Molecular homogeneity in diverse geographical populations of Phlebotomus papatasi (Diptera, Psychodidae) inferred from ND4 mtDNA and ITS2 rDNA Epidemiological consequences.

Abstract

An intraspecific study on Phlebotomus papatasi, the main proven vector of Leishmania major among the members of the subgenus Phlebotomus, was performed. The internal transcribed spacer 2 (ITS 2) of rDNA and the ND4 gene of mt DNA were sequenced from 26 populations from 18 countries (Albania, Algeria, Cyprus, Egypt, Greece, India, Iran, Israel, Italy, Lebanon, Morocco, Saudi Arabia, Spain, Syria, Tunisia, Turkey, Yugoslavia and Yemen), and compared. Samples also included three other species belonging to the subgenus Phlebotomus: P. duboscqi, a proven vector of L. major in the south of Sahara (three populations from Burkina Faso, Kenya and Senegal), P. bergeroti, a suspected vector of L. major (three populations from Oman Sultanate, Iran and Egypt), and one population of P. salehi from Iran. A phylogenetic study was carried out on the subgenus Phlebotomus. Our results confirm the validity of the morphologically characterized taxa. The position of P. salehi is doubtful. Variability in P. papatasi contrasts with that observed within other species having a wide distribution like P. (Paraphlebotomus) sergenti in the Old World or Lutzomyia (Lutzomyia) longipalpis in the New World. Consequently, it could be hypothesized that all populations of P. papatasi over its distribution area have similar vectorial capacities. The limits of the distribution area of L. major are correlated with the distribution of common rodents acting as hosts of the parasites.

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Molecular homogeneity in diverse geographical populations
of Phlebotomus papatasi (Diptera, Psychodidae) inferred
from ND4 mtDNA and ITS2 rDNA
Epidemiological consequences
Je ´ro ˆme Depaquita,*, Emmanuel Lienarda, Astrid Verzeaux-Griffona,
Hubert Ferte ´a, Azzedine Bounamousb, Jean-Charles Gantierc,
Hanafi A. Hanafid, Raymond L. Jacobsone, Michele Marolif,
Vahideh Moin-Vazirig, Fre ´de ´rique Mu ¨llera,c, Yusuf O¨zbelh,
Milena Svobodovai, Petr Volfi, Nicole Le ´gerj
aUSC ‘‘VECPAR’’, AFSSA, Faculte ´ de Pharmacie, Universite ´ de Reims Champagne-Ardenne, Reims, France
bLaboratoire de Biosyste ´matique et e ´cologie des Arthropodes, Universite ´ de Constantine, Algeria
cLaboratoire de Parasitologie, Faculte ´ de Pharmacie, Cha ˆtenay-Malabry, France
dU.S. Naval Medical Research Unit n83, Cairo, Egypt
eDepartment of Parasitology, The Hebrew University, Jerusalem, Israel
fVectorborne Diseases and International Health, MIPI Department, ISS, Rome, Italy
gSchool of Public Health and Institute of Public Health Research, Tehran University of Medical Science, Tehran, Iran
hDepartment of Parasitology, Ege University Medical School, Bornova, Izmir, Turkey
iLaboratory of Parasitology, Charles University, Prague, Czech Republic
j63 Avenue Pierre Se ´mart, 94210 La Varenne Saint Hilaire, France
Received 3 December 2007; accepted 4 December 2007
Available online 14 December 2007
Abstract
An intraspecific study on Phlebotomus papatasi, the main proven vector of Leishmania major among the members of the subgenus
Phlebotomus,wasperformed.Theinternaltranscribedspacer2(ITS2)ofrDNAandtheND4geneofmtDNAweresequencedfrom26populations
from 18 countries (Albania, Algeria, Cyprus, Egypt, Greece, India, Iran, Israel, Italy, Lebanon, Morocco, Saudi Arabia, Spain, Syria, Tunisia,
Turkey, Yugoslavia and Yemen), and compared. Samples also included three other species belonging to the subgenus Phlebotomus: P. duboscqi, a
proven vector of L. major in the south of Sahara (three populations from Burkina Faso, Kenya and Senegal), P. bergeroti, a suspected vector of L.
major(three populationsfromOman Sultanate,Iran andEgypt),andonepopulationofP.salehifromIran.Aphylogeneticstudywascarriedouton
the subgenus Phlebotomus. Our results confirm the validity of the morphologically characterized taxa. The position of P. salehi is doubtful.
Variability in P. papatasi contrasts with that observed within other species having awide distribution like P. (Paraphlebotomus) sergenti in the Old
World or Lutzomyia (Lutzomyia) longipalpis in the New World. Consequently, it could be hypothesized that all populations of P. papatasi over its
distribution area have similar vectorial capacities. The limits of the distribution area of L. major are correlated with the distribution of common
rodents acting as hosts of the parasites.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Phlebotomus papatasi; Phlebotomus duboscqi; Phlebotomus bergeroti; Phlebotomus salehi; Vector; Leishmania major; Nested clade phylogeographic
analysis; Molecular systematics
www.elsevier.com/locate/meegid
Available online at www.sciencedirect.com
Infection, Genetics and Evolution 8 (2008) 159–170
* Corresponding author at: ‘‘VECPAR’’, AFSSA, Universite ´ de Reims Champagne-Ardenne, Faculte ´ de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex,
France. Tel.: +33 3 26 91 37 23; fax: +33 3 26 91 35 97.
E-mail address: jerome.depaquit@univ-reims.fr (J. Depaquit).
1567-1348/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.meegid.2007.12.001

