis or Chagas disease is endemic in Latin America and caused by the flagellate Trypanosoma cruzi, which exhibits broad genetic variation. In various areas, the transmission of Chagas disease is ensured by sylvatic vectors, mainly carrying the evolutionary lineage I of T. cruzi. Despite its epidemiological importance, this lineage is poorly studied. Here, we investigated the genetic variability and the phylogenetic relationships within T. cruzi I using sequences of the non-transcribed spacer of miniexon genes. The variability was firstly analysed between 10 repeats of spacer-miniexon genes in two strains of T. cruzi I and in the CL Brener strain, showing lower intra-strain variability than inter-strain. Furthermore, the phylogenetic analysis of 19 T. cruzi I strains (49 copies in total) clusters the copies into at least three groups. Two evolutionary phenomena can be proposed to explain the partition of the strains: (i) an association between strains and Didelphis sp. hosts and (ii) geographical clustering between the North American and South American strains. Thereby, the miniexon gene is an attractive marker to establish the phylogeny of lineage I and explore relationships between T. cruzi and mammal hosts. # 2007 Elsevier B.V. All rights reserved. Keywords: Trypanosoma cruzi; Polymorphism; Phylogeny; Miniexon gene; T. cruzi I

1. Introduction Trypanosoma cruzi, the causative agent of Chagas disease, affects 18 million people in endemic areas with 20,000–50,000 deaths reported each year (WHO, 2002). Although described as single specie, isolates of T. cruzi show an important genetic and phenotypic diversity. Population genetics analyses have shown that T. cruzi has a predominantly clonal mode of replication (Tibayrenc et al., 1986). Two main lineages, named T. cruzi I and T. cruzi II, have been identified (Momen, 1999). T. cruzi I is preferentially associated with the sylvatic transmission cycle, although it can be found in the domestic one, whereas T. cruzi II is mainly associated with the domestic cycle. Lineage II can be

§ Nucleotide sequence data reported in this paper are available in the GenBank, EMBL and DDBJ databases under the accession numbers EF576816 to EF576849. * Corresponding author at: 911 avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France. Tel.: +33 4 67 41 63 72; fax: +33 4 67 41 61 00. E-mail address: [email protected] (S.F. Brenie`re).

1567-1348/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2007.05.003

divided into five Discrete Typing Units (DTUs): three are specific for domestic cycles (DTU IIb, IId and IIe) and the two others for the sylvatic cycle and thereby probably distributed over all of the endemic area (Brisse et al., 2000). The hybrid origin of some clones (DTUs IId and IIe) and the observation of horizontal genetic transfers show that the diversity observed could be the result of recombination events as well as punctual mutations. In fact, exchanges of DNA have been experimentally observed between two T. cruzi strains belonging to lineage I (Gaunt et al., 2003). The type of exchange observed would generate aneuploidy, which arises from nuclear hybridizations followed by loss of alleles. Several evolutionary scenarios have recently been proposed on the basis of housekeeping gene sequences that would explain the emergence of the subdivisions that follow hybridization events; however, the number of ancestral clades (two or three) at the origin of the current diversity is still discussed (Westenberger et al., 2005; de Freitas et al., 2006). Among genetic markers aimed to elucidate the structure of T. cruzi populations, the miniexon gene and intergenic regions

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O. O’Connor et al. / Infection, Genetics and Evolution 7 (2007) 587–593

were sequenced in several T. cruzi strains and specific primers for the DTUs were selected (Souto et al., 1996; Fernandes et al., 2001). The miniexon genes (sequences of 39 bp highly conserved in the Kinetoplastids), which are spliced leaders of mRNA genes, are tandemly arranged in 1–200 copies per genome. The intergenic region (spacer) is composed of a moderately conserved intron and a variable non-transcribed zone (De Lange et al., 1984); the intron is followed by a poly(T) zone which would be used as a signal for the end of transcription. The gene promoter is present at the final part of the intergenic region. This zone has a variable sequence which correlates with the two principal lineages of T. cruzi (Nunes et al., 1997). Recent sequence analyses of a 350 bp region of spacer-miniexon (post-poly(T)) revealed a microsatellite motif allowing the identification of four haplotypes among Colombian T. cruzi I strains (Herrera et al., 2007). All these data show that the non-transcribed region is highly variable and should be particularly interesting to explore, since T. cruzi I is described as a single group. In this study, we first evaluate the intra-strain variability in two T. cruzi I strains and the CL Brener reference strain which sequences are available in gene bank. Then, the genetic

