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BMC Evolutionary Biology
BioMed Central
Open Access
Research article
Identification of geographically distributed sub-populations of
Leishmania (Leishmania) major by microsatellite analysis
Amer Al-Jawabreh1,2, Stephanie Diezmann1, Michaela Müller1,
Thierry Wirth3, Lionel F Schnur4, Margarita V Strelkova5,
Dmitri A Kovalenko6, Shavkat A Razakov6, Jan Schwenkenbecher1,
Katrin Kuhls1 and Gabriele Schönian*1
Address: 1Department of Parasitology, Institute of Microbiology and Hygiene, Charité University Medicine Berlin, Dorotheenstr. 96, D-10098
Berlin, Germany, 2Leishmania Research Unit, Jericho, The Palestinian Authority, 3Ecole Pratique des Hautes Etudes, Muséum National d'Histoire
Naturelle, 16 rue Buffon, 72231 Paris cedex 05, France, 4Department of Parasitology, Hebrew University-Hadassah Medical School, P. O. Box
12272, Jerusalem 91120, Israel, 5Department of Medical Protozoology, Martsinovsky Institute of Medical Parasitology and Tropical Medicine,
Sechenov Moscow Medical Academy, M. Pirogovskaya 20, 119830 Moscow, Russia and 6Isaev Research Institute of Medical Parasitology,
Department of Leishmania Epidemiology, ul Isaeva 38, 703005 Samarkand, Uzbekistan
Email: Amer Al-Jawabreh - islahjr@yahoo.com; Stephanie Diezmann - sd21@duke.edu; Michaela Müller - mixmue@gmx.de;
Thierry Wirth - wirth@mnhn.fr; Lionel F Schnur - schnurl@cc.huji.ac.il; Margarita V Strelkova - mstrelkova@mail.ru;
Dmitri A Kovalenko - kovdmi@yahoo.com; Shavkat A Razakov - isaevins2@yahoo.com;
Jan Schwenkenbecher - janschwenkenbecher@yahoo.de; Katrin Kuhls - katrin.kuhls@charite.de;
Gabriele Schönian* - gabriele.schoenian@charite.de
* Corresponding author
Published: 24 June 2008
BMC Evolutionary Biology 2008, 8:183
doi:10.1186/1471-2148-8-183
Received: 27 November 2007
Accepted: 24 June 2008
This article is available from: http://www.biomedcentral.com/1471-2148/8/183
© 2008 Al-Jawabreh et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Leishmania (Leishmania) major, one of the agents causing cutaneous leishmaniasis (CL) in
humans, is widely distributed in the Old World where different species of wild rodent and phlebotomine
sand fly serve as animal reservoir hosts and vectors, respectively. Despite this, strains of L. (L.) major
isolated from many different sources over many years have proved to be relatively uniform. To investigate
the population structure of the species highly polymorphic microsatellite markers were employed for
greater discrimination among it's otherwise closely related strains, an approach applied successfully to
other species of Leishmania.
Results: Multilocus Microsatellite Typing (MLMT) based on 10 different microsatellite markers was
applied to 106 strains of L. (L.) major from different regions where it is endemic. On applying a Bayesian
model-based approach, three main populations were identified, corresponding to three separate
geographical regions: Central Asia (CA); the Middle East (ME); and Africa (AF). This was congruent with
phylogenetic reconstructions based on genetic distances. Re-analysis separated each of the populations
into two sub-populations. The two African sub-populations did not correlate well with strains'
geographical origin. Strains falling into the sub-populations CA and ME did mostly group according to their
place of isolation although some anomalies were seen, probably, owing to human migration.
Conclusion: The model- and distance-based analyses of the microsatellite data exposed three main
populations of L. (L.) major, Central Asia, the Middle East and Africa, each of which separated into two subpopulations. This probably correlates with the different species of rodent host.
Page 1 of 13
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BMC Evolutionary Biology 2008, 8:183
Background
Leishmania (Leishmania) major is one of the agents causing
Old World cutaneous leishmaniasis (CL) in humans,
which, in the case of L. (L.) major, is a rural, zoonotic, vector-borne disease, involving different species of wild
rodents as animal reservoir hosts and different species of
phlebotomine sand flies as vectors, depending on the geographical location where it occurs (reviewed, [1].