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1. Introduction
The phlebotomine sand fly Phlebotomus papatasi (Scopoli,
1786) is widely spread in Europe, Africa and Asia, particularly
in those areas that lie between20 and 45 degrees in the northern
latitudes. It breeds as far West as Portugal, throughout the
Mediterranean littoral, the Adriatic region, the Balkans, the
Middle East, North Africa, Central Asia and India.
P. papatasi is the main provenvector of Leishmania major in
the Old World, North of the Sahara where zoonotic cutaneous
leishmaniasis is established in reservoir hosts. In areas Southern
of the Sahara, P. duboscqi Neveu-Lemaire, 1906 is the main
proven vector. In several foci, P. bergeroti Parrot, 1934 is
suspected to have a role in the transmission of this parasite, as P.
salehi Mesghali, 1965 the fourth species belonging to the
subgenus Phlebotomus which has also been found infected in
Iran with promastigotes which could be L. major (Kasiri and
Javadian, 2000). In most areas, P. papatasi, like other sandfly
species, can also transmit arboviruses (Tesh, 1989), and can
sometimescausethedermatologicalconditionknownasHarara.
Only three species in the genus Phlebotomus have a wide
distribution area (from Morocco and Spain to Central Asia): P.
(Phlebotomus) papatasi, P. (Paraphlebotomus) sergenti and P.
(Pa.) alexandri. A previous study, carried out on rDNA ITS2,
emphasized a molecular heterogeneity in P. sergenti, the main
vector of L. tropica (Depaquit et al., 2002) which could be
associated to a ‘‘patchwork’’ of populations with different
vectorial capacities. Previous studies also showed a low
mutation rate within P. papatasi populations from different
biotopes in Iran (Parvizi et al., 2003) as well as in populations
from different countries (Esseghir et al., 1997). Hamarsheh
etal.(2007)bysequencingcytochromebmtDNAemphasizeda
variability within Mediterranean populations of P. papatasi
without prove the existence of a species complex. It has been
shown that there are genetic factors controlling the suscept-
ibility or refractoriness of P. papatasi to L. major infections,
which can be selected for by breeding (Wu and Tesh, 1990a,b).
However,thevectorialcapacity forL.majorofP.papatasifrom
diverse environmental habitats has been shown to be dissimilar
(Schlein and Jacobson, 2001) No molecular phylogeny studies
has been reported for P. alexandri, the third Phlebotomus
species, which has an even wider distribution.
Consequently, the aim of this study was to sequence P.
papatasi molecular markers differing from cytochrome b mt
DNA used by Hamarsheh et al. (2007) previously used
successfully to highlight molecular variation inside species
having a large geographical range. We selected the ND4 gene
from the mitochondrial DNA, previously used by Soto et al.
(2001) on Lutzomyia longipalpis complex, and the second
internal transcribed spacer of the ribosomal DNA (an
independent marker of the mitochondrial one) used to show
variation within P. sergenti (Depaquit et al., 2002). Twenty-
seven different populations were sampled and we explored the
specific status of P. papatasi and P. bergeroti, including
Egyptian specimens from colonies where interbreeding
experiments were carried out, although hybrids were not
available (Fryauff and Hanafi, 1991).
2. Materials and methods
Our sampling included 22 populations of P. papatasi from
16 countries, 2 populations of P. bergeroti from 2 countries and
3 populations of P. duboscqi from 3 countries (Table 1). To
appreciate the existence of putative intrapopulational variation,
a pilot study was carried out on Israeli specimens coming from
colonies started with specimens caught in two distinct
localities: Jordan Valley (Schlein and Jacobson, 2002) and
Kfar Adumim (Schnur et al., 2004). The studied specimens
were caught using CDC miniature light traps or taken from
laboratory colonies (Table 1).
Phlebotomine sandflies were stored into ethanol or liquid
nitrogen. They were dried before DNA extraction. The head
and genitalia of a single sandfly were cut off in a drop of
ethanol, then cleared in boiling Marc-Andre ´ solution, and
mounted between slide and cover slide for identification (these
mountings are available upon request to the senior author).
Genomic DNAwas extracted from the thorax, wings, legs and
abdomen using the QIAmp DNA Mini Kit (Qiagen, Germany)
following the protocol used by Depaquit et al. (2004).
Polymerase chain reactions (PCR) were performed in a
50 ml volume using 5 ml of extracted DNA solution and
50 pmol of each of the primers C1a and JTS3 for ITS2
(Depaquit et al., 2002), ND4c and ND4ar for ND4 (Soto et al.,
2001). Purification of the PCR products involved agarose-gel
fractionation, then extraction by means of a QIAquick Gel
Extraction Kit (Qiagen, Germany). Direct sequencing in both