variability and structure of T. cruzi I lineage were investigated by sequencing various copies of the spacer-miniexon gene in several other strains. The whole variability was important and phylogenetic analysis detected a sub-structuring within T. cruzi I. 2. Materials and methods 2.1. Origin of the studied strains Twenty T. cruzi I strains (DTU I), selected from a broad geographic area extending from the USA to northern Argentina as well as the reference strain CL Brener, were analysed (Table 1). Previous typing of the strains by isoenzyme and/or RAPD clearly classified them in DTU I. Parasites (epimastigote forms) were cultured at 28 8C in LIT liquid medium supplemented with 10% fetal calf serum, collected in the exponential phase of growth, and parasite pellets were stored at 80 8C. 2.2. DNA extraction The cells were resuspended in lysis buffer (50 mM Tris–HCl pH 7.5, 0.4 M NaCl, 1% SDS, 10 mM EDTA) and incubated at

Table 1 Geographical and host origins of T. cruzi strains belonging to the DTU I Strains

Code

MHOM/BO/1983/P/209 cl1 a MHOM/BO/0000/P/11 cl3 a

P/209 cl1a P/11 cl3 a

MHOM/BO/1984/P/217 IINF/BO/1986/So40 MDAP/BR/0000/Cutia MDID/AR/2001/Palda1cl9a

P/217 So40 Cutia Palda1cl9a

MDID/BO/1985/86/2021 MDID/BR/1988/G38.1 MDID/CO/0000/RTD IPAP/MX/1998/800383 IINF/AR/2000/Tev91

86/2021 G38.1 RTD 800383 Tev91

MHOM/MX/1993/JJO

JJO

1993

Mexico

ILON/MX/2003/Gue536

Gue536

2003

Mexico

ILON/MX/1998/Tep23 cl4a

Tep23 cl4 a

1998

Mexico

ILON/MX/2004/L3 033

L3 033

2004

Mexico

IPHY/MX/1999/Cari137

Cari137

1999

Mexico

IPIR/MX/1999/Pla20

Pla20

1999

Mexico

IBAR/MX/1998/Sba54

Sba54

1998

Mexico

MHOM/MX/1993/H1 MPRC/US/1970/Raccoon70

H1 Raccoon70

1993 1970

Mexico USA

a

Laboratory clones; (–) missing data.

Year

Country

Localisation (Statedepartment-province, locality)

Host

Cycle

Current phylogenetic group

1983 –

Bolivia Bolivia

Human Human

Domestic Domestic

pan-American pan-American

1984 1986 – 2001

Bolivia Bolivia Brazil Argentina

Human Triatoma infestans Dasyprocta aguti Didelphis albiventris

Domestic Domestic Sylvatic Sylvatic

pan-American pan-American pan-American Didelphis

1985 1988 – 1998? 2000

Bolivia Brazil Colombia Mexico Argentina

Chuquisaca, Sucre Cochabamba, Cochabamba La Paz, Yungas Potosi, Vitichi Espiritu Santo, Colatina Departamento 12 de Octubre, Chaco, El palmar Beni Paraiba, Camala Coyaima Morelos, Jiutepec Chacabuco, Chaco, Tres estacas Jalisco, San Martin de Hidalgo Jalisco, San Martin de Hidalgo, Los Guerrero Jalisco, San Martin de Hidalgo, Tepehuaje Jalisco, San Martin de Hidalgo, Tepehuaje Nayarit, Compostela, Carrillo Puerto Nayarit, Compostela, Platanito Oaxaca, San Bartolo de Coyotepec, San Bartolo de Coyotepec Yucatan New Orleans, Covington

Didelphis marsupialis Didelphis albiventris Didelphis marsupialis Triatoma pallidipennis Triatoma infestans

