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ORIGINAL PAPER
Species discrimination and phylogenetic inference of 17
Chinese Leishmania isolates based on internal transcribed
spacer 1 (ITS1) sequences
Bin-Bin Yang &Xian-Guang Guo &Xiao-Su Hu &
Jian-Guo Zhang &Lin Liao &Da-Li Chen &
Jian-Ping Chen
Received: 24 April 2010 / Accepted: 18 June 2010 /Published online: 9 July 2010
#Springer-Verlag 2010
Abstract Leishmaniasis is a geographically widespread
disease, caused by protozoan flagellates of the genus
Leishmania. This disease still remains endemic in China,
especially in the west and northwest frontier regions. To
date, the phylogenetic relationships among Chinese Leish-
mania isolates are still unclear, and the possible taxonomic
diversity remains to be established. In this study, the ITS1–
5.8S fragments of ten isolates collected from different foci
in China were determined. To infer the phylogenetic
relationships among them, seven sequences of Chinese
Leishmania isolates retrieved from GenBank were also
included. Both parsimony and Bayesian analyses reveal an
unexpected but strongly supported clade comprising eight
newly determined isolates, which is sister to other members
of subgenus Leishmania. In combination with genetic
distance analysis, this provides evidence of the occurrence
of an undescribed species of Leishmania. Our results also
suggest that (1) the isolate IPHL/CN/77/XJ771 from Bachu
County, Xinjiang Uygur Autonomous Region is not
Leishmania infantum but Leishmania donovani; (2) the
status referring to an isolate MRHO/CN/88/KXG-2 from a
great gerbil in Karamay as Leishmania turanica, formerly
based on multilocus enzyme electrophoresis, is recognized;
(3) an earlier finding demonstrating the L. donovani identity
of isolate MHOM/CN/80/801 from Kashi city is corrobo-
rated; (4) the three isolates from eastern Jiashi County,
Xinjiang Uygur Autonomous Region, causing desert type
of zoonotic visceral leishmaniasis (see Wang et al., Para-
sitol Int (in press), 2010), belong to L. donovani instead of
L. infantum. In addition, the results of this study make an
important contribution to understanding the heterogeneity
and relationships of Chinese Leishmania isolates, further
indicating that the isolates from China may have had a
more complex evolutionary history than expected.
Introduction
Leishmaniasis is a vector-borne disease transmitted by sand
flies, caused by a protozoan flagellate of the genus
Leishmania. It is endemic in 88 countries on four continents
and occurs in at least four major forms: cutaneous, diffuse
cutaneous, mucocutaneous, and visceral (Desjeux 2004).
The current global estimate of 350 million people at risk of
leishmaniasis and of an incidence per year of 500,000 for
visceral leishmaniasis (VL) and 1.5 million for cutaneous
leishmaniasis (CL) belies higher burdens of disease in
endemic foci. In the current context of worldwide (re)
emergence and spreading of leishmaniasis, the relevance of
species identification further gains importance.
The authors wish it to be known that, in their opinion, the first two
authors, Bin-Bin Yang and Xian-Guang Guo, should be regarded as
joint first authors.
B.-B. Yang :X.-S. Hu :J.-G. Zhang :L. Liao :D.-L. Chen :
J.-P. Chen
Department of Parasitology,
West China School of Preclinical and Forensic Medicine,
Sichuan University,
Chengdu 610041 Sichuan, China
B.-B. Yang :X.-S. Hu :J.-G. Zhang :L. Liao :D.-L. Chen (*):
J.-P. Chen (*)
Animal Disease Prevention and Food Safety Key Laboratory
of Sichuan Province, Sichuan University,
Chengdu 610064 Sichuan, China
e-mail: cdl1978119@sina.com
e-mail: jpchen007@163.com
X.-G. Guo
Chengdu Institute of Biology, Chinese Academy of Sciences,
Chengdu 610041 Sichuan, China
Parasitol Res (2010) 107:1049–1065
DOI 10.1007/s00436-010-1969-9
As with many parasites, the taxonomy of the genus
Leishmania is very complex because species definitions
and boundaries are hard to define. The links between
clinical disease, geographic origin, and nomenclature of
Leishmania species have long been controversial and
confusing. It has been recognized that the gold standard
for taxonomy and strain typing of Leishmania based on
multilocus enzyme electrophoresis (MLEE, also known as
zymodeme typing) has several disadvantages: It requires
large culture volumes and specialized equipment, it is costly
and laborious, and it has relatively poor discriminatory
power (Kuhls et al. 2005; Bañuls et al. 2007). To overcome
these problems, DNA-based methods have been widely
used for Leishmania spp. identification and/or phylogenetic
reconstruction with a variety of targets such as protein-
coding genes, non-coding segments, microsatellites, and
restriction fragment length polymorphisms (e.g., Piarroux et
al. 1995; Noyes et al. 1997; El Tai et al. 2001; Ibrahim and
Barker 2001; Kuhls et al. 2005; Asato et al. 2009; Fraga et
al. 2010; Montalvo et al. 2010). Notably, Lukešet al. (2007)
went a long way to resolving the issues for the Leishmania
donovani complex. They proposed that this species complex
consists of only two species, L. donovani sensu stricto (with
the synonym Leishmania archibaldi) and L. infantum (with
the synonym Leishmania chagasi), by using a combination
of widely used DNA analysis techniques and further
demonstrated that geographic origin of a strain is a more
important predictor of genetic relatedness than the type of
disease caused (visceral versus cutaneous leishmaniasis).
Few isolates from China, however, were included in these
studies, and no work has yet focused on the phylogenetic
relationships among different isolates from different epi-
demic areas in China.
Leishmaniasis remains endemic in China, especially in the
west and northwest frontier regions. Both human VL and CL
occur in China, with most VL cases, rare CL cases (Guan et al.
1992a; Zheng et al. 2009;Wangetal.2010). According to
different geographical origin, infective agent, and clinical
evidences, epidemic foci of VL in China were classified into
three types, i.e., plain foci, hill foci, and desert foci (Lu et al.
1994). Different foci and types of leishmaniasis in China
have brought forth the difficulty of identifying the strains of
Leishmania, as the etiological pathogens cannot be distin-
guished easily on the basis of morphological characteristics.
On the basis of MLEE of six representative isolates from the
plain, mountainous, and desert regions, Xu et al. (1984)first
identified the causative agents responsible for VL as L.
donovani sensu lato and L. infantum.Xuetal.(1984)also
provided support for the specific status of Leishmania
gerbilli, a nonpathogenic parasite of the great gerbil
Rhombomys opimus in the desert areas of Gansu Province
and Xinjiang Uygur Autonomous Region in N.W. China
(Wang et al. 1964a,b). Interestingly, Xu et al. (1989)further
found that two isolates from kala azar patients in Kashi city
of Xinjiang could not be designated definitely as L. infantum
or L. donovani by isoenzyme electrophoresis. Subsequently,
on the basis of zymodeme typing, Guan et al. (1995)first
reported that L. turanica had been found in great gerbils
from Xiaoguai Farm in Karamay, Xinjiang Uygur Autono-
mous Region. Although in recent years considerable work
has been done to determine the heterogeneity of different
isolates from three types of foci in China by analyzing
kDNA and/or nDNA (e.g., Lu et al. 1994;Luetal.1997,
1998;Luetal.2001;Huetal.2002), the phylogenetic and
taxonomical relationships among isolates covering China are
still unclear. On the basis of kDNA and nDNA heterogene-
ity, Lu et al. (1994)classified19Leishmania isolates from
epidemiologically different foci in China into five genotypes
(groups I–V). They found that members of group II,
tentatively designated as L. infantum sensu lato, displayed
much heterogeneity in both kDNA and nDNAs. They further
inferred that the isolates in China were more heterogeneous
than previously thought, necessitating the reassignment of
some isolates into different groups. Such a perspective is
therefore still far from clearly promoting us to pursue further
studies of the molecular phylogenetics of Leishmania.