directions was performed by Qiagen (Hilden, Germany) using
the primers used for DNA amplification.
2.1. Phenetic and cladistic analyses
Sequence alignment and distance analysis using Neighbor-
Joining (NJ) were performed using the MUST software
package (Philippe, 1993). Maximum parsimony (MP) using
the branch and bound option, maximum likelihood (ML) and
bootstrap (1000 replicates) were performed using PAUP*
version 4.0 software (Swofford, 2002). Gaps were treated for
parsimony as fifth character state. The best-fit evolutionary
model for the maximum likelihood analyses were estimated in
Modeltest version 3.7 (Posada and Crandall, 1998). The best-fit
maximum likelihood parameters were used to estimate the ML
tree using the quartet puzzling option. Branches were collapsed
if the branch length was less than or equal to zero.
2.2. Population genetic analysis
A nested clade analysis was performed to examine the
population structure and population history (Templeton et al.,
1995). We first used the program TCS 1.21 (Clement et al.,
2000) to construct haplotype networks using ND4 and ITS2
sequence data with a confidence level above 95%. Indels were
treatedasafifth state.Nestingofhaplotypes wasmadeby hand,
according to the rules outlined in Templeton and Sing (1993)
and Templeton (1998). Ambiguous loops were solved by using
the empirical predictions derived from coalescent theory,
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summarized by three criteria by Pfenninger and Posada (2002):
frequency, topological and geographic criteria.
The program GEODIS 2.5 was used to test significant
associations between haplotypes and geography using a nested
permutational contingency analysis and a nested geographical
distanceanalysis(TempletonandSing,1993;Templeton,1995).
Two main statistics were computed in the second analysis: the
clade distance, Dc, which represents geographic range for the
respective clade level and the nested clade distance, Dn, which
measures the average distance of samples with a certain
haplotype compared to the geographic centre of the clade with
respect to its sister clades (Templeton, 1998). The difference
between these two measures for interior and tip clades in the
network (I-T Dcand I-T Dn, respectively) was then calculated,
and the null hypothesis of spatial panmixia tested. Statistically
significant patterns were interpreted using the inference key
presented by Templeton (2004) and upgraded in 2005 (available
on line from http://darwin.uvigo.es/software/geodis.html).
Nucleotidediversity(p)(JukesandCantor,1969),haplotype
diversity (h) (Nei, 1987) and their standard deviation were
quantified with DNAsp version 4.10.9 (Rozas et al., 2003). The
demographic history of the populations was also investigated
by mismatch distribution of pairwise nucleotide differences of
the ND4 sequence data with Arlequin version 3.1 (Excoffier
et al., 2005). If a population had undergone rapid expansion, a
unimodal mismatch distribution approximating a Poisson’s
curve was expected (Slatkin and Hudson, 1991; Rogers and
Harpending, 1992). Tests for neutrality can also assess the
demographichistory.WeusedFu’sFstestwhichhasbeenshown
to be one of the best statistics for detecting population growth
(Fu, 1997; Ramos-Onsins and Rozas, 2002). Fu and Li’s D* and
F* statistics were also computed (Fu and Li, 1993). These three
tests are implemented in DNAsp using the program Neutrali-
tyTest, kindly provided by H. Li (available online at http://
www.hgc.sph.uth.tmc.edu/neutrality_test/) which includes the
indels during the computation. We tested their significativity by
coalescent simulations (10,000 permutations). Population
growth or range expansion were distinguished from effects of
background selection by the pattern of significance between Fs,
D*andF*(Fu,1997).IfonlyFswassignificantwhereasD*and
F* were not, then hitchhiking, population growth or range
expansion could be suggested, whereas the reverse suggests
background selection (Fu, 1997).
3. Results
The sequences have been deposited in Genbank (accession
numbers EF408752 to EF408802, AF218321 and AF218316).
Table 1
Specimens sequenced
SpeciesOrigin (country, area or locality and catching) Number of studied specimens
P. papatasi (Scopoli, 1786)Albania
Algeria
Cyprus
Kruje
Constantine
Astromeritis
Vassileya
Lapithos
Sinaı ¨
Skopi, Crete
Poona
Sistan-Baluchistan
Jordan Valley
Kfar Adumim
Rome
Rocca Priora
Raas-Balbeck
Sefrou
Hofouf
Orgiva
Rhuhaibe
Utique
Denizli
Urfa
Izmir area
Light trap
Sticky paper
Light trap
Light trap
Colony
Colony
Light trap
Colony
Sticky paper
Colony
Colony
Colony
Colony
Light trap
Light trap
Colony
Light trap
Light trap
Light trap
Light trap
Light trap
Light trap
Light trap
Light trap
2 males
2 males
1 male
2 males
2 males
2 males; 2 females
2 males
2 males
2 males
2 males; 1 female
2 males; 1 female
2 males