Sylvatic Sylvatic Sylvatic Domestic Domestic

Didelphis Didelphis Didelphis Didelphis Mexico

Human

Peridomestic

Mexico

Triatoma longipennis

Peridomestic

Mexico

Triatoma longipennis

Peridomestic

Mexico

Triatoma longipennis

Sylvatic

Mexico

Triatoma sp (Phyllosoma complex) Triatoma picturata

Sylvatic

Mexico

Domestic

Mexico

Triatoma barberi

Peridomestic

Mexico

Human Procyon lotor

Domestic Sylvatic

Mexico Mexico

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56 8C for 2 h with 400 mg ml1 proteinase K. DNA was extracted sequentially with phenol–chloroform–isoamylalcohol (25:24:1) and chloroform–isoamylalcohol (24:1) at room temperature and precipitated with ethanol in 3 M sodium acetate. The concentration and purity of DNA was determined by spectrophotometry (260 and 280 nm). 2.3. Miniexon intergenic region amplification PCR amplification of the spacer-miniexon gene was achieved with primers complementary to the exon previously defined (Murthy et al., 1992), which amplify a 582 bp fragment. DNA was amplified in 20 ml of reaction mixture containing 10 ml of PCR Master Mix 2X (Promega, Charbonnie`res-lesBains, France), 0.5 ml of each primer (100 pmol), 7 ml of DNA free water and 2 ml of DNA template (10 ng ml1). Amplification was performed on a MyCyclerTM Personal Thermal Cycler (Bio-Rad, Marne-la-Coquette, France) under the following conditions: five cycles (94 8C, 30 s; 45 8C, 2 min; 65 8C, 30 s) followed by 25 cycles (94 8C, 30 s; 50 8C, 1 min; 72 8C, 3 min). PCR products were separated by electrophoresis in a 2% agarose gel and stained with ethidium bromide. The 582 bp fragment was gel purified with the ‘‘MinEluteTM Gel Extraction Kit’’ following the manufacturer’s recommendations (Qiagen, Courtaboeuf, France). 2.4. Cloning and sequencing Purified PCR products were cloned using the ‘‘TOPO TA Cloning1 Kit for Sequencing’’ (Invitrogen, Cergy-Pontoise, France). The sequencing was carried out commercially by the firm ‘‘GENOME Express’’ (Meylan, France) using the universal primers M13 (forward and reverse) which are complementary to the cloning vector. 2.5. Phylogenetic analysis Sequences were manually aligned using the program ‘‘Seaview’’ (Galtier et al., 1996). The maximum likelihood (ML) approach was applied for phylogenetic construction (Felsenstein, 1981). The ML method is based upon explicit models of sequence evolution, and it has been proven to be robust to a number of systematic biases during the tree inference. The best model of evolution was selected with ‘‘Modeltest v3-06’’ (Posada and Crandall, 1998) and its significance tested by the Akaike Informative Criterion (AIC).

589

Phylogenetic trees were built with the software, ‘‘PHYML v2.4.4’’ (Guindon and Gascuel, 2003), and drawn with ‘‘TREEDYN v189-3’’ (Chevenet et al., 2006) applying the outgroup conformation option. The robustness of the nodes was evaluated by bootstrap on 1000 replications with a heuristic research (Felsenstein, 1985). In the absence of true outgroup, the robustness of each branch was calculated taking successively as outgroup a strain belonging to each group identified in the tree. 3. Results 3.1. Intra-strain polymorphism between repeats in two strains of T. cruzi I and CL Brener strain For each of the two strains (laboratory clones) belonging to T. cruzi I (P/209 cl1 and Tep23 cl4), 10 clones of complete spacer-miniexon gene, obtained after cloning of PCR products in our laboratory, were sequenced, aligned and compared. All the sequences were different but they shared more than 96% similarity among each strain. The average percentage of homology between the copies of the two strains was 92.7%  0.71. The complete genome of the reference strain CL Brener strain (T. cruzi II) being available, intra-strain polymorphism was also investigated. Copies of the spacer-miniexon were obtained from the genome bank of T. cruzi (32746 sequences; TIGR, Karolinska & SBRI; http://www.ncbi.nlm.nih.gov.). After a BLAST search, the first twenty sequences exhibiting an alignment with DTU I (X62674 strain Sylvio), DTU IIa (AY367123 strain CanIII), DTU IIb (AY367125 strain Tu18), DTU IIc (AY367126 strain M5631) and DTU IId (AY367128 strain MN) were retained. After elimination of identical sequences (same accession number), 44 were best aligned with the DTU IIb among 45 sequences (complete or partial) and only one sequence was best aligned with DTU I. Among these sequences, 11 homologous to the DTU IIb were complete and they were included in the current analysis; they had 95.16% similarity among them. Then all the copies of T. cruzi I strains and those of CL Brener (T. cruzi II) were aligned together. The homology between copies of T. cruzi I and CL Brener reached 62.6%  0.80. Table 2 summarizes the polymorphisms observed. The intron, which has a constant size in all copies, was much less variable among the T. cruzi I and T. cruzi II copies than the other part of the intergenic region: the average

