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Universidade Federal de Santa Catarina (UFSC), Centro de Cincias da Sade (CCS), Departamento de Anlises Clnicas (ACL), Setor E, Bloco K, Florianpolis, SC 88.040-970, Brazil
Universidade Federal de Santa Catarina (UFSC), Centro de Cincias Biolgicas (CCB), Departamento de Microbiologia, Imunologia e Parasitologia (MIP), Setor F, Bloco A,
Florianpolis, SC 88.040-970, Brazil
c
Laboratorio de Investigaciones en Parasitologa Tropical, Universidad del Tolima, Altos de Santa Helena, A.A. 546, Ibagu, Colombia
b
a r t i c l e
i n f o
Article history:
Received 3 July 2014
Received in revised form 12 November 2014
Accepted 24 November 2014
Available online xxxx
Keywords:
Genetic polymorphism
Vectorparasite coevolution
Parasite ancestry
Intra-specic variability
Microsatellite and SNP analysis
a b s t r a c t
Assessment of the genetic variability and population structure of Trypanosoma rangeli, a non-pathogenic
American trypanosome, was carried out through microsatellite and single-nucleotide polymorphism
analyses. Two approaches were used for microsatellite typing: data mining in expressed sequence tag
/open reading frame expressed sequence tags libraries and PCR-based Isolation of Microsatellite Arrays
from genomic libraries. All microsatellites found were evaluated for their abundance, frequency and usefulness as markers. Genotyping of T. rangeli strains and clones was performed for 18 loci amplied by PCR
from expressed sequence tag/open reading frame expressed sequence tags libraries. The presence of single-nucleotide polymorphisms in the nuclear, multi-copy, spliced leader gene was assessed in 18 T. rangeli strains, and the results show that T. rangeli has a predominantly clonal population structure, allowing
a robust phylogenetic analysis. Microsatellite typing revealed a subdivision of the KP1() genetic group,
which may be inuenced by geographical location and/or by the co-evolution of parasite and vectors
occurring within the same geographical areas. The hypothesis of parasitevector co-evolution was corroborated by single-nucleotide polymorphism analysis of the spliced leader gene. Taken together, the
results suggest three T. rangeli groups: (i) the T. rangeli Amazonian group; (ii) the T. rangeli KP1() group;
and (iii) the T. rangeli KP1(+) group. The latter two groups possibly evolved from the Amazonian group to
produce KP1(+) and KP1() strains.
2015 The Authors. Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc. This is
an open access article under the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-sa/4.0/).
1. Introduction
Trypanosoma rangeli and Trypanosoma cruzi are kinetoplastid
protozoan parasites infecting humans and a variety of wild and
domestic mammals in South and Central America. Due to the
non-pathogenic nature of T. rangeli to its mammalian hosts, this
parasite is a unique and interesting biological model for addressing
hostparasite interactions, and the genome has been recently
unveiled (Snoeijer et al., 2004; Grisard et al., 2010; Stoco et al.,
2014). Furthermore, considering the wide and superimposed
geographical distribution and sharing of reservoirs, vectors and
q
Sequence data reported in this paper are available in GenBank under accession
numbers KJ525754KJ525771.
Corresponding author at: Universidade Federal de Santa Catarina, Cx. Postal
476, Florianpolis, SC 88.040-970, Brazil. Tel.: +55 (48) 3721 2955.
E-mail addresses: thais.sincero@ufsc.br (T.C.M. Sincero), edmundo.grisard@ufsc.
br (E.C. Grisard).
soluble antigens with T. cruzi, the etiological agent of Chagas disease, T. rangeli is of epidemiological and medical importance, often
leading to a misdiagnosis of South American trypanosomiasis and
resulting in social and economic losses (Stevens et al., 2001;
Guhl and Vallejo, 2003; Grisard et al., 2010).
Polymorphism among the T. rangeli strains isolated from mammals and triatomines from different geographical regions has been
demonstrated by molecular and biochemical methods. Two main
genetic lineages based on kinetoplast DNA (kDNA) mini-circle typing have been described: KP1(+) and KP1() (Vallejo et al., 2002,
2003, 2009). Comparative sequence analysis of the ssrDNA, internal transcribed spacer 1 (ITS-1) and spliced leader intergenic
regions (SL-IRs) noted increased variability within this taxon and
resulted in the proposal of the existence of ve lineages (AE)
(Maia da Silva et al., 2004, 2007, 2009).