Humans, though infected in large numbers, are considered to be incidental hosts not directly implicated in the
transmission cycle [2].
The parasite and, thus, the disease are geographically
widely distributed in the arid and semi-arid areas of:
North-West, North, Central sub-Saharan and East Africa;
the Near and Middle East; Central Asia; and Rajasthan,
India. Despite the very broad geographical distribution
and large variety of types of wild animal host and sand fly
vector, the different strains of L. (L.) major isolated from
many sources over many years have proved to be relatively
uniform when studied by most of the classical and more
modern methods used for characterizing leishmanial
strains. Serological tests like excreted factor (EF) serotyping and the application of Leishmania species-specific
monoclonal antibodies have shown that antigenic differences exist among different strains of L. (L.) major [3].
Multilocus enzyme electrophoresis (MLEE) exposed some
enzyme electrophoretic variation with some isoenzyme
variant profiles showing a degree of geographical subdivision within a general enzyme electrophoretic uniformity [4-6].
The more recent application of various molecular biological methods revealed geographically distributed genetic
polymorphism among different strains of L. (L.) major.
Analysis of sequence polymorphisms in seven coding,
non-coding and anonymous nuclear DNA sequences [1]
as well as a partial sequence from the kinetoplast DNA
maxicircle divergent region [7] of strains of L. (L.) major
showed that the strains from Central Asia and the Middle
East were genetically distinct. None to very little variation
was seen among the Central Asian strains and somewhat
more among the Middle Eastern ones. Only a few East
African strains of L. (L.) major were included in these studies, which tended to be genetically closer to the Middle
Eastern than to the Central Asian ones. Different patterns
in the most variable sequence were attributed to variations in complex microsatellite repeats. This prompted a
search for highly polymorphic microsatellite markers that
would allow greater discrimination of otherwise closely
related strains of L. (L.) major.
Microsatellites are repeated simple motifs of a few nucleotides (<6) flanked by unique sequences. They are ubiquitous in prokaryotes and eukaryotes and have been shown
http://www.biomedcentral.com/1471-2148/8/183
to exhibit a substantial level of polymorphism in a
number of eukaryotic genomes [8,9]. They are becoming
one of the principal genetic marker systems in phylogenetic, population genetic and molecular epidemiological
studies. The leishmanial genome is relatively rich in microsatellite sequences, about 600 (CA)n loci per haploid
genome, with CA being the most abundant dinucleotide
repeat as in all vertebrates and fungi investigated so far [911]. Microsatellite markers have been used successfully for
characterizing and detecting genetic variation in other Old
World leishmanial strains and species, i. e., L. (Leishmania) donovani and L. (Leishmania) infantum [12-14] and L.
(Leishmania) tropica [15].
This study adds the species L. (L.) major. Ten informative
microsatellite loci based on nucleotide sequence information of L. (L.) major obtained from the Leishmania genome
project were used to determine polymorphism and microheterogeneity, and their geographical and epidemiological distribution.
Results
Microsatellite analysis
Twenty-three sequences located on chromosomes 1, 3, 5,
21 and 35 that contained nucleotide repeats such as
(AT)n, (GC)n, (CA)n, (GTG)n, and (GACA)n were
selected from the genome sequence of L. (L.) major published by the European Bioinformatics Institute. PCR
primers were designed for microsatellite loci that contained at least 6 repeats in the reference strain L. (L.) major
MHOM/IL/1980/Friedlin and were located on different
chromosomes or, if located on the same chromosome,
were at least distant enough to be considered unlinked
(Table 1). Fifteen of the 23 primer pairs yielded a single
PCR fragment for the majority of the strains tested. Ten of
these 15 primer pairs amplified polymorphic fragments of
different size in different strains of L. (L.) major (Table 1).
Only 4 out of 45 pair-wise combinations (9%) were in significant linkage disequilibrium (P < 0.05) when GENEPOP was used. No linkage was observed using the
Bonferroni corrections implemented in FSTAT.
The 10 polymorphic loci were used to perform multilocus microsatellite typing (MLMT) on 106 strains of L.
(L.) major collected in 10 Asian and 9 African countries.
Four of the 10 loci showed only homozygous allele combinations. However, three GTG loci, two AT loci and the
GACA locus were heterozygous for one or more of the
strains (see Additional file 1). In some cases, no PCR
product could be obtained in repeated PCR runs, which
was treated as missing data for the statistical analyses.
