1- Dr Parvizi RTL 89 10 1_3_ Iran J Arthropod-Borne Dis, 2010, 4(2): 1–10 P Parvizi et al.: Molecular Typing and …. 1 Original Article Molecular Typing and Phylogenetic Analysis of Some Species Belonging to Phlebotomus (Larroussius) and Phlebotomus (Adlerius) Subgenera (Diptera: Psychodidae) from Two Locations in Iran *P Parvizi 1, SR Naddaf 2, E AlaeeNovin 1 1Molecular Systematics Laboratory, Pasteur Institute of Iran, Tehran, Iran 2Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran (Received 5 May 2010; accepted 5 Dec 2010) Abstract Background: Haematophagous females of some phlebotomine sandflies are the only natural vectors of Leishmania species, the causative agents of leishmaniasis in many parts of the tropics and subtropics, including Iran. We report the presence of Phlebotomus (Larroussius) major and Phlebotomus (Adlerius) halepensis in Tonekabon (Ma- zanderan Province) and Phlebotomus (Larroussius) tobbi in Pakdasht (Tehran Province). It is the first report of these species, known as potential vectors of zoonotic visceral leishmaniasis in Iran, are identified in these areas. Methods: In 2006-2007 individual wild-caught sandflies were characterized by both morphological features and sequence analysis of their mitochondrial genes (Cytochrome b). The analyses were based on a fragment of 494 bp at the 3´ end of the Cyt b gene (Cyt b 3´ fragment) and a fragment of 382 bp CB3 at the 5´ end of the Cyt b gene (Cyt b 5´ fragment). We also analysed the Cyt b Long fragment, which is located on the last 717 bp of the Cyt b gene, followed by 20 bp of intergenic spacer and the transfer RNA ser(TCN) gene. Results: Twenty-seven P. halepensis and four P. major from Dohezar, Tonekabon, Mazanderan province and 8 P. tobbi from Packdasht, Tehran Province were identified by morphological and molecular characters. Cyt b 5´ and Cyt b 3´ fragment sequences were obtained from 15 and 9 flies, respectively. Cyt b long fragment sequences were ob- tained from 8 out of 27 P. halepensis. Conclusion: Parsimony analyses (using heuristic searches) of the DNA sequences of Cyt b always showed mono- phyletic clades of subgenera and each species did form a monophyletic group. Keywords: Mitochondrial Cytochrome b, Phlebotomus (Larroussius) major, Phlebotomus (Larroussius) tobbi, Phlebotomus (Adlerius) halepensis, Iran Introduction Visceral leishmaniasis is a deadly dis- ease caused by parasitic protozoa belonging to genus Leishmania, transmitted to humans through the biting of infected female sand- flies. Three species of Leishmania including L. donovani and L. infantum from the old world and L. chagasi from the new world are known to give rise to the visceral form of leishmaniasis. The disease is endemic in Iran and the etiological egent is known to be L. infantum, which mainly affects children, with majority of cases from primary foci in North- western and Southern of the country (Mohe- bali et al. 2005). Sandflies of the subgenera Larroussius and Adlerius belonging to genus Phlebotomus are known as primary vectors of zoonotic visceral leishmaniasis (ZVL) in Iran (Nadim et al. 1978, 1992, Parvizi et al. 2008) *Corresponding author: Dr Parviz Parvizi, Email: parp@pasteur.ac.ir Iran J Arthropod-Borne Dis, 2010, 4(2): 1–10 P Parvizi et al.: Molecular Typing and …. 