The distinct molecular markers and methods used to assess
DNA polymorphisms must consider (i) the expected level of
variability, (ii) the rate of mutation, (iii) the mode of inheritance
http://dx.doi.org/10.1016/j.ijpara.2014.11.004
0020-7519/ 2015 The Authors. Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.
This is an open access article under the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-sa/4.0/).
Please cite this article in press as: Sincero, T.C.M., et al. Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1() strains
and the ancestry of the Amazonian group. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.11.004
T.C.M. Sincero et al. / International Journal for Parasitology xxx (2015) xxxxxx
(segregation during cell division), and (iv) the cost, time and need
for specialised equipment and practices. Based on the type of information generated by various loci, we may group these methods
into three categories as follows: dominant bi-allelic (e.g., random
amplied polymorphic DNA (RAPD), amplied fragment length
polymorphism (AFLP), bi-allelic co-dominant (e.g., single-nucleotide polymorphism (SNP), restriction fragment length polymorphism (RFLP), and multi-allelic co-dominant (e.g., microsatellites
(MSs) (Vignal et al., 2002).
Despite the description of several MS loci for T. cruzi (Oliveira
et al., 1998, 1999; Macedo et al., 2001; Kof et al., 2007; Njiru
et al., 2007; Lewis et al., 2009; Llewellyn et al., 2009; Venegas
et al., 2009; Simo et al., 2010), Trypanosoma brucei (Kanmogne
et al., 1997; Jamonneau et al., 2002, 2004; Truc et al., 2002; Kof
et al., 2007; Cabrine-Santos et al., 2010; McInnes et al., 2012),
and Trypanosoma evansi (Botero et al., 2010; McInnes et al.,
2012), the number of these markers described for trypanosomatids
is still small. Similarly, the number of SNP markers in kinetoplastida has revealed a few loci, but only data for T. cruzi are available at
dbSNP (http://www.ncbi.nlm.nih.gov/snp/) and TcSNP (http://
snps.tcruzi.org/) (Ackermann et al., 2009). Because the number of
both MS and SNP markers is relatively small for T. rangeli
(Grisard et al., 1999; Urrea et al., 2011), we searched for and used
MS and SNP markers to assess the genetic variability and population structure of T. rangeli strains isolated from distinct geographical regions, hosts and vectors.
Table 1
List of species, strains and clones of Trypanosoma rangeli and Trypanosoma cruzi used in this study including original host, geographical origin and classication based on the
absence () or presence (+) of KP1 mini-circles.
Species
Strain
Original host
Geographical origin
KP1 typing
T. rangeli
SC-58
SC-58 clone 1
SC-58 clone 11
SC-61
SC-68
SC-74
SC-75
SC-76
PIT-10
TRE
C23
5048
Choach
D3493
B450
H8GS
R1625
Macias
San Agustn
H9
H14
1545
Palma-2
Y
Echimys dasythrix
E. dasythrix
Panstrongylus megistus
P. megistus
P. megistus
P. megistus
P. megistus
ND
Aotus sp.
Homo sapiens
Rhodnius prolixus
R. prolixus
Rhodnius brethesi
Homo sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
R. prolixus
R. prolixus
H. sapiens
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Colombia
Colombia
Colombia
Colombia
Colombia
Brazil
Honduras
El Salvador
Venezuela
Colombia
Honduras
Honduras
Colombia
Venezuela
Brazil
+
+
+
+
+
+
+
+
+
+
+
NA
T. cruzi
Please cite this article in press as: Sincero, T.C.M., et al. Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1() strains
and the ancestry of the Amazonian group. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.11.004
T.C.M. Sincero et al. / International Journal for Parasitology xxx (2015) xxxxxx
Please cite this article in press as: Sincero, T.C.M., et al. Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1() strains
and the ancestry of the Amazonian group. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.11.004
T.C.M. Sincero et al. / International Journal for Parasitology xxx (2015) xxxxxx
3. Results
3.1. Search for MS repeats in T. rangeli genomic and cDNA libraries
The search for MS repeats on T. rangeli genomic and transcriptomic libraries by TRF revealed seven and 2,138 potential MS
markers, respectively, which after analysis by TRAP resulted in
two genome and 36 transcriptome-derived sequences. Among
these, a single genomic sequence (Tr_Di-05) and 18 cDNA
sequences (Table 2) were in accordance with formerly established
criteria (Subirana and Messeguer, 2008) and submitted to experimental validation. PCR amplication of these markers was
achieved for 16 out of the 18 pre-selected repeats (88.9%), including MCLE-03 previously described for T. cruzi (Oliveira et al., 1998).