Sequence data of the ribosomal RNA (rRNA) gene, in
particular the two highly variable internal transcribed spacer
regions (ITS1 and ITS2), have been successfully used to
resolve taxonomic questions and to determine phylogenetic
affinities among closely related Leishmania species (e.g.,
Dávila and Memen 2000; Schönian et al. 2000; El Tai et al.
2001; Kuhls et al. 2005; Parvizi et al. 2008) and other
parasites (e.g., Chen et al. 2007; Lin et al. 2007; Al-Kandari
and Al-Bustan 2010). Thus, the starting point of our work is
to obtain a better understanding of the status of different
isolates from different epidemic areas in China by DNA-
based taxonomy. The ITS1–5.8S fragments were sequenced
to analyze the phylogenetic relationships of ten isolates
collected from different foci in China, in conjunction with
seven Chinese isolates retrieved from GenBank. The aims of
the present study are (1) to report a set of original ITS1–5.8S
sequences for ten Leishmania isolates from different foci in
China, (2) to determine the level of variation among ITS1–
5.8S sequences among them, and (3) to infer the phyloge-
netic relationships among isolates in China and explore the
taxonomic implications.
Materials and methods
DNA extraction, amplification, and sequencing protocols
The ten Leishmania isolates used in this study were listed in
Tab le 1. Promastigotes were cultivated in medium 199
supplemented with 15% heat-inactivated fetal bovine serum
1050 Parasitol Res (2010) 107:1049–1065
Table 1 List of Leishmania strains, origin, and database accession numbers, including sequences of L. donovani complex retrieved from
GenBank
Sequence type
(sequence
numbers)
Sequence
length
(bp)
GenBank
accession
numbers
MLEE-based
species
assignment
WHO code Origin Reference
–313 HM130599 n.d. MHOM/CN/?/GS6 Gansu, China This study
–313 HM130600 n.d. MCAN/CN/60/GS1 Gansu, China This study
–313 HM130601 n.d. MHOM/CN/90/SC10H2 Sichuan, China This study
–313 HM130602 n.d. MHOM/CN/?/GS5 Gansu, China This study
–313 HM130603 n.d. MHOM/CN/83/GS2 Gansu, China This study
–313 HM130604 n.d. MHOM/CN/84/SD1 Shandong, China This study
–311 HM130605 n.d. MHOM/CN/84/JS1 Jiangsu, China This study
–313 HM130606 n.d. MCAN/CN/?/SC11 Sichuan, China This study
–320 HM130607 L. turinica
a
MRHO/CN/88/KXG-2 Karamay, China This study
H1 (6) 297 AM901450 n.d. MHOM/IN/1961/L13 India Alam et al. 2009a
AJ634360 L. donovani MHOM/ET/00/HUSSEN Ethiopia Kuhls et al. 2005
AJ634359 L. archibaldi
b
MHOM/SD/97/LEM3463 Sudan Kuhls et al. 2005
AJ634358 L. archibaldi
b
MHOM/SD/97/LEM3429 Sudan Kuhls et al. 2005
AJ634357 L. archibaldi
b
MHOM/SD/93/GE Sudan Kuhls et al. 2005
AJ634356 L. donovani MCAN/SD/00/LEM3946 Sudan Kuhls et al. 2005
H2 (5) 297 HM130608 n.d. IPHL/CN/77/XJ771 Bachu, China This study
GQ367487 n.d. MHOM/CN/08/JIASHI-2 Jiashi, China Wang et al. 2010
GQ367488 n.d. MHOM/CN/08/JIASHI-5 Jiashi, China Wang et al. 2010
GQ367486 n.d. MHOM/CN/08/JIASHI-1 Jiashi, China Wang et al. 2010
AJ000294 L. donovani MHOM/CN/00/Wangjie1 China Kuhls et al. 2005
H3 (31) 298 GQ444144 L. infantum MHOM/IR/04/IPI-UN10 Iran Mahmoudzadeh-Niknam
et al. (unpublished data)
FM164420 L. infantum MHOM/UZ/2007/KU Uzbekistan Alam et al. 2009b
FM164419 L. infantum MHOM/UZ/2007/OBA Uzbekistan Alam et al. 2009b
FM164418 L. infantum MHOM/UZ/2007/ERD Uzbekistan Alam et al. 2009b
FM164417 L. infantum MHOM/UZ/2007/KOM Uzbekistan Alam et al. 2009b
FM164416 L. infantum MHOM/UZ/2007/MUA Uzbekistan Alam et al. 2009b
EU326227 –MHOM/BR/74/PP75 Brazil Thakur et al.
(unpublished data)
AM502245 L. infantum MCAN/ES/98/LLM-877 Spain Peacock et al. 2007
AJ634355 L. infantum MCAN/ES/86/LEM935 Spain Kuhls et al. 2005
AJ634354 L. infantum MHOM/IT/93/ISS800 Italy Kuhls et al. 2005
AJ634353 L. infantum MHOM/IT/94/ISS1036 Italy Kuhls et al. 2005
AJ634352 L. infantum MHOM/ES/92/LLM373 Spain Kuhls et al. 2005
AJ634351 L. infantum MHOM/FR/80/LEM189 France Kuhls et al. 2005
AJ634350 L. infantum MHOM/MT/85/BUCK Malta Kuhls et al. 2005
AJ634349 L. infantum MHOM/ES/91/LEM2298 Spain Kuhls et al. 2005
AJ634348 L. infantum MHOM/FR/96/LEM3249 France Kuhls et al. 2005
AJ634347 L. infantum MHOM/ES/88/LLM175 Spain Kuhls et al. 2005
AJ634346 L. infantum MCAN/FR/87/RM1 France Kuhls et al. 2005
AJ634345 L. infantum MHOM/CN/54/Peking Shannxi, China Kuhls et al. 2005
AJ634344 L. infantum MHOM/PT/00/IMT260 Portugal Kuhls et al. 2005
AJ634343 L. infantum MHOM/ES/86/BCN16 Spain Kuhls et al. 2005
AJ634342 L. infantum MHOM/FR/97/LSL29 France Kuhls et al. 2005
AJ634341 L. infantum MHOM/ES/93/PM1 Spain Kuhls et al. 2005
AJ634340 L. infantum MHOM/FR/95/LPN114 France Kuhls et al. 2005
AJ634339 L. infantum MHOM/FR/78/LEM75 France Kuhls et al. 2005
Parasitol Res (2010) 107:1049–1065 1051
Table 1 (continued)
Sequence type
(sequence
numbers)
Sequence
length
(bp)
GenBank
accession
numbers
MLEE-based
species
assignment
WHO code Origin Reference
AJ000306 L. chagasi
c
MHOM/BR/85/M9702 Brazil Kuhls et al. 2005
AJ000304 L. chagasi
c
MHOM/BR/74/PP75 Brazil Kuhls et al. 2005
AJ000303 L. infantum MHOM/CN/78/D2 Xinjiang, China Kuhls et al. 2005
AJ000295 –MHOM/ES/87/Lombardi Spain Schöenian et al.