2 males
3 males
4 males
3 males
1 male
3 males
2 males; 1 female
1 male
2 males
2 males
1 male
2 males; 1 female
Egypt
Greece
India
Iran
Israel
Italy
Lebanon
Morocco
Saudi Arabia
Spain
Syria
Tunisia
Turkey
Yemen
MontenegroDabc ˇevic ´i
P. duboscqi (Neveu-Lemaire, 1906)Burkina Faso
Kenya
Senegal
OuagadougouLight trap
Colony
Colony
2 males; 1 female
2 males; 1 female
1 male
P. bergeroti (Parrot, 1934) Egypt
Iran
Oman Sultanate
Sinaı ¨
Sistan-Baluchistan
Salalah-Herwouib
Rowda
Colony
Sticky paper
Light trap
Light trap
2 males
2 males
2 males
1 male
P. salehi (Mesghali, 1965) IranSistan-Baluchistan Sticky paper 2 males
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3.1. Analysis of the mtND4 dataset
The size of fragments was similar for all specimens. The
alignment was 600 sites long including 156 variable sites, of
which 85 were parsimony informative and 71 parsimony
uninformative. The ND4 region was AT rich within the
Phlebotomus, with a proportion of AT greater than 71%
(71.83% for Lebanese P. papatasi to 73.67% for P. salehi).
Sequences were identical forspecimenssampled from the same
location with no intrapopulational variation. Focusing on P.
papatasi populations, no or little variation (<1%) was
observed. Comparing P. bergeroti versus P. papatasi, 4–5%
variation was observed. There was 12–14% variation regarding
P. duboscqi versus P. papatasi and 4–5% variation between P.
bergeroti and P. duboscqi. P. salehi showed variations of 9.5–
10% versus P. papatasi, 10.2–10.4% versus P. bergeroti and
6.3–7.7% versus P. duboscqi.
Topologies of the trees obtained by NJ, ML and MP are
given in Fig. 1. For ML analysis, Modeltest indicated that the
best-fit model for ND4 was TVM + G.
The two populations of P. duboscqi are sister groups,
strongly supported by high bootstrap values. P. bergeroti forms
a strong clade in which populations from Egypt and Oman are
closer to each other than to the Iranian population which is their
sister group. P. papatasi is a strong clade in which the Indian
population is the sister group of the other populations. P.
bergeroti and P. papatasi are two sister groups. The position of
P.salehi isdoubtful.Itappears asthe sistergroupofP.duboscqi
according to MP analysis. Their grouping is poorly supported
by a bootstrap value of 64%. A similar topology is obtained
with NJ analysis whereas P. salehi appears as the sistergroup of
a clade including the three other species.
Within P. papatasi, 11 haplotypes including 12 segregating
sites have been observed, which seems to be high regarding the
low nucleotide diversity and high haplotype diversity (Table 2).
About one haplotype was observed per country, except for
haplotypes1,2,3and7sharedbyseveralpopulations (Table3).
The variable sites are summarized in Fig. 2.
The network obtained after TCS analysis exhibited a star-
like pattern as shown in Fig. 3.
The network contains five 1-step clades and one 2-step
clades. The haplotype 3 is the central to which the other ones
are connected, directly or through at maximum two mutation
steps.
The results of the nested clade analysis can be found in
Tables 4 and 5.
The null association between genetic and geographic
distribution was rejected for 1-1 and the total cladogram. A
restricted gene flow, or some long distance dispersal seems to
occur for the haplotypes nested in clade 1-1 (Table 5).
The mismatch distribution closely fit unimodal curve
typically associatedwithrecent
(Fig. 4). The results for the various neutrality tests are
summarized in the Table 2. Fu’s Fsis negative and significant
whereas Fu and Li F* and D* are not statistically significant for
the whole sampling.
populationexpansion
3.2. Analysis of the rDNA ITS2 dataset
The size of fragments was species dependent, and varied
between 419 (P. duboscqi, Senegal) and 437 (P. salehi, Iran).
The alignment, including all specimens and using P. sergenti as
outgroup, was 469 characters long including 158 variable sites,
of which 56 were parsimony informative and 102 were
parsimony uninformative. The alignment is available upon
request to the senior author. The region was AT rich, with a
proportion of AT greater than 72% (72.07% for Egyptian P.
bergeroti to 73.33% for Roman P. papatasi). Sequences were
identicalforspecimenssampledfromthe samelocation with no
intrapopulational variation except in Italy, Egypt and Morocco
where two haplotypes differing by one or two mutations were
observed. No or very few mutations (<1%) were observed in P.
Fig.1. PhylogenetictreesobtainedafteranalysesofthemtDNAND4dataset.(A)NJtree.(B)Maximumlikelihoodtreewithbranchsupport,?ln L = 1796.6445.(C)
Strict consensus of the four shortest equiparcimonious trees obtained after a Branch and Bound search of PAUP. Length = 220 steps; CI = 0.8682; RI = 0.8612.
J. Depaquit et al./Infection, Genetics and Evolution 8 (2008) 159–170162

