despite a considerable progress in malaria control in iran over the past few years that led to significant reduction of cases , the disease still remains a major health problem in south and southeastern parts of the country . 2008 , afsharpad et al . 2012 ) and insecticide resistance of anopheles vectors ( enayati et al . 2012 ) are aggravated by continuous influx of imported cases , mostly with plasmodium falciparum , from neighboring countries of afghanistan and pakistan ( zakeri et al . the latest checklists of iranian mosquitoes include 28 anopheles species , identified mostly on the basis of morphological features , and a few by dna - based approaches ( azari - hamidian 2007 ) . maculipennis are known to be responsible for transmission of malaria in the country ( manouchehri et al . some important malaria vectors in iran are assumed to be members of species complexes or species groups , which are often difficult to distinguish morphologically . application of dna - based approaches has resolved some cryptic species in iranian complex species including an . fluviatilis , however , the identity of some members are still doubtful or were refuted later ( azari - hamidian 2007 , naddaf et al . this , to great extent , is due to introduction of molecular markers such as its2 and 28s - d3 genes for discriminating the members of this complex species in india ( manonmani et al . biology , variation in behaviors , and role of this species in malaria transmission in different geographical areas of iran has been extensively reviewed by others ( eshghi et al . 1976 , edalat 19971998 , hanafi - bojd et al . 2012 ) . in an early study comparison of its2 sequence of iranian specimens from various localities in south and southeastern iran revealed only species y , which is presumably species t ( naddaf et al . 2003 ) , however , rapd - pcr analysis of same specimens revealed two distinct patterns , separating representatives of fars province from other areas ( naddaf et al . analysis of 28s - d3 gene from same populations corroborated rapd results , fars province specimens showed to be identical to species u in india , while individuals from other areas exhibited heterozygocity at the only base pair position that identifies species u and t ( naddaf et al . 2010 ) . in addition , in a separate study based on 28s - d3 analysis , species t and species u were reported from jiroft of fars province and chabahar of sistan va baluchestan province , respectively ( mehravaran et al . 2011 ) . the aim of this study was to evaluate cytochrome oxidase i ( coi ) gene alongside 28s - d3 as a diagnostic tool for identification of an . coi gene sequences have been extensively used for population studies and resolving evolutionary relationship among closely related species groups of insects ( lunt et al . 1996 ) and anopheline mosquitoes ( krzywinski and besansky 2003 ) . variations in this fragment have been exploited as dna barcodes for identifications of culicidae mosquitoes including an . fluviatilis mosquitoes originated from different localities in south and southeastern areas of iran including fars , hormozgan , kerman , and sistan va baluchestan provinces . the extraction method and identity of some mosquitoes based on its2 and/or 28-d3 genes were described previously ( naddaf et al . all the dna samples were initially subjected to allele specific ( as)-pcr based on 28s - d3 gene as described by singh et al . the coi gene was amplified using universal primers , ubc6 ( 5- gga gga ttt gga aat tga tta gtt cc -3 ) and ubc9 ( 5-ccc ggt aaa att aaa ata taa act tc-3 ) , designed by simon et al . each 25l reaction contained 20 pmol of each primer , 2 mm mg cl2 , 10 mm tris - hcl , 50 mm kcl , 150m of dntps , 1u of taq , and 2l of dna . pcr products were purified using a gel band purification kit ( pharmacia , piscataway , nj , usa ) according to manufacturer s recommendations and later sequenced using the same primers as used for amplification at seqlab laboratory in germany . the sequences were manually edited and corrected using bioedit software , version 7.1.3.0 ( hall 1999 ) and fragments of 474 bp length were selected for analysis . the distances between groups and between individual sequences were calculated , and phylogenetic tree for iranian sequences was generated using the kimura two parameter ( k2p ) model of neighbor - joining method in a complete deletion procedure using mega 4 software ( tamura et al . the sequence data for the coi gene sequences were submitted to genbank with the accession numbers jx020706-jx020729 . fluviatilis mosquitoes originated from different localities in south and southeastern areas of iran including fars , hormozgan , kerman , and sistan va baluchestan provinces . the extraction method and identity of some mosquitoes based on its2 and/or 28-d3 genes were described previously ( naddaf et al . all the dna samples were initially subjected to allele specific ( as)-pcr based on 28s - d3 gene as described by singh et al . the coi gene was amplified using universal primers , ubc6 ( 5- gga gga ttt gga aat tga tta gtt cc -3 ) and ubc9 ( 5-ccc ggt aaa att aaa ata taa act tc-3 ) , designed by simon et al . ( each 25l reaction contained 20 pmol of each primer , 2 mm mg cl2 , 10 mm tris - hcl , 50 mm kcl , 150m of dntps , 1u of taq , and 2l of dna . pcr products were purified using a gel band purification kit ( pharmacia , piscataway , nj , usa ) according to manufacturer s recommendations and later sequenced using the same primers as used for amplification at seqlab laboratory in germany . the sequences were manually edited and corrected using bioedit software , version 7.1.3.0 ( hall 1999 ) and fragments of 474 bp length were selected for analysis . the distances between groups and between individual sequences were calculated , and phylogenetic tree for iranian sequences was generated using the kimura two parameter ( k2p ) model of neighbor - joining method in a complete deletion procedure using mega 4 software ( tamura et al . the sequence data for the coi gene sequences were submitted to genbank with the accession numbers