Table 2 Sequence variability between copies of the spacer-miniexon of three T. cruzi strains Strain code

CL Brener P209 cl1 Tep23 cl4

Number of clones

Length of sequence parts

Total number of mutation sites

Intron (bp)

Poly(T) (bp)

Variable non-transcribed zone (bp)

Total intergenic region (bp)

Intron

Poly(T)

Variable non-transcribed zone

Total intergenic region

11 10 10

73 73 73

16–27 15–22 14–24

480–482 454–462 468–471

569–581 548–556 554–568

8 (1 MU) 4 (1 MU) 3 (0 MU)

12 8 10

25 (5 MU) 29 (9 MU) 15 (10 MU)

45 41 28

MU: single mutation (present in only one copie); sd: standard deviation.

Homology (%  sd)

97.28  0.88 97.47  0.74 98.38  0.84

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homology between the introns of strains P/209 cl1 and Tep23 cl4 versus CL Brener was 85.13%  2.77. The length of the poly(T) was highly variable. In the T. cruzi I strains, the poly(T) was followed by a variable number of (AT), except for two copies in the P/209 cl1 strain. The (AT) repeat was absent in all CL Brener copies. The variability between the copies of a strain remained as low for the intron as for the post poly(T) part. In order to study the phylogenetic relations between the 31 sequences, the poly(T) zone, starting at the 74 bp position and ending after the (AT) repeat (motif ‘‘GGTG’’ conserved in all copies), was deleted from the sequences due to its strong variability. The best evolution model applicable to the data file was the General Time-Reversible Model (GTR), with gamma distributed rate variation among sites (G), and proportion of invariable sites (I). The maximum likelihood dendrogram obtained showed three groups, formed by the copies of the same strain (Fig. 1). Node support was high for the groups formed by the copies of strains P/209 cl1 (>88%) and Tep23 cl4 (100%). Also, these two groups were clustered together and apart from the copies of CL Brener (100%). 3.2. Phylogenetic relationships within T. cruzi I lineage One or two copies of 17 additional strains were cloned, sequenced and aligned with three copies of strains P/209 cl1 and Tep23 cl4 (copies presenting the highest level of divergence) and the reference strain Cutia (AY367129). The alignment of the 35 sequences confirmed the general organization of the miniexon intergenic region within the evolutionary lineage T. cruzi I. The intron (73 bp) had a total of seven mutation sites and the length of the poly(T) varied from 14 to 33 bp. The poly(T) was followed, in 94.3% of the copies, by (AT) repeats which varied from one to seven. The remaining

Fig. 1. Maximum likelihood phylogenetic tree of 31 sequences from three T. cruzi strains, reconstructed from spacer-miniexon gene. The model of multiple substitutions used was the GTR + I + G model (ln L = 1915). Bootstrap values obtained after 1000 replicates are shown above the branches. Phylogenetic tree is drawn using Treedyn with ‘‘outgroup’’ option and ‘‘rectangular conformation without branch length’’ (the CL Brener strain being too distant of T. cruzi I strains to apply a conformation with branch length).

