International Journal for Parasitology xxx (2015) xxxxxx

Contents lists available at ScienceDirect

International Journal for Parasitology

journal homepage: www.elsevier.com/locate/ijpara

Trypanosoma rangeli displays a clonal population structure, revealing a

subdivision of KP1() strains and the ancestry of the Amazonian group q
Thas Cristine Marques Sincero a,, Patricia Hermes Stoco b, Mrio Steindel b, Gustavo Adolfo Vallejo c,
Edmundo Carlos Grisard b,
a

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.

2. Materials and methods

2.1. Parasites and DNA extraction
All parasites and strains used in this study are shown in Table 1.
Except for T. rangeli strains C23, TRE and 5048, which were kindly
provided by Dr. Concepcin Puerta from Pontifcia Universidad Javeriana, Colombia, all other parasite strains were obtained from our
laboratory cryobank (Departamento de Microbiologia, Imunologia
e Parasitologia, Universidade Federal de Santa Catarina UFSC,
Brazil). Trypanosoma rangeli and T. cruzi strains were previously

subjected to cyclic mouse-triatomine-mouse passaging and were

re-isolated by hemoculture of infected mice in liver infusion tryptose (LIT) medium supplemented with 10% FCS at 27 C. Parasite
DNA was obtained by standard phenolchloroform extraction
(Sambrook and Russel, 2001). All primers used in this study are
described in Supplementary Table S1. All procedures involving animals were previously approved by the UFSC Ethics Committee on
Animal Use CEUA (Reference number: 23080.025618/2009-81)
and were carried out according to the guidelines of the Brazilian
National Authority (Colgio Brasileiro de Experimentao Animal
COBEA).
2.2. Identication and characterisation of MSs
2.2.1. MS identication from cDNA libraries
The transcriptomic library used in this study (2.45 Mb) was
formerly generated using cDNA from epimastigote and trypomastigote forms from both T. rangeli Choach and SC-58 strains, resulting in 2,230 and 2,127 sequences, respectively (Grisard et al.,
2010). The search for tandem repeats and the analysis of the satellite repeat contents was carried out using Tandem Repeats Finder
(TRF) (Benson, 1999) and the Tandem Repeats Analysis Program
(TRAP) (Sobreira et al., 2006), respectively.
2.2.2. MS identication from genomic libraries
Genomic libraries were constructed for this study according to
PIMA methodology (PCR-based isolation of microsatellite arrays)
(Lunt et al., 1999). Briey, RAPD proles were obtained using
10 ng of DNA from each strain and the Ready-To-Go RAPD Analysis
Beads (GE Healthcare, UK), according to the manufacturers
instructions. Amplication was performed in a Mastercycler Gradient thermocycler (Eppendorf, Germany) under the following
conditions: initial denaturation at 95 C for 5 min, followed by 45
cycles of denaturation at 95 C for 1 min, primer annealing at
36 C for 1 min, and extension at 72 C for 2 min. All PCR products
were cloned into the pGEM T easy vector (Promega, USA), resulting in approximately 4050 positive clones/strain. The detection of
MS repeats in each obtained clone was carried out by multiplex

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

NA, not applicable; ND, not determined.

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

PCR using primers specic to the cloning vector (EXCEL-R and

PGEM-F, Supplementary Table S1) and an additional primer directed to CA motif repeats, as described for T. cruzi (Oliveira et al.,
1998). The 10 lL reactions comprised 1 U of Taq DNA polymerase
(LGC Biotechnology, Brazil), 0.2 mM of each dNTP (GE Healthcare),
10 pmol of each primer in a buffer containing 10 mM TrisHCl (pH
8.5), 50 mM KCl and 1.5 mM MgCl2. The amplication was performed in a Mastercycler Gradient Thermocycler with an initial
denaturation at 94 C for 3 min, followed by 35 cycles at 94 C
for 45 s, primer annealing at 55 C for 30 s, and extension at
72 C for 1 min followed by nal extension at 72 C for 10 min.
DNA sequencing of both strands from all positive clones was carried out using the DYEnamic ET Dye Terminator kit (GE Healthcare) on a MegaBACE 1000 DNA Analysis System (GE
Healthcare). Briey, each sequencing reaction used 5 pmol of each
oligonucleotide, pGEM-F or EXCEL-R, and 800 ng of plasmid DNA
under the following thermal conditions: 95 C for 25 s, 35 cycles
of 95 C for 15 s, 50 C for 20 s, and 60 C for 90 s. After the labelling
reaction, the products were precipitated in 70% isopropanol,
injected at 2 kV for 100 s, and electrophoresed for 140 min at
8 kV. The resulting contigs were then analysed using the TRAP
and TRF software, as described in Section 2.4.2.