jx020706-jx020729 . all the dna specimens from fars province yield only a product of approximately 375 bp length indicative of species u , whereas specimens from hormozgan , kerman and sistan va baluchestan provinces amplified two bands of 375 bp and 128 bp length . phylogenetic analysis using coi gene grouped individuals from fars province in two distinct clades separate from other iranian individuals representing populations of hormozgan , kerman , and sistan va baluchestan ( fig . the mean distance between iranian and indian groups was 1.66% , while the value between fars group and the group comprising other iranian members was 2.06% . the indian group exhibited the same distance ( 2.06% ) with fars group . the highest distance ( 3.09% ) among iranian individuals was between specimens 930 from fars province and 398 from sistan va baluchestan province . six individuals belonging to two clades ( 87 , 97 , 655 , and 928 ) and ( 92 ) from fars province showed 100% identity . in addition , ten individuals from other geographical areas including four from koveh ( 170 , 172 , 173 , and 186 ) and one from minab ( 121 ) in hormozgan province , two from abchekan ( 392393 ) in sistan va baluchestan province , and three from kahnouj in kerman province ( 815 , 836 , and 839 ) were 100% identical . accurate identification of malaria vectors is not only one of the most basic requisite for success of malaria control programs , but also has become an intriguing issue for understanding speciation process and evolution of anopheles mosquitoes . in absence of cytotaxonomic evidence , rapd - pcr methodology and variation in 28s - d3 gene have resolved two potential sibling species in an . we report further evidence for the occurrence of these two sibling species by coi analysis of mitochondrial dna from the same specimens . anopheles fluviatilis james is a complex of cryptic species ; cytotaxonomic studies of polythene chromosomes has revealed three reproductively isolated species in india known as s , t , and u ( subbarao et al . 1994 ) . application of the first dna - based method using its2 gene identified two putative species of x and y that are presumably equivalent to species s and t , respectively ( manonmani et al . later , a complete as - pcr assay based on variations of 28s - d3 gene against chromosomally examined specimens identified all the members of the complex ( singh et al . fluvialitis mosquitoes of iran . however , the same specimens displayed variations in 28s - d3 gene ; the individuals from fars exhibited similarity with species u in india whereas individuals from others areas showed heterozygocity at the single nucleotide position that identifies species u and t. the identity of an . fluviatilis complex in iran became complicated as the heterozygocity in 28-d3 was not reflected in its2 fragment and individuals from fars province exhibited dual identity of t and u based on its2 and 28s genes , respectively ( chen et al . fluviatilis sibling species were not addressed in their study , however , k2p genetic distances between different species of culicidae were reported to be > 2% . in our study , the mean distance between iranian and indian populations was 1.66% , whereas the value between fars group and the group comprising other iranian individuals was 2.06% . the distance between most individuals from fars province and individuals from other areas was > 2% . two individuals ( 9192 ) from fars province exhibited almost equivalent distance with all other members including those ( 930 , 926 , 928 , 655 , 87 , and 97 ) from the same province . jf966741 , unpublished ) that showed a high divergence from other sequences and appeared as an out group beyond an . the results of present study were almost concordant with earlier results obtained by rapd - pcr methodology and 28s - d3 analysis ( naddaf et al . this study shows that coi gene can be used as a useful tool along other dna markers like 28-d3 gene for dissolving closely related taxa of an . fluviatilis mosquitoes ( by its2 and 28s - d3 genes ) from india , iran , and other geographical areas by this genetic marker can bring more insight into taxonomy of this sibling species . this study shows that coi gene can be used as a useful tool along other dna markers like 28-d3 gene for dissolving closely related taxa of an . fluviatilis mosquitoes ( by its2 and 28s - d3 genes ) from india , iran , and other geographical areas by this genetic marker can bring more insight into taxonomy of this sibling species .
background : anopheles fluviatilis , one of the major malaria vectors in iran , is assumed to be a complex of sibling species . 
 the aim of this study was to evaluate cytochrome oxidase i ( coi ) gene alongside 28s - d3 as a diagnostic tool for identification of an . 
 fluviatilis sibling species in iran.methods:dna sample belonging to 24 an . 
 fluviatilis mosquitoes from different geographical areas in south and southeastern iran were used for amplification of coi gene followed by sequencing . 
 the 474475 bp coi sequences obtained in this study were aligned with 59 similar sequences of an . 
 fluviatilis and a sequence of anopheles minimus , as out group , from genbank database . 
 the distances between group and individual sequences were calculated and phylogenetic tree for obtained sequences was generated by using kimura two parameter ( k2p ) model of neighbor - joining method.results:phylogenetic analysis using coi gene grouped members of fars province ( central iran ) in two distinct clades separate from other iranian members representing hormozgan , kerman , and sistan va baluchestan provinces . 
 the mean distance between iranian and indian individuals was 1.66% , whereas the value between fars province individuals and the group comprising individuals from other areas of iran was 2.06%.conclusion : presence of 2.06% mean distance between individuals from fars province and those from other areas of iran is indicative of at least two sibling species in an . 
 fluviatilis mosquitoes of iran . 
 this finding confirms earlier results based on rapd - pcr and 28s - d3 analysis .