Sixty-six differing MLMT profiles comprising the number
of repeats in each of the 10 markers were revealed among
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BMC Evolutionary Biology 2008, 8:183
http://www.biomedcentral.com/1471-2148/8/183
Table 1: The microsatellite markers used.
Marker
Size (bp)
4GTG
70
27GTG
75
36GTG
68
39GTG
86
45GTG
86
1GC
64
28AT
65
71AT
55
1GACA
75
1CA
87
Primer sequences
F: 5'cggtttggcgctgaaagcgg
R: 5'cgtgaggacgccaccgaggc
F: 5'ggaggtggctgtggttgttg
R: 5'gccgctgacgctgcaggct
F: 5'agcgaagaagagtcgggcag
R: 5'gcgccttcagtgcgtcgtcc
F: 5'gtcttgccgcgaggtgaccg
R: 5'ccagcaccagcaccaccatc
F: 5'acggccgggtggtcgtgggt
R: 5'cgttcgcacgcacgcacgca
F: 5'ctggcacgcacacccacaca
R: 5'atctgcgctcatctggcgag
F: 5'ttgcctatcaacacaaggct
R: 5'agtctctctctctctctata
F: 5'tcttgcgaaggtgtggtctt
R: 5'agcccacgtgtacatgtgtg
F: 5'gaaagggcaggaggacggat
R: 5'cacacacacatacacacata
F: 5'ttagttccatcatacacccg
R: 5'cgttcgacatggagaataag
TA
Chromosome
Location at bp
Repeat number*
No. of alleles
58°C
35
6460-530
7
3
58°C
3
1440-515
9
3
62°C
1
140895-963
9
4
58°C
1
202765-851
9
5
58°C
1
59751-890
12
7
60°C
3
10323-386
7
2
42°C
5
27966-031
9
6
50°C
21
22113-168
13
9
54°C
1
68459-534
7
3
48°C
35
30151-238
14
7
* in L. (L.) major MHOM/IL/1980/Friedlin
TA annealing temperaturea
the 106 strains (see Additional file 1). The MLMT profiles
were named Lmj, thus indicating that these microsatellite
profiles are unique to L. (L.) major, and numbered from 1
to 66. Figures 1 to 3 show their genetic inter-relationship.
Most profiles were represented by a single strain; eleven
were present in more than one strain. Nine microsatellite
variant profiles were exposed among the 23 strains in the
sub-population CA1; six among the 16 strains in the subpopulation CA2; thirteen among the 26 strains in the subpopulation ME1; fifteen among the 17 strains in the subpopulation ME2; eleven among the 11 strains in the subpopulation AF1; and twelve among the 13 strains in the
sub-population AF2 (see Table 2, and Figure 2 for origins
and geographical distributions).
Table 3 shows that the number of alleles varies between
loci, ranging from 3 to 10. An increased degree of inbreeding within loci (Fis) was detected, ranging from
0.874–1.00 (P < 0.05) with a mean of 0.976. Observed
heterozygosity (Ho) among loci was extremely low compared with the heterozygosity (He) expected under
assumption of the Hardy-Weinberg equilibrium. These
results clearly show that L. (L.) major is largely clonal and
that hybridization only occurs at very low frequencies.
Population structure
The population structure was investigated, using the Bayesian-model based clustering approach implemented in
the software STRUCTURE [16]. The most probable
number of populations in this data set by calculating ΔK
was three: Central Asia (CA, comprising strains from
Uzbekistan, Turkmenistan and Kazakhstan; the Middle
East (ME), comprising strains from Israel, The Palestinian
Authority, Egypt, Saudi Arabia, Kuwait and eastern Turkey; and Africa (AF), comprising strains from North, East
and West Africa, western Turkey, Iran and Iraq (Figure 1A
and Table 2).
STRUCTURE analyses were re-done with each of the three
main populations, CA, ME and AF, to expose further subdivision; and each main population did separate into two
sub-populations: CA1 and CA2; ME1 and ME2; AF1 and
AF2 (Figures 1B, C and 1D, Table 2). Although calculations of ΔK do not allow for differentiation between one
or two groups in the dataset, the strains' assignment
(Table 2) was most congruent with the existence of two
sub-populations. The distribution of the different subpopulations is shown in Figure 2.
