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Molecular analyses reveal two geographic and genetic lineages for tapeworms,Taenia solium and Taenia saginata, from Ecuador using mitochondrial DNA

Danilo Solano, Juan Carlos Navarro, Antonio León-Reyes, Washington Benítez-Ortiz,Richar Rodríguez-Hidalgo

PII: S0014-4894(16)30251-X

DOI: 10.1016/j.exppara.2016.10.015

Reference: YEXPR 7325

To appear in: Experimental Parasitology

Received Date: 20 May 2016

Revised Date: 30 September 2016

Accepted Date: 14 October 2016

Please cite this article as: Solano, D., Navarro, J.C., León-Reyes, A., Benítez-Ortiz, W., Rodríguez-Hidalgo, R., Molecular analyses reveal two geographic and genetic lineages for tapeworms, Taeniasolium and Taenia saginata, from Ecuador using mitochondrial DNA, Experimental Parasitology (2016),doi: 10.1016/j.exppara.2016.10.015.

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http://dx.doi.org/10.1016/j.exppara.2016.10.015

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Molecular analyses reveal two geographic and genetic lineages for tapeworms, Taenia solium and 1

Taenia saginata, from Ecuador using mitochondrial DNA. 2

Danilo Solanoa,e, Juan Carlos Navarroa,c Antonio León-Reyesd, Washington Benítez-Ortiza,b, Richar 3

Rodríguez-Hidalgoa,b* 4

a Centro Internacional de Zoonosis (CIZ), Universidad Central del Ecuador, Quito, Ecuador. 5

b Facultad de Medicina Veterinaria y Zootecnia, Universidad Central del Ecuador, Quito, Ecuador. 6

c Universidad Internacional SEK, Facultad de Ciencias Naturales y Ambientales. Quito, Ecuador 7

d Laboratorio de Biotecnología Agrícola y de Alimentos, Universidad San Francisco de Quito, Quito, 8

Ecuador. 9

e Carrera Biotecnología, Universidad de las Fuerzas Armadas (ESPE), Quito, Ecuador. 10

*Corresponding Author: Tel.: +593 98 5028 169. 11

E-mail: [email protected] (R. Rodríguez-Hidalgo) 12

13

Abstract 14

Tapeworms Taenia solium and Taenia saginata are the causative agents of taeniasis / cysticercosis. 15

These are diseases with high medical and veterinary importance due to their impact on public health 16

and rural economy in tropical countries. The re-emergence of T. solium as a result of human migration, 17

the economic burden affecting livestock industry, and the large variability of symptoms in several 18

human cysticercosis, encourage studies on genetic diversity, and the identification of these parasites 19

with molecular phylogenetic tools. Samples collected from the Ecuadorian provinces: Loja, Guayas, 20

Manabí, Tungurahua (South), and Imbabura, Pichincha (North) from 2000 to 2012 were performed 21

under Maximum Parsimony analyses and haplotype networks using partial sequences of mitochondrial 22

DNA, cytochrome oxidase subunit I (COI) and NADH subunit I (NDI), from Genbank and own 23

sequences of Taenia solium and Taenia saginata from Ecuador. Both species have shown reciprocal 24

monophyly, which confirms its molecular taxonomic identity. The COI and NDI genes results suggest 25

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phylogenetic structure for both parasite species from south and north of Ecuador. In T. solium, both 26

genes gene revealed greater geographic structure, whereas in T. saginata, the variability for both genes 27

was low. In conclusion, COI haplotype networks of T. solium suggest two geographical events in the 28

introduction of this species in Ecuador (African and Asian lineages) and occurring sympatric, probably 29

through the most common routes of maritime trade between the XV-XIX centuries. Moreover, the 30

evidence of two NDI geographical lineages in T. solium from the north (province of Imbabura) and the 31

south (province of Loja) of Ecuador derivate from a common Indian ancestor open new approaches for 32

studies on genetic populations and eco-epidemiology. 33

Keywords: mtDNA lineages, cysticercosis, taeniasis, COI, NADH-I, Taenia solium, Taenia saginata, 34

Ecuador. 35

1. Introduction 36

Infestations by Taenia spp., and Echinococcus spp., are common, not only in developing countries but 37

also in industrialized countries, where apart from being a threat to health, they represent a socio-38

economic impact. Cysticercosis causes a debilitating disease in humans as well as losses in the meat 39

industry due to the condemnation of meat from infected animals (Jiménez et al., 2002; Raether and 40