2.2.3. Selection and genotyping of MS loci and phylogenetic inferences

Following size (P24 bp) and quality assessment (Phred >20)
and the TRF and TRAP analyses, high-quality sequences were
selected according to the criteria described by Subirana and
Messeguer (2008), whereby a given sequence of 24 bp has a very
low chance of occurring randomly (105) and is unlikely to generate a coding sequence (Subirana and Messeguer, 2008). Primers for
each selected MS locus were designed based on the obtained
sequences, and the sense oligonucleotide of each primer pair was
50 -labelled with uorescein (FAM) (Supplementary Table S1).
These dened MS loci were then used in genotyping assays of
all T. rangeli strains and the control T. cruzi strain (Table 1). PCR
was performed using the GoTaq Green Master Mix (Promega)
in a nal volume of 12.5 lL containing GoTaq DNA polymerase,
1 lM of each primer, 50 ng of template DNA in a buffer containing
200 lM dNTPs and 1.5 mM MgCl2 (pH 8.5). Amplication was performed in a Mastercycler Gradient Thermocycler with an initial
denaturation at 94 C for 3 min, followed by 35 cycles at 94 C
for 45 s, primer annealing at 55 C for 30 s, extension at 72 C for
1 min, and a nal extension at 72 C for 10 min. The amplicons
were precipitated in 70% isopropanol and eluted in 10 lL of ultrapure water. The genotyping reaction was set up using 7.75 lL of
0.1% Tween, 0.25 lL of ET-ROX 400 or 900 (GE Healthcare), and
2 lL of diluted PCR product (1:51:15 in water), as instructed by
the manufacturer. The samples were gently homogenised, denatured at 95 C for 1 min, and kept on ice until genotyping using a
MegaBace 1000 DNA Analysis System (GE Healthcare). The samples were injected for 80 s at 3 kV and electrophoresed for 80 min
at 10 kV. Determination of the allele size and quality, as well as the
genotypes of the strains for each marker, was performed by analysis of the obtained electropherograms using the Fragment Proler
software (GE Healthcare).
Phylogenetic inferences from the obtained MS data were based
on the Stepwise Mutation Model (SMM) (Kimura and Ohta, 1978).
The MS multilocus genotypes were analysed using the Factor, Mix
and Seqboot software of the Phylip package (v. 3.69) (Felsenstein,
2005). Briey, binary characters (0/1) generated by Factor were
loaded into Mix and analysed by the Wagner Parsimony Method,
allowing a Seqboot bootstrap (1,000 bootstraps) estimation of the
evolutionary relationships among the strains expressed as a Wagner network tree.