2 and also P. (Paraphlebotomus) alexandri re- cently reported as the vectors ZVL in Iran (Azizi et al. 2006). Females of the subgenus Adlerius and some females of subgenus Larroussius cannot be differentiated based on morphological fea- tures. Recently, the females of the two sub- genera were identified using sequence analysis of Cytochrome b gene (Cyt b) (Killick-Ken- drick 1990, 1999, Esseghir et al. 1997, 2000, Parvizi and Amirkhani 2008). The objective of this report was to iden- tify and type molecularly some species be- longing to P. larroussius and P. adlerius sub- genera from two locations in Iran. This paper reports the presence of sandflies of subgenera Adlerius and Larroussius in Caspian Sea lit- toral and Pakdasht area. We could differenti- ate the female specimens of subgenera Adlerius and Larroussius spiecies using analysis of Cyt b gene. It is noteworthy that up to present no record of any sandfly species from Caspian Sea littoral was avilable Materials and Methods The study area included two villages of Meyan Kooh and Imamzadeh Ghasem in Do- hezar area, (about 35 km west of Tonekaboon), Mazanderan Province (Caspian littoral) and two villages of Geshlagh and Mamazand in Pakdasht area (about 30 km south of Tehran City, Tehran Province. Sandflies were col- lected by aspirators and sticky papers (A4 papers soaked in castor oil) from inside and outside of animal shelters, and miniature CDC light traps (Sudia and Chamberland 1962), placed overnight in animal shelters. All col- lected sandflies first were processed to remove oil then stored at -20º C until used. The sand- flies were identified based on morphological features of heads and last abdominal segments to the extent possible according to the keys described by Nadim and Javadian (1976) and Lewis (1982) and then thorax and abdomen of sandfly were individually subjected to DNA extraction as described by Parvizi et al. (2003). Three pairs of primers designed by Par- vizi and Ready (2006) were used to amplify the Cyt b gene. CB1-SE (forward) and CB3- R3A (reverse) were used to amplify a more 5' fragment of 439 bp (CB1 fragment), CB3- FC (forward) and N1N-FA (reverse) amplified an overlapping 3' fragment of 499 bp (CB3 fragment) and CB1-SE (forward) and CB- R06 (reverse) amplified the Cyt b long frag- ment as one piece of 717 bp length. The PCR condition and reagents for all amplifications were according to Parvizi and Ready (2006) except for the Cyt b long fragment in which the annealing was performed in one stage at 48 ºC. PCR products were directly sequenced in both directions to identify sandflies haplo- types associated with individual female and male sandflies. All haplotypes were identified to species by phylogenetic analysis. DNA se- quences were edited and aligned using Se- quencherTm 3.1.1 software (Gene Codes Cor- poration). Multiple alignments of new DNA sequences and GenBank sequences were made