Hänel, 2003; Rodriguez Hidalgo, 2007; Schantz, 1999; Tsai et al., 2013) 41

The Andean region of Ecuador was described as hyperendemic for taeniasis / cysticercosis, with 42

prevalences in rural communities up to 1.60% for taeniasis and 14.4% for T. solium-cysticercosis by. 43

The prevalence of T. saginata in Ecuador is not well established, however, a moderate prevalence of 44

bovine cysticercosis with 0.37% on veterinary inspection and 4.03% by serological techniques has been 45

reported (Cayo-Rojas et al., 2011; Cruz et al., 1989; Rodriguez-Hidalgo et al., 2006; Rodríguez-46

Hidalgo et al., 2010, 2003; Rodriguez Hidalgo, 2007). 47

As a result of their great importance in Ecuador, these two cestodes species have been extensively 48

studied. However, the strategies aiming to control these parasitoses have major limitations i.a. it has not 49

been possible to accurately identify the prevalence of Taenia spp. and the determination of strains by 50

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means of ecological, biological or morphological criteria is difficult (Ito et al., 2003). The efficiency of 51

control depends on detailed epidemiological information including identification and precise 52

characterization of the causative agent in each endemic area (Gasser et al., 1999; Jia et al., 2010; 53

McManus, 1990). 54

The genus Taenia has been successfully identified using, enzyme electrophoresis and mitochondrial 55

molecular markers to differentiate between species and also to infer phylogenies (Hoberg et al., 2000; 56

Nakao et al., 2010; Queiroz and Alkire, 1998). However, little is known about the genetic intra-specific 57

variation in cestodes (Pawlowsky Zbigniew, 2002) and considerable research indicates a great 58

heterogeneity in pathology caused by Taenia solium but there is a misunderstanding about the role of 59

genetic diversity and adaptability of species (Del Brutto, 2013; Finsterer and Auer, 2012; Marquez and 60

Arauz, 2012; Román, 2014; Sotelo, 2011). Based on this issue, mitochondrial DNA analysis provides 61

complementary tools for characterization of a population. Gene fragments or complete genome of 62

mitochondrial DNA have been successfully used in population genetics, ecology and identification of 63

tapeworms (Jia et al., 2010). Genetic variation associated with different hosts is a well-known fact in 64

several cestodes species e.g. Echinococcus granulosus and Ito et al., (Ito et al., 2003) suspected that for 65

Taenia saginata and Taenia solium it may well be equally the case. 66

Nakao et al., (Nakao et al., 2002), using the complete genes Cytochrome Oxidase subunit I and 67

Cytochrome b of the mitochondrial DNA, showed the existence of two lineages of Taenia solium 68

worldwide: an Asian group and an African and Latin-American group of strains. A sequence from 69

Ecuador was included in these studies, showing minimal variation with the rest of the sequences in the 70

analysis, albeit this sequence represents only a small portion of the Ecuadorian gene pool. Yanagida et 71

al., (Yanagida et al., 2014) using different mitochondrial genes, report two genetic sympatric lineages 72

of T. solium in Madagascar close related with Asia and Africa/Latin America as consequence of 73

historical human migration. These authors shown the Africa/Latin America lineage connected by 74

haplotypes network with the Ecuadorian haplotype AB066491 here also used. 75

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It is important to complete more detailed variability studies for this species, even more so when genetic 76

variation of tapeworms from various geographic locations might be linked to clinical and pathological 77

differences found in human cysticercosis (Maravilla et al., 2003; Vega et al., 2003). 78

The aim of this research is to determine the genetic diversity within populations of Taenia solium and 79

Taenia saginata collected in six locations, from the north and the south of Ecuador. We also 80

hypothesized that different geographical origins or introductions, can be assessed by means of presence 81

of geographic phylogenetic structuration within sequences-populations and/or haplotypes networks 82

analysis. The results in this study can underpin epidemiological research and the control of these 83

parasites. Furthermore, it provides a great source of information and support for new diagnostic 84

methods and reinforces vaccines developing in the fight against these important diseases (Assana et al., 85

2010; Fernadez et al., 2006; Maravilla et al., 2008; Sciutto et al., 2013; Tsai et al., 2013). 86

2. Materials and methods 87

2.1. Source of Taenia spp. specimens. 88

Specimens used in this research belong to the biological bank of the Centro Internacional de Zoonosis 89