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papatasi populations. When comparing P. bergeroti and P.
papatasi, 3–5.5% variation was discerned. Therewas 7.9–8.4%
variation between P. duboscqi and P. papatasi, and 10–11.5%
variation between P. bergeroti and P. duboscqi. P. salehi differs
from P. papatasi, P. bergeroti and P. duboscqi by 1.9–2.8%,
5.3–7.6% and 9%, respectively.
Topologies of the trees obtained by NJ, ML and MP are
given in Fig. 5. For ML analysis, Modeltest indicated that the
best-fit model for ND4 was F81 + G.
P. bergeroti is a strong clade in which populations from
Egypt and Oman are close to each other.The Iranianpopulation
is their sister group. P. papatasi seems to be a clade. However,
regarding the NJ tree, P. salehi is included within the P.
papatasi branch, whereas it appears as the sister group of P.
duboscqi in the light of ML reconstruction, and as the sister
group of P. papatasi according to MP analysis, a position
supported by a strong bootstrap value.
Inorder tomeasure theweight of gaps inthe topologies, new
analyses (NJ, ML and MP) have been carried out on the ITS2
database excluding all the positions containing at least one gap.
This new database contains the haplotypes of P. sergenti (used
as outgroup), P. duboscqi, P. salehi, P. bergeroti (n = 3), and
four haplotypes of P. papatasi. Regarding the MP approach, P.
salehi and P. papatasi form a polytomy, whereas P. salehi
appears as the sister group of P. papatasi according to NJ
analysis,andasthesistergroupofP.duboscqiaccordingtoML.
Consequently, the ITS2 is informative between species without
gaps.
Table 2
Diversity (?S.D.) and neutrality estimates for ND4 and ITS2 Phlebotomus papatasi sequences from our sampling
P. papatasi Expectation
ND4 ITS2Selection Expansion
Number of haplotypes
Segregating sites
Nucleotide diversity, p
Haplotype diversity, h
Fu’s (1997), Fs
Fu and Li (1993), F*
Fu and Li (1993), D*
11
12
0.003 (?0.0002)
0.869 (?0.023)
?3.781*
?0.171
?0.371
16a; 6b
18a; 7b
0.002 (?0.0004)b
0.528 (?0.075)b
?2.341a; ?1.322b
1.201a; ?0.064b
0.989a; 0.443b
Low
Low
Not significant
Significant
Significant
Low
High
Significant
Not significant
Not significant
*Significant at the p < 0.05 level after coalescent simulations (10,000 replicates).
aIncluding indels.
bExcluding indels.
Table 3
ND4 haplotype frequencies of Phlebotomus papatasi by locality
CountryLocality Haplotypes TotalLatitudeLongitude
123456789 1011
Albania
Algeria
Cyprus
Kruje
Constantine
Astromeritis
Vassileya
Lapithos
Sinaı ¨
Skopi, Crete
Poona
Sistan-Baluchistan
Jordan Valley
Kfar Adumim
Rome
Rocca Priora
Raas-Balbeck
Dabc ˇevic ´i
Sefrou
Hofouf
Orgiva
Rhuhaibe
Utique
Denizli
Urfa
Izmir area
22
2
1
2
2
4
2
2
2
3
3
2
2
3
3
4
3
1
3
3
1
2
2
1
41828031N
36818020N
35808024N
35810015N
35819048N
28841028N
35816059N
24859019N
26841053N
32829035N
31846025N
41853033N
41847030N
34813012N
42802050N
33841049N
25840010N
36856020N
35848004N
37804027N
37852057N
37805058N
38827009N
15819009N
19836021E
06831012E
33802024E
32835009E
33809036E
33859040E
25852036E
74825022E
60820040E
35834049E
35813042E
12829022E
12845038E
36813050E
19812043E
04841054 W
49815010E
03828055 W
36843050E
09850001E
28857020E
38851008E
27804042E
47809020E
2
1
2
2
Egypt
Greece
India
Iran
Israel
4
2
2
2
3
3
Italy2
2
Lebanon
Montenegro
Morocco
Saudi Arabia
Spain
Syria
Tunisia
Turkey
3
3
4
3
1
3
3
1
2
2
Yemen1
Total7 101442273411 55
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Within P. papatasi, 6 haplotypes with 7 segregating sites
have been observed, which gives 16 different sequences and 18
segregating sites including gaps, after alignment (Fig. 6). In
order to maximize the genetic information, the gaps have not
been excluded from the following analyses, except for the
computation of the low nucleotide diversity and the 18
segregating sites (Table 2).
Haplotype 2 is shared by 7 populations from 4 countries.
Haplotype14issharedbytwogeographicallyclosepopulations
(Albania and Montenegro). We can note that specimens
sequenced from Egypt, Italy and Morocco Exhibit 2 haplotypes
per country (Table 6).
The network obtained after TCS analysis is shown in Fig. 7.
The network contains 12 1-step clades, 5 2-step clades, and 2 3-
step clades. The clade 2-1 includes an ambiguous loop which
cannot be resolved according to the usual methods.
The results of the nested clade analysis can be found in
Tables 7 and 8.
Although, the null association between genetic and
geographic distribution was rejected for 1-5, 1-8, 2-1, 2-3, 2-
4, 3-1, 3-2, and 4-1, many cases give inconclusive outcomes
probably due to an inadequate sampling. A past fragmentation
and/or long distance colonization is suggested for haplotypes
nested in the clade 1-8 (Albania, Montenegro, Spain and
Lebanon). A restricted gene flow with isolation by distance is
inferred for clade 3-2 grouping haplotypes from Iran, Egypt,
Syria, Yemen and Turkey (Table 8).
The mismatch distribution does not fit strongly with the
unimodal curve expected by the sudden expansion model
(Fig. 8). The results for the various neutrality tests are
summarized in the Table 2. They include or exclude gaps. In
both cases, Fu’s FsFuand Li F* and D*were neverstatistically
significant.
4. Discussion
Our results confirm the potential of ITS2 and ND4 to
distinguish sand flies at the specific level for a Phlebotomine
subgenus. The use of rDNA ITS2 and ND4 mtDNA has many
advantages, such as a high mutation rhythm, speed and ease of
use, multiple target sites, predefined marker systems, known
PCR primers, combined with a small database for sandflies
Fig. 2. Observed ND4 sequence variations of Phlebotomus papatasi. Identical bases are represented by a dot (.). Only variable sites, with sequence positions given
above, are shown. Refer to Table 3 for the references of haplotypes.
Fig. 3. Haplotype network estimated from the ND4 data of P. papatasi.
Haplotypes are indicated by number designations in Table 3. On the graph,
‘‘0’’ indicates intermediate haplotypes states, which are missing in the sample.
Each solid line without any number represents a single mutation change that
interconnects two haplotypes states for which parsimony is supported at the
95% level. Step 1 clades are designated as 1-X (with X the code number of a
given clade), and similarly Step 2 clade is labelled 2-X.
Table 4
Nested clade distance analysis of observed Phlebotomus papatasi
Haplotypes 1-Step clades2-Step clade
CladeDc
Dn
CladeDc
Dn
I
IV
V
VI
VII
I-T
1321
0S
0S
0
291L
1202L
1407
996
2762L
3437
1446
?477
1231
0
?1269
803
278
525
1-117121693
II
XI
I-T
1060
0
1060
1-3 15481490 2-1
III
X
I-T
772
0
772
1-4671S
965
VIII
IX
1-2
1-5
0S
0S
639
4439L
I-T 779L
239
ND4 haplotypes. Dcand Dnare clade and nested clade distances (Templeton
et al., 1995). Interior vs. tip contrasts for Dcand Dnare indicated with ‘I-T’ in
the corresponding clade. Interior clades are given in italic and bold type.
Superscripts S and L indicate that distance measures are significantly smaller
and larger, respectively, than expected under random distribution of haplotypes.
J. Depaquit et al./Infection, Genetics and Evolution 8 (2008) 159–170164