using PAUP* software (Swofford 2002) for phylogenetic analysis. Results A total of 43 female and male sand flies from 4 different collection areas were studied, from which three phlebotomine species were morphologically identified. Tonekabon Phlebotomus major (4) and P. halepen- sis (28) were the only prevalent species in Tone- kabon. The identity of 27 sand flies, all from Dohezar area, including 21 males and 6 females were determined as Phlebotomus (Adlerius) halepensis sandflies) based on both morphologi- cal features and sequencing data of Cyt b gene. Sequence analysis of Cyt b 5´ fragment obtained from 15 sandflies showed that 8 Iran J Arthropod-Borne Dis, 2010, 4(2): 1–10 P Parvizi et al.: Molecular Typing and …. 3 (53.3%) were haplotype IRN279, 2 (13.3%) haplotype IRN277, 2 (13.3%) haplotype IRN 282, 2 (13.3%) haplotype IRN285 and 2 the unique haplotypes IRN293 and IRN294 (Ta- ble 1). Genetic distances between haplotypes were as low as 0.00262 – 0.00581. All the 7 (there is only 4) Cyt b 3´ sequences fell in the same haplotypes, named as IRN277 (Table 1). Cyt b Long fragment sequences were obtained from 8 out of 27 P. halepensis with the five (62.5%) revealing haplotype IRN279, 2(25%) haplotype IRN381 and 1 (12.5%) the unique haplotype IRN279 (Table 1). Genetic distances between haplotypes were as low, 0.00139 – 0.00278. Four Phlebotomus (Larroussius) major (2 male, 2 female) were identified by morpho- logical and molecular characters from Dohezar area of Tonekabon in Mazanderan Province. Cyt b 5´ sequences were obtained from 3 out of 4 flies. Two (66.7%) were haplotype IRN385, and one was a unique haplotype (IRN 287) (Table1). The genetic distance between haplotypes was as low as 0.01597. Cyt b 3´ sequences were obtained from 3 out of 4 flies, 2 were haplotype IRN385, and 1 was haplotype (IRN287) (Table 1). The genetic distance between haplotypes was as low, as 0.01597. Cyt b Long fragment sequences were obtained from all four P. major. Two (50%) were haplotype IRN385, and two (50%) were haplotype (IRN287) (Table 1). The genetic dis- tance between haplotypes as low as 0.02234. Pakdasht The majority of sandflies of Pakdasht were P. papatasi and some species belong to Paraphlebotomus and Sergentomyia. Sequenc- ing data obtained from Cytochrome b gene offe- male specimen revealed that the only prevalent species of subgenera Larroussius was P. tobbi. Based on both morphological and mo- lecular features all the 8 male sandflies col- lected from Pakdasht (5 from Mamazand re- gion using sticky papers and 3 from Ghesh- lagh using CDC light traps) were identified as Phlebotomus (Larroussius) tobbi. No Cyt b 5´ fragment sequences was obtained from P. tobbi specimens as the prod- ucts of PCR amplification were too weak to be sequenced. Cyt b 3´ fragment sequences were obtained from 7 out of 8 P. tobbi (Table 1). Two (28.6%) were haplotype IRN 334, 2 (28.6%) haplotype IRN335, 2(28.6%) haplotype IRN 338 and 1 was the unique haplotype IRN336. Genetic distances between haplotypes were as low as 0.00319-0.01597. Cyt b long frag- ment sequence was obtained only