A total of 2,127 contigs clustered from 2.45 Mb of sequence
from the T. rangeli transcriptome (Grisard et al., 2010) were used
for MS mining. The TRF analysis revealed 2,132 simple-sequence
repeats (SSRs) distributed in 418 non-redundant classes at a frequency of one repeat every 1.24 Kb. The ratio of various classes
of MSs (dimers to hexamers) is not equally distributed throughout
the sequences. Hexameric repeats were the most abundant class of
MSs, with an average of 37.2% of all loci, followed by trimers
(18.5%), pentamers (11.1%), dimers (10.4%), and tetramers (7.7%).
The TRAP analysis revealed that a majority of the 2,132 contigs
revealed by TRF were not suitable for MS typing due to either (i)
reduced size of the regions anking the repeat motifs did not allow
the design of PCR primers or (ii) the MS size was less than 24 bp in
length, which could have an increased chance of occurring randomly and thus did not represent a robust locus. The selected
markers found in both the SC-58 and Choach strains are shown
in Table 2 and reveal simple and composed repeats, perfect or
imperfect, formed by two to six nucleotides. The efciency of the
transcriptome mining approach is indicated by the successful
amplication of 18 of the 23 loci tested.
All the T. rangeli MS loci described in the present study were
revealed to be specic because attempts to amplify these loci from
T. cruzi (Y strain), Leishmania infantum and Leishmania braziliensis
were unsuccessful (data not shown). Furthermore, the stability of
Table 2
Characteristics of microsatellite markers identied and selected from Trypanosoma rangeli genomic and/or transcriptomic sequences from Choach and SC-58 strains.
Marker
Motif
No. of alleles
Ho
He
Strain
Sequence
Origina
Reference
TR_Di-01
TR_Di-03
TR_Di-04
TR_Di-05
TR_Di-06
TR_Di-07
TR_Di-09
TR_Tri-01
TR_Tri-01b
TR_Tetra-02b
TR_Hexa-01
TR_Hexa-01b
TR_Hexa-02
TR_Hexa-03
TR_Hexa-04
TR_Hexa-05
TR_Hexa-06
(CA)21
(CT)10(GT)10
(AC)14AG(AC)6
(AC)13
(AC)17
(GT)15
(CT)13
(GCC)10
(GCC)10
(TTTG)8
(TGTGCG)4
(TGTGCG)4
(CTCCTT)4
(TGTGCG)4
(ATCCGC)4
(CCTTTT)4
(ATAAAT)4
174247
313321
195241
366374
165183
266288
109129
221242
316364
108230
379396
334347
229260
307388
358376
359366
205235
11
4
9
3
3
7
7
3
4
5
3
3
3
7
4
2
3
0.5556
0.0000
0.8500
0.0000
0.0000
0.8500
0.6842
0.0000
0.0000
0.4118
0.0000
0.0000
0.0500
0.1500
0.6000
0.0667
0.2308
0.8222
0.7100
0.8282
0.6694
0.6205
0.8462
0.7838
0.2495
0.3256
0.7522
0.5947
0.6032
0.5141
0.7179
0.6564
0.0667
0.4400
Choach
Choach
Choach
Choach
Choach
Choach
SC-58
SC-58
SC-58
Choach
SC-58
SC-58
Choach
SC-58
Choach
Choach
Choach
t
t
t
g/t
t
t
t
t
t
t
t
t
t
t
t
t
t
CHEG204007B06.b
CHEG202001E11.b
CHEG203001A03.b
CHEG205001C03.b
CHEG205003A01.b
CHEG205005A10.b
SCEG216003B10.b
SCEG216009G07.b
SCEG216009G07.b
CHEG004010A02.b
SCEG212007E09.b
SCEG212007E09.b
CHEG201009E03.b
SCEG212007E09.b
CHEG204015E05.b
CHEG203003A12.b
CHEG204001A07.b
MCLE-03
(CT)4(GT)2CTAT(GT)15
408418
0.2000
0.5244
CL Brenerb
Please cite this article in press as: Sincero, T.C.M., et al. Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1() strains
and the ancestry of the Amazonian group. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.11.004
T.C.M. Sincero et al. / International Journal for Parasitology xxx (2015) xxxxxx
Table 3
Distribution of Trypanosoma rangeli strains according to genotypes identied by microsatellite (MS) region analysis of the spliced leader intergenic region (SL-IR), their KP1(+/)
lineage classication and the frequency of repeats per genotype.