(unpublished data)
AJ000289 L. infantum MHOM/TN/80/IPT1 Tunisia Kuhls et al. 2005
AJ000288 L. infantum MHOM/FR/62/LRC-L47 France Kuhls et al. 2005
H4 (1) 304 AM901452 L. donovani MHOM/IQ/1981/SUKKAR2 Iraq Alam et al. 2009a,2009b
H5 (1) 299 AJ276260 L. donovani –Sudan El Tai et al. 2001
H6 (21) 300 GQ367489 n.d. MHOM/CN/80/801 Kashi, China Wang et al. 2010
AM901448 n.d. MHOM/LK/2002/L60b Sri Lanka Alam et al. 2009a
AM901447 n.d. MHOM/LK/2002/L60c Sri Lanka Alam et al. 2009a
EU753232 –NICD/IN/30/A India Thakur (unpublished data)
EU753231 –NICD/IN/28/A India Thakur (unpublished data)
EU753230 –NICD/IN/24/A India Thakur (unpublished data)
EU753229 –NICD/IN/23/A India Thakur (unpublished data)
EU753228 –NICD/IN/19/A India Thakur (unpublished data)
AJ634378 n.d. MHOM/IN/01/BHU20140 India Kuhls et al. 2005
AJ634377 L. donovani MHOM/IN/96/THAK35 India Kuhls et al. 2005
AJ634376 L. donovani MHOM/IN/00/DEVI India Kuhls et al. 2005
AJ634375 L. donovani MHOM/IN/54/SC23 India Kuhls et al. 2005
AJ634374 L. donovani MHOM/KE/83/NLB189 Kenya Kuhls et al. 2005
AJ000297 L. donovani MHOM/KE/85/NLB323 Kenya Kuhls et al. 2005
AJ000296 n.d. MHOM/KE/84/NLB218 Kenya Kuhls et al. 2005
AJ000292 L. donovani MHOM/IN/80/DD8 India Kuhls et al. 2005
AJ000290 n.d. MHOM/IN/71/LRC-L51a India Kuhls et al. 2005
H7 (5) 301 AJ634377 L. donovani MHOM/IN/96/THAK35 India Kuhls et al. 2005
AJ634373 L. donovani MHOM/ET/67/HU3 Ethiopia Kuhls et al. 2005
AJ634372 L. donovani MHOM/SD/93/9S Sudan Kuhls et al. 2005
AJ634371 L. infantum
d
MHOM/SD/93/452BM Sudan Kuhls et al. 2005
AJ634370 L. infantum MHOM/SD/97/LEM3472 Sudan Kuhls et al. 2005
H8 (1) 300 AJ249616 L. donovani –Sudan El Tai et al. 2000
H9 (2) 301 AJ249615 L. donovani –Sudan El Tai et al. 2000
AJ276259 L. donovani –Sudan El Tai et al. 2001
H10 (1) 303 AM901449 L. donovani MHOM/IN/1983/CHANDIGARH India Alam et al. 2009a
H11 (1) 298 AJ249621 L. donovani –Sudan El Tai et al. 2000
H12 (14) 303 AJ276258 L. donovani –Sudan El Tai et al. 2001
AM901453 L. donovani MCAN/MA/2002/AD3 Morocco Alam et al. 2009a
AJ634369 L. infantum
d
MHOM/SD/82/GILANI Sudan Kuhls et al. 2005
AJ634368 L. donovani MHOM/SD/93/338 Sudan Kuhls et al. 2005
AJ634367 L. archibaldi
b
MHOM/ET/72/GEBRE1 Ethiopia Kuhls et al. 2005
AJ634366 L. archibaldi
b
MHOM/SD/93/35-band Sudan Kuhls et al. 2005
AJ634365 n.d. MHOM/SD/62/LRC-L61 Sudan Kuhls et al. 2005
AJ634364 L. infantum
d
MHOM/SD/93/597-2 Sudan Kuhls et al. 2005
AJ634363 L. infantum
d
MHOM/SD/93/762L Sudan Kuhls et al. 2005
AJ634362 L. infantum
d
MHOM/SD/93/45-UMK Sudan Kuhls et al. 2005
AJ634361 L. infantum MHOM/SD/62/3S Sudan Kuhls et al. 2005
AJ249612 L. donovani –Sudan El Tai et al. 2000
1052 Parasitol Res (2010) 107:1049–1065
at 25°C. Approximately 1–5×10
9
promastigotes were collect-
ed at room temperature by centrifugation at 4,000 rpm for
10 min and washed with distilled water. Total genomic DNA
was extracted from the promastigotes using a standard sodium
dodecyl sulfate-proteinase K procedure, as described by
Sambrook and Russell (2001). The primers of LITSR (5′-
CTGGATCATTTTCCGATG-3′) and L5.8S (5′-TGATAC-
CACTTATCGCACTT-3′; El Tai et al. 2000)wereusedto
amplify ITS1–5.8S segments. The PCR protocols were 94°C
for 3 min followed by 30 cycles of 94°C for 30 s, 49°C for
30 s, 72°C for 1 min, and then a final elongation step at 72°C
for 10 min. The PCR products were purified on a 2.0%
agarose gel stained with ethidium bromide, using a commer-
cial DNA purification kit following the manufacturer’s
protocol. Sequencing was performed using the same PCR
primers with ABI Big Dye Terminator chemistry on an ABI
3730 automated sequencer. The sequences have been depos-
ited in GenBank under accession numbers HM130599–
HM130608 (Table 1).
Sequence alignment and analyses
A set of ITS1–5.8S sequences of Leishmania were retrieved
from GenBank, including nine species of subgenus Leish-
mania and two species of subgenus Viannia (see Tables 1
and 2). The sequences were first aligned using Clustal X
1.83 (Thompson et al. 1997) with a gap-opening penalty of
5 and gap-extension penalty of 1, following the recom-
mendation (use of small gap costs) of Hickson et al. (2000).
The aligned matrix from this procedure was checked by
eye, and minor adjustments were made manually with
SeaView v.4.2.5 (Gouy et al. 2010). The data matrices are
available from the corresponding author.