from one P. tobbi specimen (IRN413). However, the se- quence was too short to be analysed (Table 1). Nucleotide sequence data reported in this paper are available in GenBank, EMBL and DDBJ databases under accession numbers from HQ391905 to HQ391913. Table 1. All DNA haplotypes of Cyt b of subgenera Larroussius /Adlerius species identified in two locations in Iran (I.H = inside house, Ash= animal shelter, S.P= sticky paper, CDC= CDC miniature light traps) Cyt b haplotype Provinces location habit at trap type CB1-SE CB3R3A CB3FC NINFA CB1SE CB-R06 speci- men N. sex P. tobbi Ash S.P Not done IRN334 Not done IRN334 M Ash S.P Not done IRN335 Not done IRN335 M Tehran Pakdasht-Gheshlagh Ash S.P Not done IRN336 Not done IRN336 M Iran J Arthropod-Borne Dis, 2010, 4(2): 1–10 P Parvizi et al.: Molecular Typing and …. 4 Ash S.P Not done IRN335 Not done IRN337 M Ash S.P Not done IRN338 Not done IRN338 M Ash CDC Not done IRN338 Not done IRN339 M Ash CDC Not done IRN334 Not done IRN340 M Pakdasht-Mamazand Ash CDC Not done weak band IRN413 IRN413 M P. major Tonekabone- Meian kooh I.H Asp IRN287 IRN287 Not done IRN287 M Tonekabone- Imamzade ghasem I.H Asp IRN291 IRN287 Not done IRN291 M Tonekabone- Meian kooh I.H Asp Not done Not done IRN385 IRN385 F Mazanderan Tonekabone- Imamzade ghasem I.H Asp Not done Not done IRN385 IRN390 F P. halepensis I.H Asp IRN277 IRN277 Not done IRN277 M Tonekabone- Imamzade ghasem I.H Asp IRN277 IRN277 Not done IRN278 M I.H Asp IRN279 IRN277 Not done IRN279 M I.H Asp IRN279 Not done Not done IRN280 M I.H Asp IRN282 Not done Not done IRN281 M I.H Asp IRN282 Not done Not done IRN282 M I.H Asp weak band IRN277 Not done IRN283 M I.H Asp IRN279 Not done Not done IRN284 M I.H Asp IRN285 Not done Not done IRN285 M I.H Asp IRN279 Not done Not done IRN286 M Tonekabone- Meian kooh I.H Asp IRN279 Not done Not done IRN288 M I.H Asp IRN279 Not done Not done IRN289 M I.H Asp IRN285 Not done Not done IRN290 M I.H Asp IRN279 Not done Not done IRN292 M I.H Asp IRN293 Not done Not done IRN293 M I.H Asp IRN294 Not done Not done IRN294 M Tonekabone- Imamzade ghasem I.H Asp bad sequence IRN277 Not done IRN295 M I.H Asp IRN279 Not done Not done IRN296 M I.H Asp weak band Not done IRN297 IRN297 M I.H Asp Not done Not done IRN380 IRN380 F I.H Asp Not done Not done IRN381 IRN381 F Tonekabone- Meian kooh I.H Asp Not done Not done IRN381 IRN384 F I.H Asp Not done Not done IRN381 IRN386 F Tonekabone- Imamzade ghasem I.H Asp Not done Not done IRN381 IRN388 F I.H Asp Not done IRN277 weak band IRN389 F I.H Asp Not done Not done IRN381 IRN392 F I.H Asp Not done IRN277 weak band IRN393 F Mazanderan Tonekabone- Meian kooh I.H Asp Not done Not done IRN380 IRN394 M I.H Asp Not done bad sequence IRN383 IRN383 F I.H Asp Not done bad sequence weak band IRN387 F Mazanderan Tonekabone- Meian kooh I.H Asp Not done Not done Not done IRN391 F Table 1. Countinued… Iran J Arthropod-Borne Dis, 2010, 4(2): 1–10 P Parvizi et al.: Molecular Typing and …. 