(CIZ) at Universidad Central del Ecuador from localities of a former study and control program. 90

Samples have been conserved in ethanol solution 70%, and kept frozen at -20°C. Samples were 91

collected from the Ecuadorian provinces of: Loja, Guayas, Manabí, Tungurahua (South), and 92

Imbabura, Pichincha (North) from 2000 to 2012 (Supplementary data 1). Specimens were isolated form 93

human faecal material after anthelmintic treatment. Patients became from both urban and rural areas. 94

The vouchers of remains specimens are deposited in the bank of CIZ. 95

2.2. DNA extraction, amplification and sequencing of COI and NDI genes. 96

For DNA extraction of Taenia’s proglottids, the Wizard Genomic DNA Purification of Promega® 97

commercial kit was used (Promega, 2010) following the manufacturer's protocol. The DNA samples 98

obtained from proglottids of Taenia spp. were analysed in agarose gel 0.8% to ascertain presence, 99

quality and size of the extracted material. The quantification of genomic DNA extracted from Taenia 100

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spp. was performed using the Invitrogen fluorometer QUBIT®. We used the Quant-iT™ Broad-Range 101

DNA Assay Kit according to the manufacturer's instructions (Data not shown). 102

The reactions were performed in 25 µL, using a thermo cycler TECHNE TC-412, using the primers for 103

NDI sequence (Bowles and McManus, 1993) and JB3-JB4.5 for COI (Bowles et al., 1992). For each 104

reaction buffer PCR 1X, 3mM of MgCl2, 0.5 µM of oligonucleotide, 0.15 mM of dNTP´s and 0,5 U of 105

Taq Polymerase (Invitrogen) was added. The procedure was as follows: 92 °C for 5 minutes for initial 106

denaturation; 35 cycles of 94 °C, 30 s (denaturation); 55°C (COI)/ 57 °C (NDI), 30 s (annealing); 72 °C, 107

30 s/ 1 min (extension), followed by a final extension at 72 °C for 5 minutes. For each batch of 108

reactions, a negative control (molecular biology grade water) and a positive control (DNA from T. 109

saginata or T. solium) were included. 110

Amplified fragments of COI and NDI were purified using PureLink® PCR Purification Kit (Invitrogen) 111

according to the manufacturer's instructions, subsequently the fragments were sent to Macrogen Inc. 112

(Korea) for sequencing in duplicate (forward and reverse). 113

2.3. Phylogenetic analysis and haplotype networks 114

Own sequences were contig assembly performed using the Sequencer 4.2.2 (Gene Codes, Ann Arbor, 115

MI) software and identity confirmed by BLAST in NCBI resources. The consensus sequences of gene 116

cytochrome oxidase subunit 1 (COI) and NAD dehydrogenase subunit 1 (NDI) from the two groups 117

(Taenia solium and Taenia saginata) were edited in MacClade software (Maddison and Wayne, 2011), 118

resulting sequences of 404 bp (COI) and 459 bp (NDI). Sequences deposited in GenBank NCBI from 119

other species of Taenia were included as reference/external groups and outgroups (Nixon and 120

Carpenter, 1993) (Supplementary data 2) in order to get a wide geographic diversity covering distinct 121

continents and to test the problem species monophyly. DNA sequences were aligned using MacVector 122

7.2 (Accelrys, Madison, WI) by ClustalW algorithm with gap creation and extension penalties by 123

default (Supplementary data 3 and 4). Parsimony analyses were implemented in PAUP 4.0b10 124

(Swofford, 2001) using the heuristic search option with a Tree Bisection Reconnection branch-125

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swapping algorithm with at random stepwise addition of 10 replicates for each search and 100-1,000 126

replications per analysis. Gaps were treated both as missing data and as a fifth character state. The 127

characters were treated as unordered, and equally weighted, after that the characters were weighted by 128

consistence index. The robustness of the trees was estimated using parsimony bootstrap with 500 129

pseudoreplicates after excluding uninformative characters (Carpenter, 1996). We also performed a 130

Maximum Likelihood (ML, substitution model estimated by ModelTest, Posada and Crandall, (1998) 131

on PAUP) and a distance analysis (Neighbour-joining, NJ) using PAUP 4.0b10 (Swofford, 2001). 132

Later, a Nexus Matrix of sequences for each species was used to construct haplotype networks for TCS 133