Compositional heterogeneity was evaluated using Chi-
square (χ
2
) tests implemented in PAUP* 4.0b10 (Swofford
2002) and assessed using the software SeqVis v.1.3 (Ho et
al. 2006) to visualize and to conduct matched-pairs tests of
symmetry (Ababneh et al. 2006). Evidence of evolution
under conditions more complex than that assumed by
commonly applied models (i.e., stationary, reversible, and
homogeneous conditions) was inferred if the scatter of dots
in the tetrahedral plots was widely dispersed and if x%of
the matched-pairs tests of symmetry produced pvalues
greater than or equal to x; this procedure is consistent with
that advocated by Jermiin et al. (2008). Substitution
saturation was tested by inspecting a new entropy-based
index as implemented in DAMBE (Xia and Xie 2001). For
this approach, if I
ss
(i.e., index of substitution saturation) is
not smaller than I
ss.c
(i.e., critical I
ss
), then we can conclude
that the sequences have experienced severe substitution
saturation (Xia et al. 2003; Xia and Lemey 2009). The K80
+G distance matrices (Kimura, 1980) were computed with
MEGA v. 4.1 (Tamura et al. 2007), with the gamma shape
of 0.5780.
Phylogenetic analyses
Phylogenetic hypotheses of Leishmania were generated with
ITS1–5.8S rRNA segments using two types of commonly
applied phylogenetic method: heuristic searches using equally
weighted maximum parsimony (MP) analyses performed with
the program PAUP* and Bayesian inference (BI) with the
program MrBayes v.3.2 (Ronquist and Huelsenbeck 2003). In
both MP and BI analyses, gaps were treated as missing data.
For heuristic searches under parsimony, invariant char-
acters were removed from the dataset, and all remaining
characters were treated as equally weighted. Each search
involved ten random addition replicates, one tree held at
each step, TBR branch swapping, steepest descent on, and a
maximum of 10,000 saved trees; all other search settings
were left at default values. Non-parametric bootstrapping
was used to generate phylogeny confidence values
(Felsensten 1985), with 1,000 pseudoreplicates using a
heuristic tree search for each pseudoreplicate. Leishmania
panamensis (FJ948422) was used to root the trees. Because
intraspecific gene evolution cannot always be represented
Table 1 (continued)
Sequence type
(sequence
numbers)
Sequence
length
(bp)
GenBank
accession
numbers
MLEE-based
species
assignment
WHO code Origin Reference
AJ000293 n.d. MHOM/SD/68/1S Sudan Kuhls et al. 2005
AJ000291 n.d. MHOM/SD/75/LV139 Sudan Kuhls et al. 2005
H13 (1) 301 AJ249614 L. donovani –Sudan El Tai et al. 2000
a
Named previously on the basis of zymodeme analysis (Guan et al. 1995)
b
As synonym of L. donovani according to Lukešet al. (2007)
c
As synonym of L. infantum according to Lukešet al. (2007)
d
Identified as L. infantum according to the zymodeme, MON30, whereas recent analyses have shown that it is L. donovani (Jamjoom et al. 2004;
Zemanova et al. 2004)
Parasitol Res (2010) 107:1049–1065 1053
Table 2 List of the other strains, origin, and database accession numbers retrieved from GenBank
Species GenBank accession
number
WHO code Origin Sequence
length (bp)
Reference
L. mexicana AF466383 MNYC/BZ/62/M379 Brazil 320 Berzunza-Cruz et al. 2002
FJ948436 ––320 de Almeida et al. (unpublished data)
FJ948433 ––321 de Almeida et al. (unpublished data)
FJ948435 ––318 de Almeida et al. (unpublished data)
L. amazonensis AF339753 ––314 Berzunza-Cruz et al. 2002
DQ182536 MHOM/BR/73/M2269 Brazil 315 Rotureau et al. 2006
FJ753371 ––316 de Almeida et al. (unpublished data)
L. tropica FJ948459 –India 305 de Almeida et al. (unpublished data)
FJ948461 –India 306 de Almeida et al. (unpublished data)
FJ948460 –India 305 de Almeida et al. (unpublished data)
FJ948464 –India 304 de Almeida et al. (unpublished data)
FJ948465 –India 301 de Almeida et al. (unpublished data)
HM004586 –Isfahan 309 Mahmoudzadeh-Niknam
(unpublished data)
AJ000302 IROS/NA/76/ROSSI-II –303 Schöenian et al. (unpublished data)
AJ300485 MHOM/TN/88/TAT3 Tunisia 308 Schöenian et al. (unpublished data)
AJ000301 MHOM/KE/84/NLB297 Kenya 306 Schöenian et al. (unpublished data)
FJ948452 ––310 de Almeida et al. (unpublished data)
FJ948456 ––310 de Almeida et al. (unpublished data)
FJ948451 ––313 de Almeida et al. (unpublished data)
GQ913688 MHOM/AF/88/KK27 Afghanistan 303 de Almeida et al. (unpublished data)
FJ948458 –India 302 de Almeida et al. (unpublished data)
FJ460459 MHOM/EG/06/RTC-67 India 305 Shehata et al. 2009
FJ948457 –India 301 de Almeida et al. (unpublished data)
L. major AY260965 MHOM/Ir/02/PIICC1 Iran 320 Tashakori et al. (unpublished data)
AJ300482 MTAT/KE/??/NLB089A Kenya 320 Schöenian et al. (unpublished data)
DQ295824 IPAP/EG/89/SI-177 –322 Fryauff et al. 2006
FJ753395 ––319 Schöenian et al. (unpublished data)
AJ272383 –Turkmenistan 319 Chendrik et al. (unpublished data)
AJ300481 MHOM/SD/90/SUDAN3 Sudan 320 Schöenian et al. (unpublished data)
GQ471900 MRHO/IR/75/ER Iran 322 Mahmoudzadeh-Niknam et al. (unpublished data)
L. turanica
a
EF413079 –Iran 320 Parvizi and Ready 2008
AJ272378 –Uzbekistan 320 Chendrik et al. (unpublished data)
AJ272379 –Turkmenistan 320 Chendrik et al. (unpublished data)
AJ272380 –Turkmenistan 320 Chendrik et al. (unpublished data)
AJ272381 –Turkmenistan 320 Chendrik et al. (unpublished data)
AJ272382 –Kazakhstan 320 Chendrik et al. (unpublished data)
L. gerbilli AJ300486 –Uzbekistan 319 Schöenian et al. (unpublished data)
L. aethiopica GQ920677 ––301 de Almeida et al. (unpublished data)
GQ920674 ––321 de Almeida et al. (unpublished data)
GQ920673 ––322 de Almeida et al. (unpublished data)
GQ920675 ––324 de Almeida et al. (unpublished data)
L. panamensis FJ948442 ––286 de Almeida et al. (unpublished data)
L. braziliensis DQ182537 MHOM/BR/84/LTB300 Brazil 279 Rotureau et al. 2006
a
The six strains share a common allele of ITS1–5.8S with the isolate MRHO/CN/88/KXG-2, as shown in Table 1
1054 Parasitol Res (2010) 107:1049–1065
by a bifurcating tree, haplotype networks may more
effectively portray the relationships among haplotypes
within species (reviewed by Posada and Crandal (2001)).