T. rangeli strains
MS
Genotypes
No. of sequences
attributed
Repeats frequency
KP1(+)
KP1()
CA
TA
TG
TATG
TA
Legeri, 4176
AM80, AAA, AAB, Saimiri, Preguici
Choach, B450, D3493, H8GS, H14, H9, Macias, Palma2, San_Agustin, R1625, 1545, SC75, SC58, P19,
Duran, 444, 3123, Rp500, Rp528, D99, VE00, Lobita, AEI, ROma01, P21, VE9, VE3, ROR20, ROR62,
ROR85
Tra643
R.col, P53, 44, 43, 201
2715
55
LDG
RGB
SC76, PIT10
G5
401, 1141
TRE
PG
5048
C23
GAL 47, SO 29, SO 48
Peru1, Peru1A, Peru2
G1
G2
G3
2
6
29
0
0
0
1
2
3
0
0
0
0
0
1
0
0
0
0
0
2
G4
G5
G6
G7
G8
G9
G10
G11
G12
G13
G14
G15
G16
G17
G18
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
5
1
1
1
1
2
1
2
1
1
1
1
3
3
6
6
6
6
7
7
7
8
9
9
9
10
10
11
12
6
9
9
10
4
8
14a
5
4
5
13
4
4
7
9
1
9
9
9
1
1
1
1
1
1
1
1
1
1
2
1
3
3
3
5
3
8a
4
3
4
6
3
5
1
2
2
0
0
0
2
2
1a
2
2
2
2
2
2
2
2
Imperfect repeats.
Please cite this article in press as: Sincero, T.C.M., et al. Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1() strains
and the ancestry of the Amazonian group. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.11.004
T.C.M. Sincero et al. / International Journal for Parasitology xxx (2015) xxxxxx
4. Discussion
The search for SSRs in T. rangeli expressed sequence tag (EST)/
open reading frame expressed sequence tags (ORESTES) libraries
proved to be an efcient method for selecting MS markers, also
allowing the assessment of repetitive content within the coding
regions of the T. rangeli genome. Although less polymorphic than
the MSs found within non-coding regions (Li et al., 2004), the
repeats found in coding regions are abundant, easily detectable
by computational screening, with no bias concerning the types of
repetition (except A/T monomers), and allow the direct mapping
of genes representing the transcribed part of the genome.
The assessment of the MS polymorphism in T. rangeli, as dened
by the number of alleles per locus, revealed that 18 out of the 20
loci tested (Table 3) showed polymorphism that is comparable
with other MS genotyping of phylogenetically related organisms
such as T. cruzi and Leishmania spp. (Oliveira et al., 1999; Fakhar
et al., 2008), and a consequent reduction in Ho of the studied
strains.
Our studies of T. rangeli population structure were based on MS
genotyping. In general, the Ho values were lower than the He values
(Table 2), a result that is consistent with other MS-based studies of
L. infantum (Ferreira et al., 2012) and T. cruzi populations (Oliveira
et al., 1998, 1999).
Assessment of a given population structure, which varies
according to the species studied, may be inuenced by the numbers and types of markers used. For T. rangeli, as few as three
hypervariable MS loci (TR_Di-04, TR_Di-07, TR_Di-09) were sufcient to distinguish the populations according to the KP1(+) and
KP1() genetic groups.
FST is calculated as the variance that distinguishes a population
over the total variation present. Porter (1990) suggested the following: FST < 0.2 indicates a negligible population subdivision;
0.2 < FST < 0.3 indicates a moderate subdivision; FST values >0.3
are indicative of effective isolation. More recently, Hey and Pinho
(2012) suggested an FST value >0.35 to identify a distinct or isolated
population. With regard to the question of whether FST tends to
reect mostly gene ow or the time since population separation,
the studies compiled by Hey and Pinho (2012) suggest a fairly even
balance between the two.
Considering all T. rangeli strains in a single analysis, a moderate
subdivision is shown (FST = 0.30380 0.00203, P = 0.00293), in
accordance with the KP1() and KP1(+) lineages. However, considering strains from each group in a separate analysis, the KP1()
strains show a genetic diversity almost two times higher
(0.4718 0.2652) than the KP1(+) strains (0.2572 0.1981), indicating a further subdivision of the KP1() strains (FST > 0.35). These
data are consistent with those observed in the phylogenetic trees
(Figs. 1 and 2) and are in accordance with the geographical distribution of the strains, whereby the KP1() strains are grouped into
two branches: the strains isolated in Colombia and the strains isolated in Brazil. The KP1(+) population appears to have some degree
of subdivision but at a more subtle level (0.2 < FST > 0.3).