On using a predefined clustering approach, the total area
from which the strains of L. (L.) major were collected was
divided into five geographical sectors: Central Asia (CA),
the Middle East (ME), North, West and East Africa with
the last three being referred to collectively as Africa (AF).
The boundaries between these sectors are mainly significant geographical barriers like seas and deserts. Each
strain was assigned to one of the sectors, according to its
place of collection without regard to underlying genetic
relationships (Figure 3). The Central Asian cluster was
highly homogenous, genetically and according to predefined geographical placement, with all the strains having
a very high membership coefficient of 0.999. The Middle
Page 3 of 13
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BMC Evolutionary Biology 2008, 8:183
http://www.biomedcentral.com/1471-2148/8/183
Table 2: The strains of Leishmania (L.) major analysed in this study.
WHO code
Origin country, location, source
Microsatellite profile
Population
General region
MHOM/TM/1973/5ASKH
MHOM/TM/1987/Rod
IPAP/TM/1991/M-97
MRHO/TM/1995/T-9537
MHOM/TM/1982/Lev
MHOM/TM/1986/ER
MRHO/KZ/1988/Tur-27R
MHOM/UZ/1987/BUR
MRHO/UZ/1987/KK29
MHOM/UZ/1987/Kurb
MHOM/UZ/1999/Nuriya
MHOM/UZ/2002/Isv M-22h
MHOM/UZ/2002/Isv M-17h
MHOM/UZ/2002/Isv M-12h
MHOM/UZ/2002/Isv M-30h
MHOM/UZ/2002/Isv M-28h
MHOM/UZ/2002/Isv M-25h
MHOM/UZ/1998/Isv M-09h
MHOM/UZ/1998/Isv M-01h
MHOM/UZ/2002/Isv M-10h
MHOM/UZ/2002/Isv M-29h
MHOM/UZ/1998/Isv M-04h
MHOM/UZ/1998/Isv M-08h
Turkmenistan, Ashkhabad: Human
Turkmenistan, Bakharden: Human
Turkmenistan, Tezeel: Ph. papatasi
Turkmenistan, Serags:R. opimus
Turkmenistan, Geok-Depe: Human
Turkmenistan, Tejen: Human
Kazakhstan, Turkestan:R. opimus
Uzbekistan, Karaulbasar: Human
Uzbekistan, Takhtakupyr: R. opimus
Uzbekistan, Karshi: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Lmj 01
Lmj 01
Lmj 01
Lmj 02
Lmj 03
Lmj 04
Lmj 01
Lmj 01
Lmj 01
Lmj 02
Lmj 01
Lmj 05
Lmj 06
Lmj 06
Lmj 06
Lmj 06
Lmj 06
Lmj 06
Lmj 06
Lmj 07
Lmj 08
Lmj 08
Lmj 09
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
CA1
Central Asia (CA)
MHOM/UZ/1998/Isv M-02h
MHOM/UZ/2002/Isv M-27h
MHOM/UZ/2002/Isv M-26h
MRHO/UZ/1959/NealP
MHOM/UZ/2000/Isv T-03h
MHOM/UZ/2003/Isv T-21h
MHOM/UZ/2003/Isv T-24h
MHOM/UZ/2003/Isv T-29h
MHOM/UZ/2003/Isv T-32h
MHOM/UZ/2003/Isv T-35h
MRHO/UZ/2003/Isv T-6g
MRHO/UZ/2003/Isv T-20g
MRHO/UZ/2003/Isv T-23g
MRHO/UZ/2003/Isv T-38g
MRHO/UZ/2003/Isv T-44g
MRHO/UZ/2003/Isv T-37g
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Mubarak: Human
Uzbekistan, Karakul: R. opimus
Uzbekistan, Termez: Human
Uzbekistan, Termez: Human
Uzbekistan, Termez: Human
Uzbekistan, Termez: Human
Uzbekistan, Termez: Human
Uzbekistan, Termez: Human
Uzbekistan, Termez: R. opimus
Uzbekistan, Termez: R. opimus
Uzbekistan, Termez: R. opimus
Uzbekistan, Termez:R. opimus
Uzbekistan, Termez: R. opimus
Uzbekistan, Termez: R. opimus
Lmj 13
Lmj 10
Lmj 11
Lmj 14
Lmj 12
Lmj 15
Lmj 15
Lmj 15
Lmj 15
Lmj 15
Lmj 15
Lmj 15
Lmj 15
Lmj 15
Lmj 15
Lmj 15
CA2
CA2
CA2
CA2
CA2
CA2
CA2
CA2
CA2
CA2
CA2
CA2
CA2
CA2
CA2
CA2
MHOM/MA/1995/LEM2983
MHOM/MA/1981/LEM265
MHOM/SN/1977/DK74
MHOM/SD/2004/MW1
MHOM/SD/2004/MW38
MHOM/SD/2004/MW94
MTAT/KE/195?/T4
MTAT/KE/????/NLB089A
MHOM/??/1987/NEL2
MHOM/IQ/1986/BH012
MRHO/IR/1976/vaccine-strain
Morocco, Er Rachidia: Human
Morocco, Tata: Human
Senegal, M'bour: Human
Sudan, location not known: Human
Sudan, location not known: Human
Sudan, location not known: Human
Kenya, Baringo:Tatera sp.