Therefore, we constructed unrooted parsimony networks of
haplotypes for L. donovani complex and Leishmania sp.
(see below) using TCS v.1.21 (Clement et al. 2000), with
gap treated as a fifth state.
Prior to Bayesian analyses, the best-fit model of evolution,
K80 + G, was selectedusing jModeltest v. 0.1.1 (Posda, 2008)
under the Bayesian information criterion (Schwarz 1978),
following recent recommendations (Posada and Buckley
2004). We estimated posterior probability distributions by
allowing four incrementally heated Markov chains (default
heating values) to proceed for four million generations, with
samples taken every 200 generations. Analyses were
repeated beginning with different starting trees to ensure
that our analyses were not restricted from the global
optimum (Huelsenbeck et al. 2002). Convergence was first
tested by examining the average deviation of the split
frequencies of the two runs, in order to determine whether
the two runs had converged. MCMC convergence was also
explored by examining the potential scale reduction factor
(PSRF) convergence diagnostics for all parameters in the
model (provided by the sump and sumt commands) and
graphically using the cumulative, compare, and absolute
difference options of the program AWTY (Nylander et al.
2008). The first one million generations, before this chain
reached apparent stationarity, were discarded, and the
remaining samples from the independent runs were pooled
to obtain the final approximation of the posterior distribution
of trees. To yield a single hypothesis of phylogeny, the
posterior distribution was summarized as a 50% majority-
rule consensus.
In addition, as gap (or “indel”) characters have been widely
recognized as a valuable source of data for phylogenetic
inference across the tree of life (e.g., Dessimoz and Gil 2010),
phylogenetic information from indel events of ITS1 was also
included in MP and BI by coding indel events into a separate
data matrix with the program SeqState (Müller 2005)using
the simple indel coding method (Simmons and Ochoterena
2000). In the latter, all indels are scored as binary characters
regardless of their length. In BI, a discrete model employing
identical rates of forward and backward transitions (Lewis
2001) was applied to the indel matrix.
Bayesian hypothesis testing
We used Bayes factors to compare our preferred Bayesian tree
topology (see below) to Bayesian trees with constraint. This
method differs from traditional hypothesis testing because it
does not offer a criterion for absolute rejection of a null
hypothesis but instead an evaluation of the evidence in favor
of the null hypothesis (Kass and Raftery 1995). The
phylogeny inferred from the ITS1–5.8S data set was
constrained to alternative hypotheses. Constraint analyses
were conducted in MrBayes v.3.2 using the command prset
topologypr=constraint. All analyses consisted of two
simultaneous runs each with an abbreviated three MCMC
chains run for four million generations or more (as
necessary). The Bayes factor was determined by calculating
the marginal likelihood for both unconstrained and constraint
analyses using Tracer v.1.5 (Rambaut and Drummond 2009).
The difference in these ln-transformed marginal likelihoods
was compared to the table provided by Jeffreys (1935,1961)
and further modified by Raftery (1996). Based on these
tables, we consider a 2ln Bayes factor ≥10 as significant
evidence for a hypothesis (Kass and Raftery 1995).
Results
Base composition and nucleotide substitution patterns
The newly determined ITS1–5.8S fragments ranged in size
from 297 bp for isolate IPHL/CN/77/XJ771, 311 bp for
isolate MHOM/CN/84/JS1, and 313 bp for the remaining
isolates with exception of isolate MRHO/CN/88/KXG-2
(320 bp). Specifically, we found that the isolate MRHO/
CN/88/KXG-2 shared the same ITS1 sequence with six
strains of Leishmania turanica retrieved from GenBank, as
listed in Table 2. The 5.8S rRNA segment was 69 bp in
length. The alignment of the Leishmania taxa required
accommodation of 85–112 gaps in the ITS1 region per
sequence. Indels (insertion/deletion events) represented
between 20.7% and 27.4% of the aligned sequence length.
Most indels were 3–4 bp in length, and the maximum indel
length was 19 bp. Of the 409 aligned characters, 111 were
GG
CC
TT
AA
Fig. 1 Tetrahedral plots for ITS1–5.8S dataset, which were obtained
using the Select Sites command from the View menu in the program
SeqVis (Ho et al. 2006)
Parasitol Res (2010) 107:1049–1065 1055
variable, with 89 parsimony-informative. Percentage base
compositions were A, 31.76; C, 20.01; G, 22.54; T, 25.69.
The average maximum likelihood estimated Ti/Tv ratio
was 1.45.
A base stationarity test showed insignificant differences
among taxa in base composition bias in the data (χ
2
=22.34,
df=171, p=1.00). Figure 1presented the tetrahedral plot
from the ITS1–5.8S rRNA. Clearly, there was no conspic-
uous compositional heterogeneity in the alignment. The
implication of this plot was that these sites were likely to
have evolved under the same stationary, reversible, and
homogeneous conditions. To corroborate whether this was
the case, the matched-pairs test of symmetry was used in
conjunction with the alignment. Table 3summarized the
distribution of pvalues. The distribution of pvalues clearly
showed that the evolutionary process was likely to have
been stationary, reversible, and homogeneous, implying
that it would be wise to analyze this data using a
phylogenetic approach that assumes a stationary, reversible,
and homogeneous evolutionary process. The observed I
ss
value of 0.764 was not significantly different from the I
ss.c
value of 0.692 for a symmetrical topology (p= 0.4757, two-
tailed test) and was significantly greater than the I
ss.c
value
of 0.362 for an asymmetrical topology (p= 0.0001, two-
tailed test), suggesting that the ITS1–5.8S might have
experienced substitution saturation.
K80 distances among the Leishmania species except
Leishmania sp. ranged from near zero (between Leishmania
braziliensis and L. panamensis) to 0.224 (between Leish-
mania mexicana and L. panamensis). Most pairwise
comparisons mentioned above had divergence values of
less than 0.224, with 0.102 on average. Meanwhile, the
divergence between Leishmania sp. and other species
ranged from 0.104 (Leishmania sp. versus Leishmania
aethiopica) to 0.231 (Leishmania sp. vs. L. panamensis),
with an average of 0.147 (Table 4).
Phylogenetic relationships
The heuristic search of the ITS1–5.8S matrix resulted in
10,000 equally parsimonious trees of 149 steps, with
high values of CI (0.8456) and RI (0.9599). In the strict
consensus phylogram (Fig. 2), eight isolates in China
formed a strongly supported clade (clade A; Leishmania
sp.; BP= 100%) that was sister to the remaining members
of subgenus Leishmania (BP= 100%). Leishmania amazo-
nensis and L. mexicana formed a robust clade (BP=100%)
that was basal to all remaining subgenus Leishmania species
(BP= 100%). Within the other members of subgenus
Leishmania,L. donovani complex clustered with Leishmania
tropica (BP=66%), next joined by L. turanica plus L.