These results reinforce the classication of T. rangeli strains into
distinct genetic groups according to the presence/absence of the
KP1 kDNA mini-circle as well as the vectorparasite co-evolution
theory that proposes the isolation of vectors in nature, thus preventing gene ow between the KP1(+) and KP1() strains
(Steindel et al., 1994; Vallejo et al., 2007; Urrea et al., 2011).
The phylogenetic analyses (Fig. 1) suggest that the population
structure of T. rangeli can be partly explained by classication
based on the presence or absence of the KP1 mini-circle. However,
two highly signicant branches were observed among the KP1()
strains, one comprising strains from southern Brazil (PIT10, SC58, SC-61, SC-68, SC-74, SC-75, and SC-76) and another with strains
from Colombia (C23, 5048, and TRE), corroborating the results of a
possible population subdivision among KP1() strains as evidenced by FST discussed above.
Analyses of SNPs are accurate but require high quality
sequencing from well-characterised biological material. For this
study, we initially subjected all T. rangeli strains to a cyclical
mouse-triatomine-mouse passage to assure the biological characteristics of T. rangeli (anterior transmission). After amplication of
the SL gene using a high delity proof-reading Taq DNA polymerase, all high quality reads (Phred P30) were considered for clustering, which resulted in a majority of sequences with Phred
quality P90 (1 error/109 bases). MEGA 4.0 analysis revealed a
total of 77 parsimony informative sites based on the alignment
of the 18 sequences of the T. rangeli SL gene by ClustalW
(Supplementary Table S6).
A phylogram based on the SNPs found within the SL gene
allowed interspecic clustering, clearly separating T. rangeli from
T. cruzi and T. vivax (Fig. 2). Even with no resolution for intraspecic clustering, the T. rangeli KP1() strains isolated in Brazil
remained clustered closer to the Colombian strains and outside
the main KP1(+) clade of strains, as was also observed in former
studies using the same strains but distinct markers such as the Histone H2A intergenic region (Puerta et al., 2009) and PTP2 gene
(phosphotyrosine phosphatase) (Prestes et al., 2012).
In contrast to the SNP analysis of the SL-IR gene, the analysis of
MS repeats allowed a clear assembly of T. rangeli strains according
to the KP1(+) and KP1() genetic groups, with high bootstrap support (Fig. 1), as previously reported by other groups (Cuervo et al.,
2006; Suarez et al., 2007). These results indicate a recent divergence between the strains due to the high mutation rate of MS
markers (Wilson and Balding, 1998), in contrast to SNPs. The separate clustering of the Brazilian and Colombian KP1() strains also
reinforces the vectorparasite co-evolution theory and indicates
that the T. rangeli population structure should be more complex
than the classication according to the type of kDNA mini-circle,
as proposed by Maia da Silva et al. (2007, 2009). These authors suggest the existence of at least ve genetic groups for T. rangeli (AE).
Group A is composed of isolates from Colombia, Venezuela, Honduras and Brazil (Rondnia and Maraj Island), group B of Brazilian
isolates from Acre, Amazonas and Par States, and group C of
strains isolated in Panama, El Salvador and Colombia. Group D is
composed exclusively of strain SC-58 isolated from southern Brazil
and group E of isolates from bats (Platyrrhinus lineatus) from central Brazil.
Using RAPD and sequencing analyses of the SL-IR from 25 T.
rangeli strains, Urrea et al. (2011) recently reported results that
support a strict association of T. rangeli genotypes and Rhodnius
spp., suggesting a possible co-evolutionary association between
parasites and their vectors. The authors suggested associations
between T. rangeli KP1(+) strains and Rhodnius prolixus, Rhodnius
robustus and Rhodnius neglectus, and between T. rangeli KP1()
strains and Rhodnius pallescens, Rhodnius colombiensis and Rhodnius
ecuadoriensis.
Considering this vectorparasite clustering and aiming to assess
the evolutionary history of T. rangeli, we performed a comparative
analysis including the 18 sequences of the SL-IR generated in the
present study and formerly described sequences (Maia Da Silva
et al., 2007, 2009; Urrea et al., 2011) (Fig. 3). It is worth mentioning
that despite the lack of resolution for some branches, the clustering
of the strains was maintained and corroborated the analysis using
complete sequences (Fig. 2), showing the robustness of this
approach and reinforcing the KP1(+/) classication, which
appears to be more general than proposed by Maia da Silva et al.