2.1.1 with 95% of connection limit (Clement et al., 2000). We use the monophyly (based on % of 134

bootstrapping, location in clades on the tree, and a genetic divergence intra species bigger than inter 135

species) and phylogenetic species concept to delimit the different taxa. Then, the paraphyletic position 136

in the tree topology of sequences from GenBank under with the same label means a different species 137

and as consequence a misidentified specimen. 138

3. Results and Discussion 139

3.1. PCR amplification of genes COI and NDI 140

A total of 53/68 specimens of T. solium and T. saginata were successfully amplified. Most samples 141

were complete adult specimens (without scolex), with one third of the samples were proglottids only. 142

Tapeworms were previously characterized using morphology and RFLP analysis of 12S gene 143

(Rodriguez-Hidalgo et al., 2002). 144

3.2 Phylogenetic Analysis 145

3.2.1 Cytochrome oxidase subunit I gene (COI) 146

The maximum parsimony analysis of the 67 sequences used, characters re-weighted, yielded a single 147

parsimonious tree whose length values, consistency index and retention index values of L= 364.82, CI= 148

0.84 and RI= 0.91 (Fig. 1). The ML and NJ yielded the same topology of MP. The substitution model 149

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that fit wit the matrix alignment calculated by ModelTest with Bayesian Information Criteria (BIC) for 150

ML was TrN+G (Supplemtary data 5). 151

The topologies of phylogenetic trees obtained using COI gene (weight and reweighted) reveal the 152

internal grouping of clades, corresponding to sequences of a monophyletic derivated clade Taenia 153

solium, Taenia saginata in a monophyletic clade plus T. asiatica and the outgroups sequences in a 154

basal location. The Taenia saginata group does not show a phylogenetic structure, therefore samples 155

from distant locations like Japan, Belgium, Kenya and Ecuador present values of divergence between 156

0% and 0.26%, evidenced in the tree as a polytomy. A more detailed analysis of the alignment showed 157

two changes: E42.Tsag. Tungurahua, G by A in position 303, and Tsag. FRANCE T by C in position 158

243. 159

In a similar fashion, the internal distribution of the Taenia solium clade shows a phylogenetic structure. 160

Within the internal or derivated clade of T. solium, the sequences showed two subclades with 161

geographical correspondence: an ancestral Asian subclade + Peruvian sequence (Fig. 1, in yellow and 162

green colours) and a derivated African + America subclade (Fig. 1, in blue colour) with Ecuadorian 163

sequences included in a politomy (not-resolved branches). These findings are consistent with the results 164

found by Nakao et al. (Nakao et al., 2002). The distance (p-uncorrected) matrix (Table 1, 165

Supplementary data 6) shows that genetic divergence of the COI gene in samples from Imbabura 166

(North Ecuador) and Loja (South Ecuador), varies between 0 % and 1.06%, therefore, no significant 167

difference was observed among Ecuadorian samples from different geographic locations. 168

The difficulty of taxonomic identification of metacestodes of Taenia spp. is evidenced when some 169

particular sequences found in gene databases do not correspond to the species labelled based on their 170

location in our trees. In our study, two COI sequences: T. solium-Mexico (EU747650) and T. solium-171

Peru (AF360866), appear as different species from those originally published. In the first case 172

(EU747650), the sequence shows a clear homology and sister relationship with T. hydatigena, which 173

has pigs as a common intermediate host (Conlan et al., 2009) suggesting a misidentify. The sequence 174

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(AF360866) is presented as a particular case, the topology analysis locates it as an ancestor of T. 175

solium, however the divergence with this species is considerably high: 11.94%. The divergence 176

between T. solium and the closest sequence T. hydatigena is 12.40%, therefore, this specimen 177

(AF360866) could be a genetic variation of T. solium from Peru (conservative point of view), or an 178

intermediate species between T. solium and T. hydatigena, this however, requires further research. 179

In order to resolve the polytomies observed in the tree and to detect mutational ancestor-descendant 180

changes between geographical sequences, we have performed the haplotype network (TCS) shown in 181

Fig. 2. We found eleven haplotypes of worldwide distribution grouped into two different (not 182

connected) networks with Ecuadorian haplotypes located in both linked with African-American and 183