Kenya, Marigat:T. robusta
Africa, location not known
Iraq, location not known: Human
Iran, location not known: R. opimus
Lmj 18
Lmj 19
Lmj 20
Lmj 22
Lmj 23
Lmj 24
Lmj 21
Lmj 17
Lmj 16
Lmj 38
Lmj 37
AF1
AF1
AF1
AF1
AF1
AF1
AF1
AF1
AF1
AF1
AF1
MHOM/MA/1992/LEM2463
MHOM/DZ/1998/CRE95
MHOM/DZ/1998/LPS13
MHOM/TN/1997/LPN162
MHOM/TN/1994/GLC7
MHOM/SN/1978/DK106
Morocco, Ain Beni Mathar: Human
Algeria, M'sila: Human
Algeria, Biskra: Human
Tunisia, Sfax: Human
Tunisia, Gafsa: Human
Senegal, Djourbel: Human
Lmj 25
Lmj 26
Lmj 27
Lmj 34
Lmj 28
Lmj 30
AF2
AF2
AF2
AF2
AF2
AF2
Africa (AF)
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Table 2: The strains of Leishmania (L.) major analysed in this study. (Continued)
MHOM/SN/1996/DPPE23
MHOM/SN/1996/LEM3181
MHOM/SN/1978/DK99
MHOM/BF/1996/LIPA538
MHOM/BF/1998/LPN166
MHOM/TR/1993/HA
MHOM/TR/1993/SY
Senegal, Thies: Human
Senegal, Thies: Human
Senegal, Matam: Human
Burkino Faso, Ouagadougou: Human
Burkino Faso, Ouagadougou: Human
WesternTurkey, Manisa: Human
WesternTurkey, Aydin: Human
Lmj 32
Lmj 31
Lmj 29
Lmj 33
Lmj 35
Lmj 36
Lmj 36
AF2
AF2
AF2
AF2
AF2
AF2
AF2
MHOM/TR/1994/HK
MHOM/PS/1998/ISLAH388
MHOM/PS/1998/ISLAH389
MHOM/PS/1998/ISLAH402
MHOM/PS/2000/ISLAH503
MHOM/PS/2000/ISLAH506
MHOM/PS/2001/ISLAH600
MHOM/PS/2001/ISLAH659
MHOM/PS/2001/ISLAH657
MHOM/PS/2001/ISLAH658
MHOM/PS/2002/ISLAH662
MHOM/PS/2002/ISLAH697
MHOM/PS/2002/ISLAH690
MHOM/PS/2002/ISLAH691
MHOM/PS/2003/ISLAH718
MHOM/IL/1967/Jericho II
MHOM/IL/1986/Blum
MHOM/IL/1990/LRC-L585
MHOM/IL/1980/Friedlin
MHOM/IL/2003/LRC-L949
MHOM/IL/2003/LRC-L964
MHOM/IL/2003/LRC-L965
MHOM/IL/2003/LRC-L962
MPSA/IL/1983/PSAM398
MHOM/IL/2000/LRC-L779
MHOM/KW/1976/P47
Eastern Turkey, Kars: Human
The Palestinian Authority, Jericho: Human
The Palestinian Authority, Jericho: Human
The Palestinian Authority, Jericho: Human
The Palestinian Authority, Jericho: Human
The Palestinian Authority, Jericho: Human
The Palestinian Authority, Jericho: Human
The Palestinian Authority, Jericho: Human
The Palestinian Authority, Jericho: Human
The Palestinian Authority, Jericho: Human
The Palestinian Authority, Jericho: Human
The Palestinian Authority, Jericho: Human
The Palestinian Authority, Jericho: Human
The Palestinian Authority, Jericho: Human
The Palestinian Authority, Jericho: Human
Israel, Jericho: Human
Israel, Jericho: Human
Israel, Jericho: Human
Israel, Almog: Human
Israel, Nabi Musa: Human
Israel, near Beersheba: Human
Israel, Qeziot: Human
Israel, location not known: Human
Israel, Arava Hatzeva: Ps. obesus
Israel, Arava, Ein Yahav, or Egypt, Sinai:Human?