2.3. Population analysis

A number of metrics were used to test for population genetic
structure such as MS allelic frequencies, Neis unbiased measures
of genetic distance, Wrights xation index (FST) and heterozygosity were calculated with GENEPOP (V. 3.4) (http://genepop.curtin.
edu.au). An Analysis of Molecular Variance (AMOVA) and the probability of occurrence of subpopulations or genetic subdivisions
were carried out using ARLEQUIN (v. 3.1) (Excofer et al., 2005)
using the original haplotype denition with the following settings:
Deletion Weight = 1, Transition Weight = 1, Transversion
Weight = 1, Epsilon Value = 1e-07, Signicant digits for output = 5
and Allowed Level of Missing Data = 0.05.
2.4. SNP analysis of the SL gene
2.4.1. PCR, cloning and sequencing
Amplication of the SL gene for all strains was performed using
primers ME-L (50 -TTC GAA CCC ATA ACT TGT TTG GT-30 ) and ME-R
(50 -AGC CAT TGT TTC AAA GTA CTC AAT CAG AAA CTG-30 ), as
described by Fernandes et al. (1997), generating an amplicon of
approximately 900 bp. The reaction was performed using the Qiagen HotStar HiFidelity system, with HiFi buffer (TrisHCl, pH
8.7), 300 lM dNTP ultrapure, BSA, Triton X-100, and 1.5 mM
MgSO4), 1 lM of each primer, 50 ng of DNA and 1 U of HotStar
HiFidelity DNA polymerase (Qiagen, USA) in a total volume of
25 lL. The amplication was performed in a Mastercycler Gradient
Thermocycler (Eppendorf) under the following thermal conditions: denaturation at 95 C for 5 min, followed by ve cycles at
95 C for 20 s, primer annealing at 60 C for 30 s, and extension
at 72 C for 30 s, 30 cycles of 95 C for 20 s, annealing at 55 C for
30 s, and extension at 72 C for 30 s, and a nal extension step of
72 C for 5 min.
The expected bands were puried using GFX PCR DNA and a Gel
Band Purication Kit (GE Healthcare), cloned into the pGEM T
Easy vector (Promega), and transformed by electroporation in Escherichia coli DH5a cells. After transformation, recombinant clones
were obtained by selective growth on LuriaBertani (LB) medium
supplemented with 100 g/mL of ampicillin and checked for the
presence of the inserts with PCR using primers pGEM-F (50 -ACG
CCA AGC TAT TTA GGT GAC ACT ATA-30 ) and EXCEL-R (50 -GTT
GTA AAA CGA CGG CCA GTG AAT-30 ).
For sequencing, ve positive clones of each strain were grown in
LB medium supplemented with 100 lg/mL of ampicillin for 20 h at
37 C with shaking. Plasmid DNA was recovered by alkaline lysis
according to standard protocols (Sambrook and Russel, 2001). Both
DNA strands were sequenced for all clones as described in Section
2.2.2.
2.4.2. SL gene sequence analysis
After quality analysis using the Phred/Phrap/Consed package
(http://www.phrap.org) (Ewing and Green, 1998; Ewing et al.,
1998), high quality sequences (Phred P30) were checked for their
identity using a BLAST search (www.ncbi.nlm.nih.gov/BLAST) and
then aligned using ClustalW (Thompson et al., 1994). Phylogenetic
reconstructions using the SL gene sequences were obtained for all
T. rangeli strains using the bootstrapped Maximum Parsimony
(1,000 replicates) and Kimura 2 parameter (K2P) models in MEGA
software (v. 5) (Tamura et al., 2011).
2.4.3. Network analysis
A reconstruction of the phylogenetic relationship among T. rangeli strains was carried out as formerly described by Herrera et al.
(2013) for T. cruzi strains. For that, the intergenic region of 63 gene

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

sequences from the T. rangeli SL-IR, corresponding to 18 new

sequences obtained in this study and 45 non-redundant sequences
retrieved from GenBank, were comparatively analysed (Supplementary Table S2, Supplementary Fig. S1). The detailed characteristics of these strains, including their genetic lineages
(KP1+/KP1 or AE groups), are presented in Supplementary
Table S3 (Vallejo et al., 2002; Maia Da Silva et al., 2007, 2009;
Urrea et al., 2011).
Sequences ranging from 230 to 311 bp were aligned and edited
manually using BioEdit (v. 7.0.90) (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and MEGA (v. 5) (Tamura et al., 2011); the 5.8S
rRNA gene was removed, generating two distinct regions of SL-IR
that were separately analysed as proposed by Herrera et al.
(2013). An analysis of the rst region, named the SNP region, was
performed by deleting the beginning of the sequence alignment,
the entire MS region, and one site where two sequences presented
a position annotated S (C/G). After trimming, alignment of the
173 bp-long SNP-enriched fraction revealed 24 polymorphic sites,
of which 14 was parsimony informative. The analysis of the second
region, named the MS region, was carried out by selecting the sites
corresponding to the MS region (CA)n (TA)n (TG)n (TATG)n (TA)n
in the alignment. Because the T. rangeli SL gene is far more conserved than that in T. cruzi, gaps were retained to allow analysis
using the Single Mutation Model.
Haplotype reconstruction was performed using the algorithm
PHASE (Stephens et al., 2001, 2003) of DnaSP (v. 5) (Librado and
Rozas, 2009) using default parameters. The phylogenetic relationship was built using the median-joining (MJ) network method in
Network version 4.5.1.6 (Bandelt et al., 1999) (http://www.uxus-engineering.com). As recommended by Herrera et al. (2013),
the network approach is preferable to conventional phylogenetic
methods for intraspecic phylogeny. The network was obtained
after haplotype reconstruction using DnaSP. After star contraction
(pre-processing), with default parameters and epsilon 10, postprocessing was used to remove unnecessary median vectors and
links (cleaning procedure). Network diagrams of haplotypes were
manually arranged considering geographical and host origin, cycle
and subgroup classication (Supplementary Table S4).