Asian haplotypes. 184

3.2.2. NAD dehydrogenase subunit I gene (NDI) 185

The maximum parsimony analysis of the 31 sequences and re-weighted yielded a single tree with 186

values of L= 344.89, CI= 0.76 e RI= 0.83 (Fig. 3). The ML and NJ yielded the same topology of MP. 187

The substitution model that fit wit the matrix alignment calculated by ModelTest with Bayesian 188

Information Criteria (BIC) for ML was TVM+G (Supplementary data 7). 189

The topology of the tree shows three consecutive basal clades corresponding to the outgroups taxa. 190

Then, internally two sister and reciprocal monophyletic clades: T. solium clade + T. asiatica (T. 191

saginata clade plus T. krabbei and T. multiceps). In the derivate group of T. saginata we found a 192

polytomy with no geographic structure. The sequences of T. asiatica, T. krabei and T. multiceps are 193

located as ancestral taxa to T. saginata. 194

The T. solium clade shows a geographical structure, forming two groups: one group with all sequences 195

of America (Fig. 3, in red colour), Ecuador included; and another group with Asian sequences (Fig. 3, 196

in yellow colour). The strict consensus from three topologies shows that the distribution of the clades 197

has the same geographical pattern. The bootstrap analysis with 1,000 repetitions shows clades with 198

strong support by statistical re-sampling of the alignment matrix (Fig. 3). 199

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In addition, topologies of phylogenetic trees obtained using NDI gene show a similar distribution to the 200

one found in COI gene trees, however, within the clade of Taenia solium two subclades were found 201

from the north (Imbabura) and from the south (Loja) of Ecuador. The distance (p-uncorrected) matrix 202

(Table 1, Supplementary data 8) shows that genetic divergence of the NDI gene in samples from 203

Imbabura and Loja is around 1,2% which is much less than in case of an interspecies divergence (e.g. 204

12%, between T. asiatica and T. saginata it is 12%). These findings demonstrate higher variability in 205

NDI than COI in the mtDNA of Taenia spp., in accordance with Jia et al. (Jia et al., 2010). The clade 206

of Taenia saginata as well as the results shown in the COI gene did not show evidence of significant 207

geographic differentiation. 208

In order to resolve the polytomies observed in the reweighed tree and to detect ancestor-descendant 209

mutational changes between geographical sequences (Fig. 4) we used the TCS software. The haplotype 210

network constructed is shown in Fig. 4. Five haplotypes worldwide distributed were grouped into a 211

single network with H4 and H5 from South Ecuador and North Ecuador respectively with the India 212

haplotype H1 as close related based on mutational changes. 213

3.3. Haplotype Networks 214

Searching for deeper lineage structure than in phylogenetic analysis, a network of haplotypes was 215

performed, in order to discriminate patterns of genetic divergence through specific mutational changes. 216

The network analysis using COI molecular marker resulted in two not connected networks of eleven 217

haplotypes. The first network consists of haplotypes from Brazil, Cape Verde (South Africa), Colombia 218

and Southern Ecuador (Loja) showing the detailed connection of mutational changes that the 219

politomies in the phylogenetic tree does not, for instance, the ancestor-descendent mutations 220

relationship among Colombia and Cape Verde haplotypes with Loja-Ecuador. The second network is 221

formed by haplotypes from France, Mexico, Haiti, South Korea, China, India and our North-South 222

Ecuador (Imbabura + Loja) including the Ecuadorian sequence in GenBank. Those results showing a 223

clear correlation of the Ecuadorian samples with African and Europe-Asian sequences both occurring 224

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sympatric in the Country similar to Yanagida et al., (2014) results. The two networks of haplotypes 225

analysed are correlated with one of the most common routes of maritime trade between the XV-XIX 226

centuries, supporting the hypothesis of Nakao et al, (Nakao et al., 2002) on the introduction of Taenia 227

solium from Europe to Africa and Latin America. 228

The haplotype network obtained from NDI shows a most probable introduction of Taenia solium to 229

Ecuador from a haplotype from India and subsequently the sequences from Ecuador (north and south) 230

show two separate lineages of mutational changes, with 2 changes to North Ecuador lineage and four 231

mutations to South Ecuador lineage. This network is consistent with the results reported by Martinez-232