5 IRN383 R06 IRN282 IRN277 IRN278 IRN279 IRN285 IRN293 IRN294 IRN295 IRN 380 IRN381 IRN336 IRN334 IRN335 IRN338 IRN287 IRN291 IRN385 P. major P. halep ensis P. unkno wn P. tobbi 1 nucleotide change Fig. 1. Unrooted consensus phylogenetic tree for DNA sequences of Cyt b Long (718 nucleotides) of Phlebotomus (Adlerius) / Phlebotomus (Larroussius), produced by branch and bound parsimony search using PAUP* Iran J Arthropod-Borne Dis, 2010, 4(2): 1–10 P Parvizi et al.: Molecular Typing and …. 6 IRN336 CB3 IRN334 CB3 IRN335 CB3 IRN338 CB3 IRN385 R06 IRN287 CB3 IRN383 R06 IRN277 CB3 10 nucleotide changes P. tobbi P. major P. halepensis P. unknown 10 nucleotide changes Mazanderan Tehran Fig. 2. Unrooted phylogenetic tree for DNA sequences of Cyt b 3' end (last 316 nucleotides) of Phlebotomus (Adlerius)/Phlebotomus (Larroussius), produced by branch and bound parsimony search using PAUP* Iran J Arthropod-Borne Dis, 2010, 4(2): 1–10 P Parvizi et al.: Molecular Typing and …. 7 Single or Nucleotide position composite DNA sequence from each specimen 1111111111111111111111111111111111111222222 11222233344445666677788999990000000111222334444455666666777777889000001 1539235814703695125737909124780134789378567690234514124567012459129012581 IRN287haplo CB3 IRN385haplo R06 IRN334haplo CB3 IRN335haplo CB3 IRN338haplo CB3 IRN336haplo CB3 IRN277haplo CB3 IRN279haplo CB3 IRN278haplo CB3 IRN295haplo CB3 IRN380haplo R06 IRN381haplo R06 IRN383haplo R06 IRN287haplo CB3 IRN385haplo R06 IRN334haplo CB3 IRN335haplo CB3 IRN338haplo CB3 IRN336haplo CB3 IRN277haplo CB3 IRN279haplo CB3 IRN278haplo CB3 IRN295haplo CB3 IRN380haplo R06 IRN381haplo R06 IRN383haplo R06 NNNATCTAACTTAAAATTGAAGAAACCTTTATACATACCCTTATTATAATTATAATAGATTTTATCAGCATAT TCTACCTAACCTAAAATTGAAGAAACCTTTATACATACCTTTATTGTAATTATAATAGATTTTATCAGCATAT TTCTTCCTTTCCAAAACCGTAGTAATTAAATTACACTACATTATTACAATTATAATAGATTTTGTTAGCCCTT TTCTTCCTTTCCAAAACCGTAGTAATTAAATTACACTACATTATTACAATTATAATAGATTTTGTTAGCCCTT TTCTTCCTTTCCAAAACCGTAGTAATTAAATTACACTACATTATCACAATTATAATAGATTTTGTTAGCCCTT TTCTTCCTTTCTGAGACCGTAGTAATTAAATTACACTACATTATTACAATTATAATAGATCTTGTTAGCCCTT TTTATTAATTTTAAATTTGTTGTAATTATATCACATAATTCTCCTCTAATTATAATAGATTTTGATAGCTTTT TTTATTAATTTTAAATTTGTTGTAATTATATCACATAATTCTCCTCTAATTATAATAGATTTTGATAGCTTTT TTTATTAATTTTAAATTTGTTGTAATTATATCACATAATTCTCCTCTAATTATAATAGATTTTGATAGCTTTT NNNATTAATTTTAAATTTGTTGTAATTATATCACATAATTCTCCTCTAATTATAATAGATTTTGATAGCTTTT TTTATTAATTTTAAATTTGTTGTAATTATATCACATAATTCTCCTCTAATTATAATAGATTTTGATAGCTTTT TTTATTAATTTTAAATTTGTTGTAATTATATCACATAATTCTCCTCTAATTATAATAGATTTTGATAGCTTTT CTTATTAACTATAGATTTATCAAGTTAATTAATATTTATTCACCTAATCAACATTATATCACCTTCTATATAA GCTTTAACAAAGTCTTTCCTATTAAAAATCTCAAATTTCTCAA GCTTTAACAGAGTCTTTCCTATTAAAAATCTCAAATTTCTCAA GCTTTAACAATATCTTCCCTATATCAATATTCAAATTTTTCAA GCTTTAACAATGTCTTCCCTATATCAATATTCAAATTTTTCAA GCTTTAACAATGTCTTCCCTATATCAATATTCAAATTTTTCAA GCTTTAACAATGTCTTCCCTATATCAATATTCAAATTTTTCAA ACACTTACCACGTCTCTTCTACATAAATATTCTAATCATATAC ACACTTACCACGTCTCTTCTACATAAATATTCTAATCATATAC ACACTTACCACGTCTCTTCTACATAAATATTCTAATCATATAC ACACTTACCACGTCTCTTCTACATAAATATTCTAATCATATAC ACACTTACCACGTCTCTTCTACATAAATATTCTAATCATATAC ACACTTACCACGTCTCTTCTACATAAATATTCTAATCATATAC AAAACGTTAATTATCCTCTATAATATTTTTAACTTAATTAATT Fig. 3. Input data matrix of variant nucleotides for