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

Size range alleles (bp)

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

Oliveira et al. (1998)

Ho, observed heterozygosity; He, expected heterozygosity.

a
Sequence origin as genome (g) and/or transcriptome (t).
b
Trypanosoma cruzi strain.

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

these loci as MS markers was assessed by the PCR amplication of

each MS repeat after 70 passages of the studied strains in axenic
culture, revealing no size or sequence changes (data not shown).
3.2. Genotyping of T. rangeli strains
The 18 polymorphic MS markers were successfully amplied
from 22 of the 23 T. rangeli strains (except strain 5048) (Supplementary Table S5). It is noteworthy to mention that the amplication of a single allele for all markers in both SC-58 strain clones (1
and 11) could be the result of monosomic chromosomes. Trypanosoma rangeli is considered a diploid parasite as is also assumed for
other trypanosomatids such as T. cruzi, T. brucei, Leishmania major,
Leishmania amazonensis and L. braziliensis (Stoco et al., 2014).
Although the T. rangeli population structure was analysed while
assuming such a premise, we cannot rule out the possibility of
aneuploidy since this phenomenon has been described in these
phylogenetically related parasites, especially for the genus Leishmania (Gaunt et al., 2003; Minning et al., 2011; Rogers et al.,
2011; Mannaert et al., 2012). Complete data of the MS genotyping
are shown in Supplementary Table S5.
The number of alleles per studied locus is shown in Table 3. All
loci showed a variable number of alleles, ranging from two to 11.
Table 2 also shows the observed (Ho) and expected (He) heterozygosity obtained for each locus, conrming the expected deciency
in heterozygosity for organisms lacking sexual reproduction, such
as T. rangeli.
An analysis of the unrooted Wagner network based on the MS
genotyping of 18 T. rangeli polymorphic loci (Fig. 1) using the
SMM (Kimura and Ohta, 1978) supports the clustering of three
populations: (i) KP1(+) strains, (ii) Colombian KP1() strains, and
(iii) Brazilian KP1() strains.
3.3. Population structure of T. rangeli
The level of population subdivision was estimated using FST
(Wrights xation index) between population pairs. The likelihood
of this subdivision was evaluated using STRUCTURE software with
100,000 interactions of the Markov chain algorithm and consider-

ing a model of mixed ancestry (admixture model) and correlated

allelic frequencies. Assessment of the population structure of all
T. rangeli strains (Table 1) revealed an FST value of 0.30380
(P = 0.00293), indicating a moderate subdivision of this taxon
(Porter, 1990). A separate analysis of the genetic diversity of the
KP1() and KP1(+) strains revealed FST values of 0.4718 (
0.2652) and 0.2572 ( 0.1981), respectively, indicating an effective
isolation for KP1() strains and a moderate subdivision for KP1(+)
strains (Porter, 1990; Hey and Pinho, 2012). As expected for asexual and clonal organisms, the Ho values were lower than the He
(Table 2).
3.4. SNP analysis of the T. rangeli SL gene
Cloning and high quality sequencing (Phred quality P90) of the
SL gene was achieved for 18 of the 23 T. rangeli strains (except for
SC-74, SC-61, SC-68 and both SC-58 strain clones). A discrete size
polymorphism was observed among the strains, as formerly
reported (Grisard et al., 1999), varying from 904 bp for strain 5048
to 1,022 bp for the PIT10 strain. All obtained sequences are available
in GenBank under accession numbers KJ525754KJ525771. All parsimonious sites are shown in Supplementary Table S6.
A phylogram resulting from the Maximum Parsimony analysis
of the SL gene from the 18 T. rangeli strains is shown in Fig. 2. Using
almost the same strains, the clustering obtained by this method
corroborates the genotyping results shown in Fig. 1 and reinforces
the classical KP1(+)/KP1() mini-circle classication (Vallejo et al.,
1994, 2002, 2003, 2009).
A further assessment of these differences was carried out by
analysing the MS region of the SL-IR (Supplementary Fig. S1) of
the 18 T. rangeli strains cited above, aggregating 23 non-redundant
sequences described by Urrea et al. (2011) and 22 sequences
described by Maia da Silva et al. (2007, 2009) which were retrieved
from GenBank. The haplotype analysis of all 63 non-redundant
sequences is shown in Fig. 3. Despite the lack of resolution for
some branches, especially among KP1(+) strains, the tree topology
corroborates the results shown in Figs. 1 and 2, emphasising with
high bootstrap support the existence of a third group formed
exclusively by T. rangeli strains isolated from the Brazilian Amazon
region.