Table 4 Pairwise genetic distances for ITS1–5.8S segments among Leishmania species in this study
1234567891011
1L. donovani complex –
2L. tropica 0.039 –
3L. turinica 0.043 0.038 –
4L. gerbilli 0.065 0.060 0.023 –
5L. major 0.054 0.047 0.051 0.063 –
6L. aethiopica 0.041 0.023 0.052 0.074 0.055 –
7L. amazonensis 0.047 0.074 0.087 0.099 0.085 0.080 –
8L. mexicana 0.053 0.070 0.083 0.094 0.081 0.078 0.008 –
9L. braziliensis 0.158 0.179 0.161 0.168 0.175 0.178 0.211 0.212 –
10 L. panamensis 0.163 0.185 0.166 0.173 0.181 0.184 0.223 0.224 0 –
11 Leishmania sp. 0.114 0.108 0.131 0.152 0.143 0.104 0.131 0.129 0.225 0.231 –
The substitution model, K80 + G, with gamma shape of 0.5780, was selected using jModeltest v. 0.1.1 (Posda, 2008) under the Bayesian
information criterion (BIC; Schwarz 1978)
Table 3 Summary of results from matched-pairs tests of symmetry
Threshold (pvalue
a
) ITS1–5.8S
Number
b
Proportion
0.05 0 0
0.01 0 0
0.005 0 0
0.001 0 0
0.0005 0 0
0.0001 0 0
0.00005 0 0
a
The smallest pvalue is 0.1290
b
The number of times that the matched-pairs test of symmetry resulted in a
pvalue below the threshold (number of tests is 1,653)
1056 Parasitol Res (2010) 107:1049–1065
gerbilli (BP=52%), L. major (BP=100%), and finally by L.
aethiopica (BP= 100%). Nevertheless, the monophyly of L.
tropica was not supported. Unexpectedly, the isolate MRHO/
CN/88/KXG-2, identified as L. turanica by MLEE, did not
cluster where expected, which appeared in the Leishmania
sp. branch. When indels of the ITS1 were treated as
additional characters, the heuristic search yielded 10,000
equally parsimonious trees of 298 steps, with high values of
CI (0.7651) and RI (0.9435). As shown in Fig. 3,the
consensus tree was similar to Fig. 2with respect to the
placements of clades A and B. The placements of other
species were incongruent with those in Fig. 2.
For the BI analyses, the likelihood value of the 50%
majority consensus tree (Fig. 4) was ln L=−1,541.09. The
average PSRF was 1.001. Overall, as well with maximum
parsimony analyses, Leishmania sp., consisting of eight
isolates from China, was sister to the subgenus species
(PP= 0.76). Similarly, L. amazonensis and L. mexicana
formed a robust clade (PP= 1.00) that was sister to all
remaining subgenus Leishmania species (PP=0.96). The
relationships within the remaining species were similar to
those of Fig. 3except for recognizing the monophyly of L.
tropica (PP= 0.56) instead of L. donovani complex. When
the ITS1 indels were incorporated as additional characters,
the resultant 50% majority consensus tree was shown as
Fig. 5,withlnLof −2,167.78 and the average PSRF of
1.001. In this context, the topology is similar to Fig. 4
except that the monophyly of L. donovani complex was
H4
H2
H1
H3
H3
H5
H6
100
C
H7
H8
H9
L. donovani complex
C
H9
H10
H11
H12
52
p
H12
H13
AJ300485
FJ948459
FJ948457
GQ913688
GQ913688
FJ948458
FJ460459
66
FJ948465
FJ948451
FJ948456 L. tropica
FJ948452
HM004586
AJ000302
52
AJ000302
AJ000301
FJ948464
FJ948461
FJ948460
HM130607
100
100
L. turanica
D
HM130607
AJ300486
AJ272383
GQ471900
52
L.
turanica
L. gerbilli
D
GQ471900
AJ300481
AY260965
100
100 E
DQ295824
FJ753395
AJ300482
L. major
AJ300482
GQ920677
GQ920674
100
76
81
100
F
GQ920673
GQ920675
DQ182536
100
L. aethiopica
F
FJ753371
AF339753
AF466383
100
100
L. amazonensis
B
AF466383
FJ948435
FJ948433
100
100
L. mexicana
B
FJ948436
HM130601
HM130599 / HM130600
100
94
HM130599
/
HM130600
HM130602 / HM130603
HM130605
100 A
HM130604
HM130606
Leishmania sp.
10
DQ182537
FJ948442
L. braziliensis
L. panamensis
10
Fig. 2 Maximum parsimony
consensus tree from 1,000 boot-
strap replicates of ITS1–5.8S
dataset by using PAUP*.
Numbers above the branch rep-
resent percent recovery in boot-
strap analysis (1,000
pseudoreplicates). `Tree length=
149, CI= 0.8456, RI = 0.9599
Parasitol Res (2010) 107:1049–1065 1057
recovered with moderate posterior probability (PP=0.88)
instead of L. tropica.
To get additional insight into the relationships among
the L. donovani complex strains, we analyzed our data set,
using the coalescent-based statistical parsimony network
approach. The network of 13 haplotypes was shown as
Fig. 6.H9andH12seemedtobecentralhaplotypes,and
the haplotype diversity was highest in Sudan. L. donovani
revealed much more polymorphism than L. infantum
despite a wider geographical distribution for the latter
(see H3 in Fig. 6). L. infantum (H3) was most closely
related to the H1 of L. donovani, with one mutational step.
H2, shared by five strains from China, was also most
closely related to H1. Having an advantage over the
bifurcating tree in detail at the intraspecific level, the
haplotype network could intuitively reflect the genetically
greater distances between the singleton (H11) and one
central haplotype (H12; five mutational steps, see Fig. 6).
AsshowninFig.7, the haplotype shared by GSH2 and
GS2 (i.e., HM130602 and HM130603) was the interior
GQ920677 F
GQ920675
GQ920673
GQ920674
100
L. aethiopic
a
F
GQ920674
HM004586
FJ948456
FJ948452
61
FJ948452
FJ948458
GQ913688
80
FJ948459
FJ948457
FJ460459
80
FJ460459
FJ948465
FJ948451
L. tropica
AJ000302
AJ000301
AJ300485
AJ300485
FJ948464
FJ948461
FJ948460
66
FJ948460
AJ272383
AJ300481
54
E
GQ471900
DQ295824
AJ300482
57
100
L. major
E
AJ300482
AY260965
FJ753395
100
100
77
70
Lt i
HM130607
AJ300486
H1
89
100
L
.
t
uran
i
ca
L. gerbilli D
H1
H2
H3
H4
57
C
H4
H5
H6 L donovani complex
C
H7
H8
H9
90
80
L. donovani complex
H9
H10
H11
H12
H13
DQ182536
61
DQ182536
FJ753371
AF339753
AF466383
100
100
61
L. amazonensis
B
AF466383
FJ948435
FJ948433
100
L. mexicana
B
FJ948436
HM130601
HM130606
HM130606
HM130602 / HM130603
100
80
Leishmania sp.
A
HM130599 / HM130600
HM130604
HM130605
p
DQ182537
FJ948442
10
Fig. 3 Maximum parsimony
consensus tree from 1,000 boot-
strap replicates of ITS1–5.8S
with indel coding by using
PAUP*. Numbers above the
branch represent percent recov-
ery in bootstrap analysis (1,000
pseudoreplicates). Tree length=
298, CI= 0.7651, RI = 0.9435
1058 Parasitol Res (2010) 107:1049–1065
haplotype of Leishmania sp. and may be older than any
other haplotypes. There were three tip haplotypes harbored
by GS6/GS1, SC10H2, and JS1, respectively. Similarly,
this network reflected a greater distance between JS1 and
the interior haplotype as four mutational steps.