PAUP* analysis of Phlebotomus (Adlerius) / Phlebotomus (Larroussius) species: 316 base pairs of Cyt b 3´ Single or Nucleotide position composite DNA sequence from each specimen 1111111111111111111111111111111111111111222222 11122223334455556666688890000001122222333333444555555566677788999000111 134925891273657890124725940136792512356034679289123467801923524369589145 IRN294haplo CB1 IRN291haplo CB1 IRN277haplo CB1 IRN282haplo CB1 IRN279haplo CB1 IRN287haplo CB1 IRN285haplo CB1 IRN380haplo R06 IRN383haplo R06 IRN381haplo R06 IRN385haplo R06 IRN293haplo CB1 IRN294haplo CB1 IRN291haplo CB1 IRN277haplo CB1 IRN282haplo CB1 IRN279haplo CB1 IRN287haplo CB1 IRN285haplo CB1 IRN380haplo R06 IRN383haplo R06 IRN381haplo R06 IRN385haplo R06 IRN293haplo CB1 AAAGTACCCCACAGCTATATATAAGATGTTTAAATACTTCTTATCCTATGCACAAAATTCACCATACCTAAT TAATTATCATTCTGATATATAAAGGTTGTCCTCACACTTTTTATTATAAGCTCTAAACTTATATTATCCGTT TTAATTCTATATTAATATAAAAGAGATGTATAGCTACTTTTCTTTTTATGCACAAAATCCTTAAATCACAAT TTAATTCTATATTAATATAAAAGAGATGTATAACTACTTTTCTTTTTATGCACAAAATCCTTAAATCACAAT TTAATTCTATATTAATATAAAAGAGATGTATAGCTACTTTTCTTTTTATGCACAAAATCCTTAAATCACAAT TAATCATCATTCTAATATACAAAAGTTGTCCTCACACTTTTTATTATGAGCTCTAAACTTATATTATCCATC TTAATTCTATATTAATATAAAAGAGATGTATAGCTACTTTCCTTTTTATGCACAAAATCCTTAAATCACAAT TTAATTCTATATTAATATAAAAGAGATGTATAGCTACTTTTCTTTTTATGCACAAAATCCTTAAATCACAAT ACTATTTTATATTGTAGATATTGATTCCATCCAATGGCTTTATCTAAATATTGTCTTTTTATAAATTTTATC TTAATTCTATATTAATATAAAAGAGATGTATAGCTACTTTCCTTTTTATGCACAAAATCCTTAAATCACAAT TAATTATCATTCTGATATATAAAGGTTGTCCTCACACTTTTTATTACGAGCTCTAAACTCATATTATCCGTC ???????????TTAATATAAAAGAGATGTATAGCTACTGTTCTTTTTATGCACAAAATCCTTAAATCACAAT ATAATTCAGATATCTCTACTCATAAATACACAAGATAACAAAAGCATTTTAGATTTCCGTTCTTCC TTCACTCAGACACCGCAGTGTGTAAATAGTCTTCTTATCTATAGCGCATCTGACTCTTATCTCCCC ACAACTCTGACTTTTTTGTTTATGAAAATATGTTTTATCTAAAGCTCATCAGGTATTCTACTTTCC ACAACTCTGACTTTTTTGTTTATGAAAATATGTTTTATCTAAAGCTCATCAGGTATTCTACTTCCC ACAACTCTGACTTTTTTGTTTATGAAAATATGTTTTATCTAAAGCTCATCAGGTATTCTACTTCCC TTYACTCAGACACCACAGTGTGTAAATAGTCTTCTTATCTATAGCACATCTGACTCTTCTCTTCCC ACAACTCTGACTTTTTTGTTTATGAAAATATGTTTTATCTAAAGCTCATCAGGTATTCTACTTCCC ACAACTCTGACTTTTTTGTTTATGAAAATATGTTTTATCTAAAGCTCATCAGGTATTCTACTTCCC ATTCTAAAATACTTATTATTAAATTTTTTAC?TTTATAATTATCTATTATAAACATCTATCTTTAT ACAACTCTGACTTTTTTGTTTATGAAAATATGTTTTATCTAAAGCTCATCAGGTATTCTACTTCCC TTCACTCAGACACCGCAGTGTGTAAATAGTCTTCTTATCTATAGCGCATCTGACTCTTATCTCCCC ACAACTCTGACTTTTTTGTTTATGAAAATATGTTTTATCTAAAGCTCATCAGGTATTCTACTTCCC Fig. 4. Input data matrix of variant nucleotides for PAUP analysis of Phlebotomus (Adlerius)/Phlebotomus (Larroussius) species: 382 base pairs of Cyt b 5´ Iran J Arthropod-Borne Dis, 2010, 4(2): 1–10 P Parvizi et al.: Molecular Typing and …. 