Kuwait, location not known: Human
Lmj 50
Lmj 39
Lmj 40
Lmj 40
Lmj 39
Lmj 40
Lmj 49
Lmj 40
Lmj 41
Lmj 42
Lmj 43
Lmj 44
Lmj 44
Lmj 45
Lmj 47
Lmj 39
Lmj 39
Lmj 39
Lmj 39
Lmj 46
Lmj 48
Lmj 39
Lmj 48
Lmj 39
Lmj 39
Lmj 51
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
ME1
MHOM/PS/2003/ISLAH720
MHOM/IL/2003/LRC-L1000
MHOM/IL/2003/LRC-L960
MHOM/IL/2001/LRC-L846
MHOM/IL/2002/LRC-L946
MHOM/IL/2002/LRC-L940
MHOM/IL/2003/LRC-L963
MHOM/IL/2003/LRC-L952
MHOM/IL/2002/LRC-L862
MHOM/IL/2003/LRC-L958
IPAP/IL/1984/LRC-L465
IPAP/IL/1998/LRC-L746
IPAP/IL/1984/LRC-L464
MHOM/EG/1984/LRC-L505
IPAP/EG/1989/RTC-13
MHOM/SA/1984/KFUH68757
MPSA/SA/1989/SABIR-1
The Palestinian Authority, Jericho: Human
Israel, Qalya, northern Dead Sea: Human
Israel, Revivim: Human
Israel, Yerucham: Human
Israel, Yerucham: Human
Israel, Yerucham: Human
Israel, Shifta: Human
Israel, Qeziot: Human
Israel, near Mitzpe Ramon: Human
Israel, En Yahav: Human
Israel, Uvda: Ph. papatasi
Israel, Uvda: Ph. papatasi
Israel, Uvda: Ph. papatasi
Egypt, Sinai: Human
Egypt, Sinai: Ph. papatasi
Saudi Arabia, Hofuf?: Human
Saudi Arabia, Hofuf: Ps. obesus
Lmj 52
Lmj 64
Lmj 62
Lmj 55
Lmj 58
Lmj 59
Lmj 63
Lmj 57
Lmj 61
Lmj 60
Lmj 53
Lmj 53
Lmj 54
Lmj 56
Lmj 53
Lmj 66
Lmj 65
ME2
ME2
ME2
ME2
ME2
ME2
ME2
ME2
ME2
ME2
ME2
ME2
ME2
ME2
ME2
ME2
ME2
Middle East (ME)
Key: Ph. = Phlebotomus; R. = Rhombomys; T. = Tatera; Ps. = Psammomys; The prefix Lmj indicates that these microsatellite profiles are unique to L. (L.)
major.
East cluster was also genetically well defined, except for
the exclusion of the two western Turkish and sole Iraqi
and Iranian strains, which grouped with the African
strains. The entire African cluster was very heterogeneous
regarding MLMT profiles and did not separate into dis-
tinct North, West and East African regional clusters as
envisaged by the predefined clustering approach.
Increasing values of K (2–6) were used to identify ancestral populations. At K = 2, the first split separated the population CA from other strains. At K = 3, three main
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1 2 3
4
5 6
7
8 9 10
Africa
1500
0
-500
-1000
-1500
-2000
-2500
-3000
AF
500
2
3
4
1
2
3
4
5
6
7
8
5
6
7
8
CA
9
CA1
9 10
CA2
60
0
-50
-100
-150
-200
-250
40
20
0
2
3
4
K
1
2
3
4
5
5
6
7
8