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.

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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.

other KP1(+) and KP1() strains, with a high bootstrap value

(Fig. 3). Because the Amazon region is considered the origin of
Rhodnius spp. (Hiraki et al., 1978) and the fact that T. rangeli evolved
together with its invertebrate hosts (Kimura and Ohta, 1978; Urrea
et al., 2011), it would be reasonable to speculate that this group represents the ancestral genotype of T. rangeli strains. This hypothesis

was further addressed through a network analysis for intraspecic

variability using two regions of SL-IR (SNP and MS regions), as
described in Section 2.4.3 (Table 3, Fig. 4, Supplementary Table S4).
The analysis of the MS region of SL-IR (Table 3, Supplementary
Table S4) shows a progressive increase in repeat number,
especially for the CA motif, starting with one and two for the

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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).

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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.

Amazonian group (G1, G2) and up to 12 for the Peruvian strains

(G18). The motifs TA and TATG were absent in the rst three genotypes, with the TG and nal TA dinucleotides starting to appear in
the KP1(+) strains (G3). The most heterogeneous group was formed
by KP1() strains (G4G18), revealing a variable number of
repeats. The SMM used to analyse all the data indicated that the
MS regions showing lower numbers of repeats could be considered
ancestral markers, thus indicating that T. rangeli strains evolved
from the Amazonian group to the KP1(+) and KP1() strains.
Fig. 4 shows the analysis of the SNP region (Haplotypes 123)
median network trees resolved from 63 sequences of T. rangeli
SL-IR, reinforcing the observations discussed above. The trunk of
the analysis is represented by N1, N2 and N3, partitioning haplotypes into three main branches: the Amazonian group, which is
more distant from the other strains; the KP1(+) strains isolated
from Robustus group triatomines, which are more similar to each
other; and the more diverse KP1() strains isolated from Colombiensis, Pallescens and Ecuadoriensis group triatomines. The data
also reveal no apparent geographical structure except for the Amazonian group. As expected there was no specic association of the
T. rangeli haplotypes with a given vertebrate host but a more visible association with the triatomine vectors (Urrea et al., 2011).
While the KP1(+) group is exclusively associated with the Robustus
genotype, the KP1() group is associated with the Pallescens, Ecuadoriensis and Colombiensis groups of triatomines (Fig. 4, Supplementary Tables S3, S4). The Amazonian haplotype (KP1?) was
not clustered along the established triatomine groups.
In conclusion, our results characterised MS markers found
within coding sequences of the T. rangeli genome, allowing an
understanding of the population structure of this parasite and sup-

porting the clonal structure of this species. Additionally, such

markers reinforce the robustness of the two major groups circulating in central/South America, KP1(+) and KP1() and their intrinsic
relationship with triatomine groups, highlighting variability within
the KP1() group composed of strains from southern Brazil and the
possibility of the ancestry of this taxon based on Amazonian
strains.
Taken together, the analysis of MS genotyping and SL sequences
suggests the following three T. rangeli groups: (i) the Amazonian
group, which is related to R. brethesi and some vertebrates; (ii)
the T. rangeli KP1() group, which is related to R. pallescens, R.
colombiensis, R. ecuadoriensis, Panstrongylus megistus and some vertebrates (although strains from Santa Catarina remain between
other KP1() and KP1(+) strains); and (iii) the T. rangeli KP1(+)
group, which is related to R. prolixus, R. robustus, R. neglectus and
some vertebrates (Vallejo et al., 2003; Urrea et al., 2011).
The analysis of the T. rangeli population structure reinforces its
use as a model organism to study the genomic organisation and
evolution of trypanosomatids. Also, the present study points out
the inuence of vector species on the clonality of these parasite
strains, which is not well understood for most of the vector-borne
human or animal trypanosomatid-related diseases.
Acknowledgments
We are grateful to Dr. LDP Rona for critical reading of the manuscript. TCMS and PHS were recipients of Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES, Brazil)
scholarships. This study was supported by grants from Conselho
Nacional de Desenvolvimento Cientco e Tecnolgico (CNPq,

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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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