part of the sequence had a total of 88 mutation sites, of which 26 (29.54%) were single mutations (present in only one copy) dispersed throughout the sequence. The overall length of the intergenic region varied from 547 to 574 bp. As previously done, the poly(T) zone was excluded from the phylogenetic analysis. The best model of multiple substitutions was the GTR + I + G model. The tree constructed with the maximum likelihood method is presented in Fig. 2. Two significant clades were identified: (i) all the strains of Mexico (except one), one from the USA, and another one from Argentina (node support = 99%) and (ii) the second clade grouped the previous clade with all the Bolivian strains (except for one) and the reference strain Cutia, isolated in Brazil (value of robustness = 81%); these clades were apart from five other strains. These last strains were isolated in Argentina, Bolivia, Brazil and Colombia, all from Didelphis sp. and one from Triatoma pallidipennis in Mexico; they formed a weakly supported monophyletic group. Furthermore, no synapomorphic mutation was observed for any of the groups, ‘‘Mexico’’, ‘‘pan-American’’ and ‘‘Didelphis’’. The introduction in the phylogenetic analysis of the 10 copies from P/209 cl1 and 10 from Tep23 cl4 did not modify the current clustering and bootstrap values (less than 5% decrease). 4. Discussion T. cruzi I, which has a very large geographical distribution from North to South America, predominates from the Amazonian basin northwards where domestic and sylvatic triatomines species ensure the transmission of Chagas disease in various countries including Venezuela, Colombia, Central America and Mexico (Barnabe´ and Brenie`re, 1999; WHO, 2002). In these regions, T. cruzi I infects patients and different serious pathologies commonly associated with Chagas disease are observed (Rosas, 2000; Monteon-Padilla et al., 2002; Sousa et al., 2006). However, megasyndromes are detected only rarely from the Amazonian basin northwards (Miles et al., 1981). Moreover, parasite typing suggests relationships between strains and pathology (Vago et al., 2000). So, the importance of Chagas morbidity in these regions does not corroborate the hypothesis of innocuity of T. cruzi I expressed by several scientists. Experimental infections showed that T. cruzi I, which can be observed in heart tissues, appeared as a heterogeneous group presenting substantial differences in biological characteristics, despite their strong genetic relatedness (Espinoza et al., 1998; Garzon et al., 2005). Thus, the analysis of genetic diversity and population structure is necessary for understanding the relationships of this group of parasites with their mammal hosts. Preliminary alignments of the spacer-miniexon gene from reference strains available in GenBank enabled us to observe an important polymorphism within the same DTU. Therefore, this marker should provide information on the genetic structure of T. cruzi I. The first step of the present analysis was to examine the variability of the spacer-miniexon gene between repeats. For the CL Brener strain (DTU IIe), all the copies were aligned with

O. O’Connor et al. / Infection, Genetics and Evolution 7 (2007) 587–593

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Fig. 2. Maximum likelihood phylogram of 35 sequences from 19 T. cruzi I strains based on spacer-miniexon gene. The model of multiple substitutions used was the GTR + I + G model (ln L = 1470.4). Bootstrap values obtained after 1000 replicates are shown above the branches. Phylogenetic tree is drawn using Treedyn with ‘‘outgroup’’ option and ‘‘rectangular conformation with branch length’’. Code strains are indicated at the end of each branch, and followed by the number of the cloned sequence and the country of strain origin in brackets.

a sequence analogous to the haplotype DTU IIb, except one which significantly aligns with the haplotype DTU I. This result is compatible with the assumption of the hybrid origin of the strain CL Brener and with the described mechanism of genetic exchanges between strains (Westenberger et al., 2005; Gaunt et al., 2003), but this result should be confirm by further experimental steps as suggested by Thomas et al. (2005). Furthermore, the two current phylogenetic trees showed that the copies of a same strain, whether it belongs to T. cruzi I or T. cruzi II, are clustered together (same clade). So, any copy of one strain should be a valuable genetic marker for population genetics. For the T. cruzi I strains examined, all the sequences aligned with the haplotype DTU I. The present phylogenetic analysis identifies a first ‘‘clade’’ which agglomerates the majority of the strains (bootstrap value of 80.9%) isolated in either North or South America (the pan-American group). This monophyletic group did not include four strains of South America (Argentina, Bolivia, Brazil and Colombia) and a Mexican strain. Except for the latter, the four strains were isolated from Didelphis sp. According to the tree, the ‘‘Didelphis-group’’ appeared as the most basal. The second ‘‘clade,’’ well supported by a bootstrap value of 99.5%, agglomerates the majority of the Mexican