Bayesian hypothesis testing
Bayes factor comparisons were summarized in Table 5.The
analyses conducted reflect our primary interests of evaluat-
ing the inclusion of Leishmania sp. in L. donovani complex.
As mentioned above, analyses of the ITS1 data resulted in a
Chinese Leishmania clade that excluded L. turanica and L.
donovani complex. Bayes factor analyses of the ITS1–5.8S
incorporating indels coding were conducted to compare
topologies with constraints to the optimal tree topology. In
all cases, there was very strong (2ln Bayes factor >10)
evidence against the constrained topologies.
Discussion
Probabilistic methods, namely maximum likelihood and BI,
have progressively supplanted the MP method for inferring
AJ000302
FJ948456
FJ948452
HM004586
0.63
0.64
HM004586
FJ948451
FJ948459
G
FJ948457
GQ913688
FJ948458
056
L. tropica
G
FJ948458
FJ460459
FJ948465
AJ000301
0
.
56
AJ000301
AJ300485
FJ948464
0.95
FJ948461
FJ948460
GQ920677
GQ920677
GQ920675
GQ920673
1.00
091
L. aethiopica
F
GQ920674
AJ272383
GQ471900
0.93
0
.
91
E
GQ471900
AJ300481
AY260965
DQ295824
1.00
075
L. major
E
DQ295824
FJ753395
AJ300482
0
.
75
0.61
Lt i
HM130607
AJ300486
1.00
H3
L
.
t
uran
i
ca
L. gerbilli D
H3
H2
H1
H4
H5
H6
0.96
L donovani
complex
H7
H8
H9
L
.
donovani
complex
H9
H10
H11
0.76
H12
H13
DQ182536
082
DQ182536
FJ753371
AF339753
0
.
82
100
L. amazonensis
B
AF466383
FJ948435
FJ948433
1
.
00
1.00
L. mexicana
B
FJ948436
HM130601
HM130602 / HM130603
0.99
A
HM130602
/
HM130603
HM130599 / HM130600
HM130606
0.89
100
Leishmania sp.
A
HM130606
HM130604
HM130605
1
.
00
02
HM130605
DQ182537
FJ948442
0
.
2
Fig. 4 The 50% majority-rule consensus tree inferred from Bayesian inference of ITS1–5.8S dataset by using MrBayes v. 3.2. Numbers at nodes
represent Bayesian posterior probabilities
Parasitol Res (2010) 107:1049–1065 1059
phylogenetic trees. One of the major reasons for this shift is
that MP is much more sensitive to the Long Branch
Attraction artifact than are probabilistic methods. Based on
simulation studies, Philippe et al. (2005) found that MP can
be affected by heterotachy and that it is much less efficient
than probabilistic methods in dealing with all other
evolutionary heterogeneities. Thus, in support of several
recent studies (e.g., Gadagkar and Kumar 2005; Gaucher
and Miyamoto 2005; Spencer et al. 2005), Philippe et al.
(2005) strongly urged the continued preference of probabi-
listic methods for inferring phylogenies from real sequen-
ces. In our study, there is no significant compositional
heterogeneity in ITS1 sequences, which are likely to
have evolved under the same stationary, reversible, and
homogeneous conditions. The MP analysis incorporating
indels coding resulted in a topology that is congruent
with the Bayesian trees. However, Bayesian support
values in the BI tree (Fig. 5) were found to be
HM004586
052
FJ948456
FJ948452
0
.
52
AJ000302
0.77
AJ000302
FJ948451
AJ300485
1.00
L. tropica
AJ0003010.74
GQ920677
GQ920675
1.00 F
GQ920675
GQ920673
GQ920674
FJ948458
0.72
100
L. aethiopic
a
FJ948458
GQ913688
FJ948459
1
.
00
FJ948457
FJ460459
FJ948465
L. tropica
FJ948465
FJ948464
FJ948461
FJ948460
0.96
FJ948460
AJ272383
AJ300481
0.53
0.68
E
GQ471900
AY260965
DQ295824
1.00
086
L. major
E
DQ295824
FJ753395
AJ300482
100
0
.
86
0.98
Ltranica
HM130607
AJ300486
1
.
00
H1
0
.
86
0.98
L
.
t
u
ranica
L. gerbilli D
H2
086
H3
0.98
H4
H4
H5
H6
L donovani
complex
C
H7
H8
H9
0.88
0.94
L
.
donovani
complex
H9
H10
H11
H12
H12
H13
DQ182536
077
FJ753371
AF339753
0
.
77
AF466383
1.00
1.00 L. amazonensis
B
AF466383
FJ948435
FJ948433
1.00
L. mexicana
B
FJ948436
HM130601
HM130602 / HM130603
0.99
A
HM130599 / HM130600
HM130606
0.91
1.00
Leishmania
sp.
A
HM130606
HM130604
HM130605
Leishmania
sp.
02
DQ182537
FJ948442
0
.
2
Fig. 5 The 50% majority-rule consensus tree inferred from Bayesian
inference of ITS1–5.8S plus indel coding by using MrBayes v. 3.2,
with indels treated in a manner similar to the simple gap coding
outlined by Simmons and Ochoterena (2000). Numbers at nodes
represent Bayesian posterior probabilities
1060 Parasitol Res (2010) 107:1049–1065
comparatively higher than bootstrap values for the clades
in that MP tree (Fig. 3), suggesting that the Bayesian
inference could be properly applied to the phylogenetic
analyses of subgenus Leishmania. Considering congruent
with the recent studies on the interrelationships of
subgenus Leishmania (Asato et al. 2009; Fraga et al.
2010), we tentatively support the relationships inferred
from BI of the dataset incorporating indels coding (Fig. 5)
as the preferred phylogeny.
As expected, the isolate MRHO/CN/88/KXG-2, previ-
ously identified as L. turanica by MLEE (Guan et al. 1995),
clusters with L. gerbilli. This result is in congruent with the
taxonomic scheme published by the World Health Organi-
zation (WHO 1990). Interestingly, a common allele was
shared by the isolate MRHO/CN/88/KXG-2 with six other
strains of L. turanica from Central Asia, as shown in
Table 2. This further lends support that the MRHO/CN/88/
KXG-2 belongs to L. turanica. On the other hand, as
highlighted by Guan et al. (1992b), Leishmania parasite of
the Karamay great gerbils (including isolate MRHO/CN/88/
KXG-2) was pathogenic to monkey and man, causing
cutaneous leishmaniasis. This medical characteristics, how-
ever, is different from that of L. turanica, being nonpatho-
genic to humans, as described by Strelkova et al. (1990).
Thus, more isolates of L. turanica from different geograph-
ical areas and multiple loci are required for phylogeo-
graphic studies in order to clarify the intra-species genetic
diversity and complex phylogeographic pattern.