8 Discussion Using branch and bound parsimony searches with equal character weighting, PAUP* analysis for DNA sequences of Cyt b 3´ fragment (last 316 nucleotides) of 8 hap- lotypes of Phlebotomus (Adlerius)/Phleboto- mus (Larroussius) produced one parsimoni- ous tree rooted using default outgroup (tree length= 145; 69 characters were parsimony- uninformative; number of parsimony-informa- tive characters= 47) (Fig. 1, 2). The haplo- types of each species formed monophyletic clades, but it was not possible to compare regions because each species was collected in unique locations. Using branch and bound parsimony searches with equal character weighting, PAUP* analysis for DNA sequences of Cyt b 5´ fragment (383 nucleotides) of 10 hap- lotypes of Phlebotomus (Adlerius)/Phleboto- mus (Larroussius) produced one most parsi- monious tree (tree length= 188; 60 charac- ters were parsimony-uninformative; number of parsimony-informative characters= 78) (Fig. 3, 4). P. major and P. halepensis were monophyletic, with two unknown species located between them. It was not possible to compare regions because each species was collected in unique locations Using branch and bound parsimony searches with equal character weighting, PAUP* analysis for DNA sequences of Cyt b Long fragment (last 718 nucleotides) of 18 haplotypes of Phlebotomus (Adlerius)/Phle- botomus (Larroussius) produced 1 most par- simonious tree rooted using default outgroup (tree length= 343; 116 characters were parsi- mony-uninformative; number of parsimony- informative characters= 144). Phlebotomus major and P. tobbi were monophyletic, and both unknown species (i.e. not identifed by morphology) were monophyletic with P. hale- pensis, not with a species of Larroussius. The longer sequence gave more phylogenetic information. Nucleotide haplotypes within Adlerius/ Larroussius species differed pairwise by < 0.1%, but absolute genetic distances were greater between some species, e.g. 0.13413- 0.14093 between P. tobbi and P. halepensis, and 0.12141-0.12828 between P. halepensis and P. major. For subgenus Adlerius (single species, P. halepensis), fixed diagnostic polymor- phisms occurred at amino acid positions 97, 101, 173 and 239 of the Cyt b Long fragment and at amino acid positions 39, 72 and 105 of the Cyt b 3´ fragment. For subgenus Larroussius, fixed diag- nostic polymorphisms occurred at amino acid positions 99, 106 and 238 of the Cyt b Long fragment and at amino acid positions 74 and 104 of the Cyt b 3´ fragment. Aransay et al. (1999) showed that the 18S rRNA gene was a useful marker for in- ferring phylogenetic relationships within the subfamily Phlebotominae, finding a clade containing the subgenera Euphlebotomus, Adlerius and Larrroussius, a second clade with Paraphlebotomus and Phlebotomus, and a third clade with Sergentomyia and American Lutzomyia species. Depaquit et al. (2000) used ITS2 rDNA gene sequences and found a clade with Paraphlebotomus and Phlebotomus. ITS2 rDNA sequences were also monophyletic for subgenus Larroussius (Muccio et al. 2000). In our study, the DNA sequences of Cyt b were shown to be good markers for finding clades of genera and subgenera. Esseghir et al. (2000) characterized Cyt b for species of the same subgenera as we studied, but they had to give different weights to the nucleo- tides in 1st, 2nd and 3rd base positions of codons to get phylogenetic results. This was not the aim of our work, which was finding diagnostic markers for the species. 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