Page 7

(Depaquit et al., 2000; Di Muccio et al., 2000; Soto et al., 2001;
Depaquit et al., 2002). The rDNA, a multicopy gene, involves
homogenization processes usually of repeated genes in a
particular multi-gene family within a given species, called
molecular drive (Dover, 1982).
Within P. papatasi, the limited number of variations of the
selected markers contrasts with their variability observed in
other widespread species (Soto et al., 2001; Depaquit et al.,
2002).
4.1. Phenetic and cladistic analysis
P.duboscqi,P.bergerotiandP.papatasiformclades, aresult
strongly supported by bootstrap analysis. There is no doubt
about their specific status.P.bergerotiandP.papatasiappearas
sister species, with P. duboscqi being their sister species.
According to morphological characters, the specific status of
the latter species is strongly supported. However, its
phylogenetic position is doubtful according to our analyses.
According to ITS2, the long branch of P. salehi observed in NJ
tree (Fig. 2) is due in part to 16 autapomorphic character states
(Table 9).
The phylogenetic position of P. salehi will be clarified after
analysis of other molecular markers like another mitochondrial
gene (cytochrome b), another ribosomal marker (ITS 1) and a
nuclear one.
The introduction in our database of the sequence of an
Indian specimen of P. papatasi (Genbank accession number
AF205529) sequenced by Di Muccio et al. (2000) showed 4–
5% divergence, contrasting with our results. India being the
eastern limit of the distribution of P. papatasi, we decided to
sequence ourselves two specimens from India (Genbank
accession numbers EF408760 and EF408797 for ND4 and
ITS2, respectively)showinglessthan 1% divergence. Witha 4–
5% divergence rate, that is to say a specific level within this
group, similar to the papatasi–bergeroti divergence, the
specimens sequenced by Di Muccio et al. (2000) could belong
to another species. Their sequences have to be confirmed and
morphology checked. If not, we could consider this margin of
divergence as artifactual.
Thereismorevariabilitybetweenpopulations ofP.bergeroti
or in P. duboscqi than between populations of P. papatasi,
although Kato et al. (2006) have observed a high degree of
similarities between two geographically distant populations of
P. duboscqi.
Esseghir et al. (1997) investigated cyt b mt DNA
sequences and concluded that the divergence of P. duboscqi
unambiguously predated that of P. papatasi and P. bergeroti.
Our results also show that P. papatasi and P. bergeroti are
more closely related than to P. duboscqi. We concur with
Esseghir et al. (1997), that the formation of the Sahara desert
in the Miocene (10–6 millions years ago (mya)) probably
separated P. duboscqi from P. papatasi and P. bergeroti. The
separation of P. papatasi and P. bergeroti could be due to the
climatic drift toward greater aridity in Africa, 2.8–1.0 mya
(de Menocal, 1985). Indeed, P. papatasi has a distribution
area much greater than that of P. bergeroti, probably because
P. papatasi is a less thermophilic and low-land species than
P. bergeroti.
If P. sergenti settled in the Mediterranean Basin during
Miocene (Depaquit et al., 2000, 2002), we could consider, in
light of the present results that the Mediterranean settlement by
P. papatasi is recent.
4.2. P. papatasi intraspecific analysis
Taking intoconsideration thedoubtful phylogeneticposition
of P. salehi, we decided not to use it within this intraspecific
analysis.
Table 5
Nested contingency results and inferred patterns of ND4 haplotypes observed in Phlebotomus papatasi
CladePermutational
x2-statistic
Probability
(1000 iterations)
Inference chainInferred pattern
Haplotypes nested in 1-1124
<0.001*
1-2-3-5-6-7-YesRestricted gene flow/
dispersal but with some
long distance dispersal
Sampling design
inadequate to
discriminate between
isolation by distance
vs. long distance dispersal
One-step clades nested in 2-1220
<0.001*
1-2-3-5-6-7-8-No
Clades are the same as in Table 4 and Fig. 3.
*Significant at the p < 0.05 level.
Fig. 4. Mismatch distribution for the ND4 haplotypes for Phlebotomus papa-
tasi. The observed frequency is represented by white boxes and the expected
frequency under the expansion model is depicted by solid line connecting dots.
J. Depaquit et al./Infection, Genetics and Evolution 8 (2008) 159–170165

Page 8

No robust intraspecific phylogeographic structure emerged
in the parsimony, maximum likelihood and Neighbor-Joining
analyses. No clear phylogeographic structure could be
discerned among the samples for both ITS2 and ND4 datasets.
The absence of geographic structure in both molecular markers
is not unexpected in this species which has no geographically
structured morphological variation as observed within P.
sergenti s.l. or L. longipalpis, having comparable wide
distribution areas. Hamarsheh et al. (2006) have found a little
polymorphism linked with microsatellite loci within P.
papatasi. According to morphological data, Belen et al.
(2004)havefoundmorphologicalvariabilitywithinP.papatasi.
In 2007, Hamarsheh et al. conclude this variability of
cytochrome b mtDNA does not support the existence of a
species complex.
Fig. 5. Phylogenetic trees obtained after analyses of the ITS2 rDNA dataset. (A) NJ tree. (B) Maximum likelihood treewith branch support, ?ln L = 1206.0172. (C)
Strict consensus of the 42 shortest equiparcimonious trees obtained after a Branch and Bound search of PAUP. Length = 226 steps; CI = 0.8717; RI = 0.8041.
Fig. 6. Sequence variations observed in the second ribosomal internal tran-
scribed spacer (ITS-2)ofPhlebotomuspapatasi.Identicalbases are represented
by a dot (.) and gap by a minus sign (?). Only variable sites, with sequence
positions given above, are shown. Refer to Table 6 for the references of
haplotypes.
Fig. 7. Haplotype network estimated from the ITS2 data of P. papatasi.
Haplotypes are indicated by number designations in Table 6. On the graph,
‘‘0’’ indicates intermediate haplotypes states, which are missing in the sample.
Each solid line without any number represents a single mutation change that
interconnects two haplotypes states for which parsimony is supported at the
95% level. Step 1 clades are designated as 1-X (with X the code number of a
given clade), andsimilarlySteps2, 3 and 4 clades are labelled2-X, 3-X and 4-X,
respectively. 1-Step clades are contained within white boxes, 2-step clades are
delineated by dashed lines, 3-step clades are contained within larger boxes. The
overall cladogram is a single 4-step clade, delineated by dashed lines.
J. Depaquit et al./Infection, Genetics and Evolution 8 (2008) 159–170 166