Hernández et al. (Martinez-Hernandez et al., 2009) using COI and Cytb, who found a strong correlation 233

among sequences from Latin America (Peru and Mexico) and India. 234

In conclusion, both networks COI and NDI, suggest that the introduction of Taenia solium to Ecuador 235

occurred probably in two different events and geographical origins. Those differences might have been 236

maintained in spite of anthropogenic pressure such as intensive human migrations, and trading of 237

animals and meat products. This meta-population seems to have undergone two colonization processes, 238

with differentiation between northern and southern Ecuador. The Southern colonization influenced 239

from Asia and Africa haplotypes introductions and the Northern Ecuador mainly influenced by Asian 240

introductions. In summary, this research reports the pattern of the genetic variability and gene flow 241

among Ecuadorian localities of both Taenia species. This study provides important genetic baseline 242

data and new approaches for studies on genetic populations, eco-epidemiology and control. 243

Acknowledgements 244

We thank Jef Brandt who provided important comments and suggestion to our manuscript. 245

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Figure legends 359

Fig. 1. Phylogenetic tree with characters reweighed from the COI gene showing the phylogenetic 360

relationships of Taenia spp. Right side brackets show the species outgroups, and the taxonomic 361

nomination of sequences. Numbers on branches are the bootstrap values. Lines-branches in colors 362

shown the geographical association into each subclades. 363

Fig. 2. Global Network haplotypes of Taenia solium for COI gene. Two not connected networks 364

shows the two geographical lineages. Ecuador haplotypes are located in both networks (Africa-Latin 365

America and European-Asia networks). 366

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Fig. 3. Phylogenetic tree with NDI gene and characters reweighted showing the phylogenetic 367

relationships of Taenia spp. Right side brackets show the species outgroups, and the taxonomic 368

nomination of sequences. Numbers on branches are the bootstrap values. Lines-branches in colours 369

shown the geographical association into each subclades. 370

Fig. 4. Global Network haplotypes of Taenia solium using data from NDI gene. Ecuador 371

haplotypes are located in two separate lines (Southern and Northern Ecuador) after mutational steps 372

from India haplotype. 373

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Gene Specie Collection Place Specie Collection Place Divergence Percentage

(%)

COI

E. granulosus Turkey (JN810792)

T.solium (E44) Southern Ecuador (Loja) 19,003

T.solium (E11) Northern Ecuador (Imbabura) 19,365

T. saginata (E40) Southern Ecuador (Guayas) 19,155

T. saginata (E55) Northern Ecuador (Pichincha) 19,155

T. parva Spain (EU544580)





T. solium

India (AF360869) T.solium (E44) Southern Ecuador (Loja) 1,558


Cameroon (FN995666) T.solium (E44) Southern Ecuador (Loja) 1,061

T.solium (E11) Northern Ecuador (Imbabura) 0

T. saginata

Belgium (AB107242) T. saginata (E40) Southern Ecuador (Guayas) 0

T. saginata (E55) Northern Ecuador (Pichincha) 0

China (AB533172) T. saginata (E40) Southern Ecuador (Guayas) 0


Kenya (AM503327) T. saginata (E40) Southern Ecuador (Guayas) 0


NDI

E. granulosus Turkey (HM563034)




T. saginata (E55) Ecuador North (Pichincha) 26,932

T. parva Spain (EU544633)





T. solium

India (EF076753) T.solium (E45) Southern Ecuador (Loja) 0,664


Australia (AJ239107) T.solium (E44) Southern Ecuador (Loja) 13,387


T. saginata

Australia (AJ239106) T. saginata (E40) Southern Ecuador (Guayas) 0,85


Kenya (AM503345) T. saginata (E40) Southern Ecuador (Guayas) 0,207


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Figure 1. Phylogenetic tree with characters reweighed from the COI gene showing

the phylogenetic relationships of Taenia spp.

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Figure 2. Global Network haplotypes of Taenia solium for COI gene.

?

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Figure 3. Phylogenetic tree with NDI gene and characters reweighted showing the

phylogenetic relationships of Taenia spp.

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Figure 4. Global Network haplotypes of Taenia solium using data from NDI gene.

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• Taenia solium and Taenia saginata cause taeniasis/cysticercosis, a NTD in

Ecuador.

• Maximum Parsimony analyses in Taenia solium revealed greater geographic

structure.

• COI haplotype networks suggest two geographical events in the introduction of

T. solium in Ecuador.

• Two NDI geographical lineages in T. solium derivate from a common Indian

ancestor

molecular analyses reveal two geographic and genetic ... · hidalgo, r., molecular analyses reveal...

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