The species concept has long been a matter of debate
which is far more resolved (De Meeûs et al. 2003;de
Queiroz 2007).AsnotedbyBañulsetal.(2002), any new
species of Leishmania should be based on the clearly
distinct phylogenetic approach of Tibayrenc’s discrete
typing unit (Tibayrenc 1998). Eight new sequences
reported in this work formed a robust clade (clade A;
H8
L. donovani
Iran
Uzbekistan
Brazil
Spain
Italy
France
Malta
Portugal
Tunisia
China
India
Sudan
Morocco
Kenya
Ethiopia
Sri Lanka
Iraq
H9
H6
H9
H12 H5 H7
H10
H4
Linfantum
H13
L
.
infantum
H13
H3
H1
H11
10
20
H2
1
10
strains
1
Fig. 6 Statistical parsimony
network showing genetic rela-
tionships and distance among 13
haplotypes of L. donovani com-
plex from different countries.
Numbers of haplotypes corre-
spond to Table 1. In the net-
work, solid circles indicate
sampled haplotypes; small
hollow circles indicate
unsampled or extinct haplo-
types. Each mutation step is
shown as either a short or
longer line connecting neigh-
boring haplotypes (including
observed and unobserved one).
The size of the solid circles
roughly represents the numbers
of strains carrying the haplo-
type, with the scale given beside
the network; different filled pat-
terns represent the
corresponding geographical ori-
gin from which the haplotype
was sampled
hill foci
HM130602/HM130603 hill foci
GS5
GS2
HM130602/HM130603
GS6
HM130599
GS6
GS1 SC10H2 SC11
HM130601
HM130600
HM130606
HM130604
HM130601
HM130600
HM130606
plain foci
HM130604
plain foci
HM130605
JS1
HM130605
Fig. 7 Statistical parsimony network showing genetic relationships
and distance among six haplotypes of Leishmania sp. from different
sites in China. In the network, solid circles indicate sampled
haplotypes; small hollow circles indicate unsampled or extinct
haplotypes. Each mutation step is shown as either a short or longer
line connecting neighboring haplotypes (including observed and
unobserved one)
Parasitol Res (2010) 107:1049–1065 1061
Leishmania sp.) that is sister to the remaining members of
subgenus Leishmania. When we constrained all the
isolates in China to form a monophyletic group, the tree
obtained differs significantly from the BI tree based on
2ln Bayes factor comparison (55.856>10; see Table 5).
Thus, we reject the monophyletic origin of Chinese
Leishmania isolates and exclude the possibility that all
the isolates in this study only belong to L. donovani
complex. In addition, the mean genetic divergence (K80
distance) between Leishmania sp. and other species is
0.147, which is higher than that among several other
species (mean 0.102). We further confirm that Leishmania
sp. is an undescribed pathogenic species endemic in
China, comprising isolates from hill foci, desert foci,
and plain foci (see Fig. 7).
Judging from the kinetoplast and nuclear DNA
heterogeneity, the isolate IPHL/CN/77/XJ771 was ten-
tatively designated as L. infantum sensulatobyLuetal.
(1994). This hypothesis, however, is challenged by our
molecular data. There is complete identity of the ITS1
sequence of the isolate IPHL/CN/77/XJ771 and that of a
strain of L. donovani (MHOM/CN/00/Wangjie1). They
share a common haplotype H2 with three isolates from
eastern Jiashi County. As shown in the haplotype
network (Fig. 6), H2 is most closely related to H1, which
is shared by four strains from Sudan, one from India, and
one from Ethiopia. Consequently, we argue that the
isolate IPHL/CN/77/XJ771 is L. donovani instead of L.
infantum.
The three isolates from eastern Jiashi County, causing
desert type of zoonotic visceral leishmaniasis, were
designated as L. infantum based on the genetic analysis
of the ITS1 sequence (Wang et al. 2010). This conclusion,
however, should be interpreted with caution, since it was
not deduced from a robust phylogenetic tree, and the MP
tree was misleading (see Fig. 3 in Wang et al. 2010). In
contrast, as mentioned above, the coalescent-based statis-
tical parsimony network approach provides additional
insight into the relationships among the L. donovani
complex. We infer that the three isolates should belong
to L. donovani. As can be seen from Fig. 6, the isolate
MHOM/CN/80/801 from Kashi city shares H6 with
several isolates from India, Kenya, and Sri Lanka, and
there is only one mutational step between H6 and a central
haplotype H9 from Sudan. In combination with the results
of Lukešet al. (2007), the demonstration that the isolate
MHOM/CN/80/801 belongs to L. donovani is corroborat-
ed, in accord with Wang et al. (2010).
In conclusion, phylogenetic analyses suggested that
Chinese Leishmania isolates do not form a monophyletic
group, but among which eight newly determined isolates
form a monophyletic group, being sister to other
members of subgenus Leishmania. The genetic distance
analysis further provides evidence of the occurrence of an
undescribed species of Leishmania.Ourresultsalso
suggest that the isolate IPHL/CN/77/XJ771 is L. dono-
vani; the three isolates from eastern Jiashi County,
Xinjiang Uygur Autonomous Region belong to L.
donovani instead of Leishmania infantum. In addition,
the results of this study make an important contribution
to understanding the heterogeneity and relationships of
Chinese Leishmania isolates, further indicating that the
isolates from China may have had a more complex
evolutionary history than expected. However, more
samples from different geographical areas and multiple
independent evolving loci are required for phylogenetic
studies in order to clarify the evolutionary history among
Chinese Leishmania isolates.Itfurthermightbeusefulin
understanding the links between clinical disease, geo-
graphic origin, and nomenclature of Leishmania species.
Table 5 Summary of 2ln Bayes factor comparisons of alternative phylogenetic hypotheses
Constraint (H
0
) ln marginal likelihood Evidence against H
0
Alternative phylogenetic hypotheses ln L: unconstrained
(Fig. 5)
ln L:
constrained
2ln Bayes factor
(2ln(B
10
))
Monophyly constraint of L. donovani complex
a
−2187.027 −2268.638 163.222 Very
strong
Monophyly constraint of isolates from China with exception
of MRHO/CN/88/KXG-2
b
−2187.027 −2214.955 55.856 Very
strong
Marginal likelihoods were calculated using the method of Suchard et al. (2001) using Tracer 1.5 (Rambaut and Drummond 2009)
2ln Bayes factors ≥10 are considered very strongly different (Kass and Raftery 1995), indicating evidence against alternative hypotheses
a
Constrained tree with Leishmania sp. except MRHO/CN/88/KXG-2 embedded within L. donovani complex (H1–H13)
b
Constrained tree with Leishmania sp. clustering with H2, H3, and H6
1062 Parasitol Res (2010) 107:1049–1065
Acknowledgments This work was supported by the National
Natural Science Foundations of China (30771883, 30800094) and
the National Project of Important Infectious Diseases (2008-ZX10004-
011). X-G Guo was supported by the National Natural Science
Foundation of China (30700062). We thank Dianmei Lu and Zhibiao
Xu for kinkly help with collecting some important references.
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