Page 9

Taking into account the vast distribution area of P. papatasi,
the largesampling of the present study, and the results shown in
Tables 5 and 8, it seems probable that P. papatasi is
characterized by a restricted gene flow. It is, however, limited
by the insularity of some populations. Considered as poor
flyers, insular Phlebotomine sandflies cannot have continuous
connections with continental populations now. The fact that
insular populations (Crete, Cyprus) share similar haplotypes
with continental ones is intriguing. The exclusive biogeogra-
phical events are the recent glaciations (1.8 mya–12,000 years
ago) which could permit the possible settlement of Crete and
Cyprus 1 million years ago. However, the molecular clock of P.
papatasi for the sequenced domains seems slower than that of
Larroussius (Esseghir et al., 1997, 2000; Aransay et al., 2003).
Despite proposing the paleogeography of P. papatasi, our
results provide no support for natural introgression between P.
papatasi and P. bergeroti, which are known to interbreed under
experimental conditions (Fryauff and Hanafi, 1991). In 1999,
Ghosh et al. obtained experimentally hybrids between P.
papatasi and P. duboscqi, but to our knowledge, hybrids
morphologically characterized have yet to be found in nature.
The increasing of the sampling (new populations, more
specimens per population) could perhaps better resolve this
point.Newmolecularmarkershavingahighermutation rhythm
could be more informative within P. papatasi than ITS2 and
ND4 to resolve intraspecific phylogeography.A few haplotypes
are shared between populations for both molecular markers.
Some of them are shared between populations without
geographic connections (i.e. Saudi Arabia, Syria, Spain,
Cyprus and Crete for ND4 or Tunisia, Israel, Crete and Cyprus
for ITS2).
Nuclear DNA and mtDNA datasets show incongruous
patterns of past demographic history. ND4 has significant
negative value for Fu’s Fs, no significant values for Fu and Li’s
D* and F*, low nucleotide diversity, high haplotype diversity
and the shape of the mismatch distribution is unimodal. These
results suggest an expansion of population (Rogers and
Harpending, 1992; Fu, 1997). However, this result was not
found for ITS2 dataset. But, the Fu’s Fs is negative and
marginally non-significant which does not clearly indicate an
opposite conclusion. Increasing the sampling could have an
impact on the indexes values of neutrality tests and the shapes
of the mismatch distribution and ITS2 haplotype network.
We note within P. papatasi, that 16 haplotypes occur for
ITS2 versus 11 for ND4. A higher number of haplotypes for
rDNAmarkerthanformitochondrialND4issurprising because
in general the mtDNA evolves more rapidly than nuclear DNA,
especially for single-copy nuclear DNA (but ITS is a multicopy
nuclear DNA). However, in fruit flies, the rates of nucleotide
substitution of mitochondrial and nuclear scnDNA are similar
(Powell et al., 1986). Moreover, the rate of nucleotide
substitution in the mitochondrial DNA differs from a gene to
another one and a few studies related to ND4 have been
published (Soto et al., 2001; Depaquit et al., 2004).
The experimental approach used in this study could ease the
use of molecular tools for identification of naturally infected
sandflies using specific primers or PCR-RFLP in foci where
several species belonging to the subgenus Phlebotomus are
Table 6
ITS2 haplotype frequencies of Phlebotomus papatasi by locality
CountryLocality HaplotypesTotal Latitude Longitude
123456789 101112 1314 1516
Albania
Algeria
Cyprus
Kruje
Constantine
Astromeritis
Vassileya
Lapithos
Sinaı ¨
Skopi, Crete
Poona
Sistan-Baluchistan
Jordan Valley
Kfar Adumim
Rome
Rocca Priora
Raas-Balbeck
Dabc ˇevic ´i
Sefrou
Hofouf
Orgiva
Rhuhaibe
Utique
Denizli
Urfa
Izmir area
22
2
1
2
2
4
2
2
2
3
3
2
2
3
3
4
3
1
3
3
1
2
2
1
41828031N
36818020N
35808024N
35810015N
35819048N
28841028N
35816059N
24859019N
26841053N
32829035N
31846025N
41853033N
41847030N
34813012N
42802050N
33841049N
25840010N
36856020N
35848004N
37804027N
37852057N
37805058N
38827009N
15819009N
19836021E
06831012E
33802024E
32835009E
33809036E
33859040E
25852036E
74825022E
60820040E
35834049E
35813042E
12829022E
12845038E
36813050E
19812043E
04841054W
49815010E
03828055W
36843050E
09850001E
28857020E
38851008E
27804042E
47809020E
2
1
2
2
Egypt
Greece
India
Iran
Israel
22
2
2
2
3
3
Italy2
2
Lebanon
Montenegro
Morocco
Saudi Arabia
Spain
Syria
Tunisia
Turkey
3
3
22
3
1
3
3
1
2
2
Yemen1
Total313 2212232252452255
J. Depaquit et al./Infection, Genetics and Evolution 8 (2008) 159–170167