strains (the Mexican group) extending from the Occident to the Yucatan (2000 km). The position of a Southern USA strain, Raccoon70, clustered with those of Mexico, inferring a common origin and a limited expansion to North America of this divergent group. Moreover, this group is composed of closely related strains, either suggesting their recent diversification, or reflecting a lack of variability of the miniexon marker. However, a strain from Argentina isolated in domestic cycle also clustered in this group. The current phylogeny tends to show two possible evolutionary phenomena: (i) specific association between T. cruzi strains and mammal genus and (ii) geographical structuring between the strains of North and South America. Nowadays, the oppossum, Didelphis sp. is one of the most important natural reservoirs widely distributed in North and South America (WHO, 2002). Based on phylogenetic analysis of several trypanosomes species spread over different continents (America, Africa, Europe and Australia), Kawashita et al. (2001) proposed a very ancient origin of T. cruzi associated with the marsupial fauna which predominated in South America during the Upper Cretaceous (100 My), when this continent was separated from other land masses. Moreover, a recent compilation of the genetic characterization of present

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strains suggests a strong association of T. cruzi I with Didelphis while T. cruzi II is associated with Armadillos (Yeo et al., 2005). The association of T. cruzi I with Didelphis was also observed in the USA and Argentina (Barnabe´ et al., 2001; Diosque et al., 2004). In the current sample of strains, the position of the Didelphis strains in the tree supports the hypothesis of a long evolution of T. cruzi I with marsupials. The identification of one Mexican strain isolated from a vector in the ‘‘Didelphis-group’’ captured in a human dwelling would be related to the important circulation of Didelphis sp. in Mexican villages. This stresses their importance as trypanosome donors as evidenced by blood feeding pattern analysis (Brenie`re et al., 2004). Assuming an association (coevolution?) between Didelphis sp. and strains of T. cruzi I lineage, the arrival of opossum from South America to North America by the Isthmus of Panama during the Pliocene (around 3 My) can explain the South–North distribution of ‘‘Didelphis-group’’ strains. The other T. cruzi I strains should be the result of adaptation to other hosts. Indeed, the emergence of the Isthmus of Panama allowed a massive North–South migration of placentals which supplanted the majority of marsupial species in South America. This great upset in mammalian fauna could give rise to adaptations of T. cruzi strains to numerous new mammal species of North American origin, resulting in increases in the genetic variability of the parasite. The clade which agglomerates mainly North American strains exhibits a low variability (founder effect) that would be explained by the South–North migration of some placental species through the Isthmus of Panama. The presence of an Argentinean strain in this clade favours the hypothesis of the introduction of these strains from the South. However, no information is available on the frequency of this type of strain in Argentina. In general the current analysis suggested genetic structuration of T. cruzi I strains and a complex evolution. The long evolution of T. cruzi I would be the result of adaptation of the strains to many species of mammals whose dynamics and geographical distribution in America have been conditioned by paleogeographical and climate changes. Acknowledgements These investigations received financial support from the ‘‘Institut de Recherche pour le De´veloppement’’. We are grateful to E. Magallo´n-Gaste´lum, M. Soto-Gutierrez, F. Lozano-Kasten and P. Diosque for their contribution to isolation of strains. The contribution of EJPD is the one N8 2007-040 of the Institut des Sciences de l’Evolution de Montpellier (UMR 5554 CNRS). References Barnabe´, C., Brenie`re, S.F., 1999. Eco-distribucio´n de los clones de Trypanosoma cruzi. In: Chagas La Enfermedad en Bolivia, Conocimientos Cientificos al Inicio del Programa de Control (1998–2002), Julio R. Alfred Cassab et al. Copyright, Editiones Gra´ficas ‘‘E. G.’’, La Paz, Bolivia pp. 209–215. Barnabe´, C., Yaeger, R., Pung, O., Tibayrenc, M., 2001. Trypanosoma cruzi: a considerable phylogenetic divergence indicates that the agent of Chagas

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