(2007, 2009).
This analysis also revealed a distinct and interesting group composed of T. rangeli strains isolated in the Brazilian Amazon region
(related to Rhodnius brethesi), which clustered separately from all
Please cite this article in press as: Sincero, T.C.M., et al. Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1() strains
and the ancestry of the Amazonian group. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.11.004
T.C.M. Sincero et al. / International Journal for Parasitology xxx (2015) xxxxxx
Fig. 1. Unrooted Wagner network of Trypanosoma rangeli strains based on the genotyping of 18 polymorphic loci. Each microsatellite allele was considered one state from a
multistate character; the genetic distance between any two strains was estimated as the number of mutational steps necessary to transform one into the other. Numbers on
the nodes represent the bootstrap values based on 1,000 replications. Bootstrap values not shown were all below 600 and are not given individually. Bar indicates KP1 (+/)
classication.
Fig. 2. Phylogram resulting from maximum parsimony analysis (1,000 replicates) (Kimura 2 parameter model using MEGA software) of the spliced leader gene from
Trypanosoma rangeli strains. The tree was rooted using Trypanosoma cruzi (Tc CL, U57984.1) and Trypanosoma vivax (Tv Desowitz, AJ250749.1) sequences. Bootstrap values are
shown in each tree node.
Please cite this article in press as: Sincero, T.C.M., et al. Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1() strains
and the ancestry of the Amazonian group. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.11.004
T.C.M. Sincero et al. / International Journal for Parasitology xxx (2015) xxxxxx
Fig. 3. Phylogram obtained with spliced leader intergenic region sequences from Trypanosoma rangeli strains described in this study (denoted by Tr prex) and from all nonredundant sequences obtained in GenBank (Maia da Silva et al., 2007, 2009; Urrea et al., 2011). The two main branches separated the Robustus genotype from Pallescens II,
Colombiensis and Ecuadoriensis II genotypes. KP1 mini-circle classications are also indicated. The bootstrap values obtained by maximum likelihood analysis are shown on
tree nodes (1,000 replicates).
Please cite this article in press as: Sincero, T.C.M., et al. Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1() strains
and the ancestry of the Amazonian group. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.11.004
T.C.M. Sincero et al. / International Journal for Parasitology xxx (2015) xxxxxx
KP1(+)
G03
KP1(-)
G17
G09
G08, 11, 12, 13, 15
G04
G14, 16
G18
N1
G17
N2
N3
G10
Colombiensis genotype
G05, 06, 07
Ecuadoriensis II genotype
Pallescens II genotype
G01 e 02
Robustus genotype
Amazonian strains
Santa Catarina strains
KP1?
Fig. 4. Distribution of Trypanosoma rangeli strains according to the single nucleotide polymorphism region analysis of the spliced-leader intergenic region. Haplotype (H_1
H_23) median network trees are resolved from 63 sequences of T. rangeli. A network program was used for the reconstruction of the trees, applying the star contraction option
before construction with the median-joining option. A cleaning procedure was done with the post-processing option. Black circles are median vectors that are hypothesised
sequences not found in the sample and required to connect the existing haplotypes. The other circles are nodes corresponding to one haplotype or contracted haplotypes
found in the current data set; their size is proportional to the number of haplotypes that form each node. Sco is the result of a star contraction of haplotypes 3 and 4. The torso
of analysis is represented by N1, N2 and N3, and partitioned haplotypes into three main branches. The different colours represent triatomine genotypes described by Urrea
et al. (2011). Localisation of genotypes obtained from microsatellite region analysis (Table 3, G01G18) are also indicated.
Please cite this article in press as: Sincero, T.C.M., et al. Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1() strains
and the ancestry of the Amazonian group. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.11.004
10
T.C.M. Sincero et al. / International Journal for Parasitology xxx (2015) xxxxxx
Brazil), CAPES and Financiadora de Estudos e Projetos (FINEP, Brazil). The funders had no role in the study design, data generation
and analysis, decision to publish, or preparation of the manuscript.
GE Healthcare Brazil is acknowledged for technical support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ijpara.2014.11.
004.
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11
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and the ancestry of the Amazonian group. Int. J. Parasitol. (2015), http://dx.doi.org/10.1016/j.ijpara.2014.11.004