Page 10

found in sympatry. Although the identification of males of the
subgenus Phlebotomus is relatively easy, the identification of
females (which can only be distinguished by pharyngeal
examination and computation of the ascoid/fourth antennal
segment lengths ratio) is more difficult for non-specialists.
4.3. Epidemiological consequences
Phlebotomine sandflies belonging to the subgenus Phlebo-
tomus are the main vectors of L. major (Dedet et al., 1982;
Killick-Kendrick et al., 1985; Lane and Fritz, 1986; Hanafi
et al., 1996; Kasiri and Javadian, 2000). Paraphlebotomus and
Synphlebotomus are suspected vectors (Killick-Kendrick,
1990). Recently, two species belonging to the subgenus
Adlerius were incriminated in the L. tropica transmission. P.
arabicus was found naturally infected (Jacobson et al., 2003)
and P. halepensis was experimentally infected (Sadlova et al.,
2003).
The distribution area of L. major is within that of the
sandflies belonging to the subgenus Phlebotomus. Some areas
Table 7
Nested clade distance analysis of observed Phlebotomus papatasi
Haplotypes1-Step clades 2-Step clades 3-Step clades4-Step clade
CladesDc
Dn
CladesDc
Dn
CladesDc
Dn
CladesDc
Dn
XIV
XV
I-T
XIII
XIV
I-T
0
0
0
0
623S
623
1633
776
838
1808L
731S
?1077S
3-11158S
1148S
4-1
1-410621241
2-3 9511061
1-5865896
I-T
1-8
?196
1081
?344
1160
X
XI
XII
I-T
IX
36S
0S
0
36
368S
1708L
1882
?1384S
2-4 1239 1365
1-9
I-T
0
Status?
1868
I-T 289304
I
II
1-1
1-11
I-T
1-2
1-10
I-T
1-6
1-3
1-7
1-12
I-T
0
0
0
0
0
Status?
0
0
427
1251
529
1262
1266
4
571
1189
3-2 17401862L
2-2 12642359
III
IV
2-5777 3097L
V
VIII
VII
VI
613
2100L
1046
1250
-310
2-11238S
1236S
I-T
?4681550L
I-T Status?
ITS2 haplotypes. Dcand Dnare clade and nested clade distances (Templeton et al., 1995). Interior vs. tip contrasts for Dcand Dnare indicated with ‘I-T’ in the
corresponding clade. Interior clades are given in italic and bold type. Superscripts S and L indicate that distance measures are significantly smaller and larger,
respectively, than expected under random distribution of haplotypes.
Table 8
Nested contingency results and inferred patterns of ITS2 haplotypes observed in Phlebotomus papatasi
CladePermutational
x2-statistic
Probability
(1000 iterations)
Inference chainInferred pattern
Haplotypes nested in 1-5
Haplotypes nested in 1-8
18
18
0.025*
0.004*
1-19-20-No
1-19-20-2-3-5-15-No
Inadequate geographical sampling
Past fragmentation and/or long
distance colonization
Inconclusive outcome
–
–
–
Restricted gene flow with isolation
by distance
Inconclusive outcome
One-step clades nested in 2-1
One-step clades nested in 2-3
One-step clades nested in 2-4
Two-steps clades nested in 3-1
Two-steps clades nested in 3-2
39
22
11
33
44
<0.001*
0.001*
0.036*
<0.001*
<0.001*
1-19-20-2-11-17-No
No significant clade analysis
No significant clade analysis
No significant clade analysis
1-2-3-4-No
Three-steps clades nested in 4-146.67
<0.001*
1-2-No
Clades are the same as in Table 7 and Fig. 7.
*Significant at the p < 0.05 level.
J. Depaquit et al./Infection, Genetics and Evolution 8 (2008) 159–170 168

Page 11

whereP.papatasiisrecordedareL.majorfree(Portugal,Spain,
Greece and Cyprus). In Turkey, a few cases of leishmaniasis
caused by L. major were recorded (Akman et al., 2000). Their
status need to be clarified at the light of isoenzymatic typing of
the strains and by collecting more details about their
autochtonous origin. Moreover, additional studies focusing
on the usual rodent hosts have to be carried out.
It has recently been found in Morocco that significant
molecular variations in P. sergenti can also occur in close foci
of cutaneous leishmaniasis (Yahia et al., 2004). Considering
thelowdivergencerateobservedinthepresentstudy,wecould
correlate it with the similar vectorial capacities of P. papatasi
to transmit L. major and phleboviruses throughout its
distribution area. The epidemiology of the leishmaniasis
due to L. major, a zoonosis, is also driven by the presence or
absence of its usual rodent hosts (Rhombomys, Psammomys,
Arvicanthis, Mastomys, Tatera, etc.), while the zoonotic host
forsandflyfeverisunproven.Aparticularattentionisrequired
in L. major free areas, which are also areas free of its usual
rodenthosts.Theintroductionofthesehostsincountrieswhere
P.papatasiisendemiccouldbefollowedbytheintroductionof
the disease.
Acknowledgements
The authors wish to thank M. Boutry and C. Grimplet for
their technical help, N. Haddad, B. Pesson and J.A. Rioux for
providing us Phlebotomine sandflies, and M. Kaltenbach for
proof-reading this manuscript.
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Number of position shared exclusively by taxa
ND4ITS2
salehi (autapomorphies)
salehi-papatasi
salehi-duboscqi
salehi-bergeroti
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