Discrimination of Iranian honeybee populations (Apis mellifera meda) from commercial subspecies of Apis mellifera L. using morphometric and genetic methods

The morphological characters of honeybees have an important role for discriminating honeybee subspecies. In the present research, Iranian populations of honeybee (Apis mellifera) were collected from 19 areas in Iran. The samples were collected from stationary beekeeping sites. Moreover, pictures of honeybee forewings held in the Bee Data Bank in Oberursel were compared with Iranian honeybee populations. 19 morphological characters were measured for each forewing of worker honeybee to evaluate differentiation of Iranian honeybee populations from the commercial honeybee subspecies A. m. mellifera, A. m. carnica, A. m. caucasica and A. m. ligustica. Additionally, part of the tRNA^leu gene, an intergenic region and part of COII was used to confirm differentiation of the commercial subspecies from Iranian honeybee populations. Results of the cluster analyses showed that 19 morphological characters of forewings differentiated Iranian populations from the commercial subspecies. Moreover, the phylogenetic tree of part of the tRNA^leu gene, an intergenic region and part of COII differentiated the commercial subspecies from Iranian honeybee populations. Results of the discriminant function analyses (DFA) indicated that the references samples of A. m. meda overlapped with Iranian populations.     

Phylogenetic relationships of Pterocarya (Juglandaceae) with an emphasis on the taxonomic status of Iranian populations using ITS and trnH-psbA sequence data

Pterocarya fraxinifolia (Lam.) Spach., a relict tree species of the Juglandaceae family, is native to the Great Caucasus, Anatolia, and to the Hyrcanian forests of the southern Azerbaijan and Northern Iran. In this study, the phylogenetic relationship of the species, sampled in selected Iranian populations, and the global biogeography of the genus Pterocarya were addressed. Leaves were collected from 8 to 10 trees from three geographically isolated habitats. The samples were analyzed with nuclear (internal transcribed spacer [ITS] regions) and chloroplast (trnH-psbA) DNA markers. The obtained results were compared and analyzed with the data registered in NCBI GenBank. It is reported that the ITS regions varied from 644 to 652 for Pterocarya genus, but we did not observe polymorphisms for Iranian Pterocarya. The phylogenetic tree divided the Pterocarya genus in three clades: clade 1 grouping exclusively the samples P. fraxinifolia, clearly separated from the East Asiatic taxa; clade 2 that includes the species P. hupehensis and P. macroptera; clade 3 clustering P. stenoptera and P. tonkinensis. Although the Iranian Pterocarya samples and P. fraxinifolia from the Caucasus were in the same clade, they presented two different secondary structures. The Iranian populations showed the maximum genetic distance with P. stenoptera and P. tonkinensis. Our analysis demonstrates that the traditional division of all the six species sampled throughout their distribution area as well as the phylogeny of the genus Pterocarya needs to be reviewed.     

Molecular phylogeny and intraspecific differentiation of the Eremias velox complex of the Iranian Plateau and Central Asia (Sauria,Lacertidae)

The Central Asian racerunner, Eremias velox, is a widely distributed lizard of the Eurasian lacertid genus Eremias. Nucleotide sequences of mitochondrial genes, cyt b and 12S rDNA from 13 geographically distant localities in Iran and Central Asia, were analysed. Phylogenetic analyses of the sequence data unambiguously recovered five major clades within the E. velox complex with a high level of genetic divergence, indicating long periods of isolation. The basal position of the Iranian clades in the phylogenetic trees suggests that the E. velox clade originated on the Iranian plateau in the Middle Miocene. According to our calibrations, the northern Iranian clade diverged first some 10–11 Ma and that the Central Asian lineages split from the northeastern Iranian lineage approximately 6 Ma, most likely as a result of uplifting of the Kopet‐Dagh Mountains in the northern margin of the Iranian plateau. Topology of the phylogenetic trees, combined with the degree of the genetic distances among the independent lineages recovered in this study, provide a solid foundation for a fundamental revision of the taxonomic status of the major clades within this species complex.     

Alpine–Himalayan orogeny drove correlated morphological,molecular, and ecological diversification in the Persian dwarf snake (Squamata: Serpentes: Eirenis persicus)

The Persian dwarf snake Eirenis (Pseudocyclophis) persicus (Anderson, 1872) has a wide distribution range in south‐western Asia. This species group was comprehensively studied here using traditional biometry, geometric morphometrics, ecological niche modelling, and genetics. Our analyses revealed that E. persicus is split into two clades. A western clade, bearing at least two different species: E. persicus, distributed in south‐western Iran, and an undescribed species from south‐eastern Turkey and western Iran. The eastern clade consists of at least three species: Eirenis nigrofasciatus, distributed across north‐eastern Iraq, and western and southern Iran; Eirenis walteri, distributed across eastern Iran, southern Turkmenistan, and western and southern Pakistan, and Eirenis angusticeps, distributed in north‐eastern Pakistan. Ecological niche modelling revealed that the distribution of the species in the western clade are mainly affected by winter precipitation, and those in the eastern clade are mainly affected by the minimum temperature of the coldest month. A molecular clock analysis revealed that the divergence and diversification of the E. persicus species group mainly correspond to Eocene to Pliocene orogeny events subsequent to the Arabia–Eurasia collision. This study confirms that specimens with the unique morphology of having 13 dorsal scale rows on the anterior dorsum, occurring in the Suleiman Mountains in central Pakistan, can be referred to Eirenis mcmahoni (Wall, 1911). However, at this moment we have insufficient data to evaluate the taxonomy of this species.     

The phylogeny,phylogeography, and diversification history of the westernmost Asian cobra (Serpentes: Elapidae: Naja oxiana) in the Trans‐Caspian region

We conducted a comprehensive analysis of the phylogenetic, phylogeographic, and demographic relationships of Caspian cobra (Naja oxiana; Eichwald, 1831) populations based on a concatenated dataset of two mtDNA genes (cyt b and ND4) across the species'' range in Iran, Afghanistan, and Turkmenistan, along with other members of Asian cobras (i.e., subgenus Naja Laurenti, 1768). Our results robustly supported that the Asiatic Naja are monophyletic, as previously suggested by other studies. Furthermore, N. kaouthia and N. sagittifera were recovered as sister taxa to each other, and in turn sister clades to N. oxiana. Our results also highlighted the existence of a single major evolutionary lineage for populations of N. oxiana in the Trans‐Caspian region, suggesting a rapid expansion of this cobra from eastern to western Asia, coupled with a rapid range expansion from east of Iran toward the northeast. However, across the Iranian range of N. oxiana, subdivision of populations was not supported, and thus, a single evolutionary significant unit is proposed for inclusion in future conservation plans in this region.     

Mitochondrial evidence uncovers a refugium for the fat dormouse (Glis glis Linnaeus, 1766) in Hyrcanian forests of northern Iran

Fat dormouse is a squirrel-like rodent which is closely tied to deciduous forest ecosystem in southwestern Eurasia. As such it is a valuable indicator of forest survival in refugia during glacial-interglacial periods. Previous phylogeographic analyses uncovered divergent fat dormouse lineages in southern refugia in Italy and the Balkans, but retrieved a surprisingly low overall genetic diversity across the majority of the species’ range. We explored 812 bp long fragment of a cytochrome b (cyt b) gene in ten fat dormice from refugial Hyrcanian forests in northern Iran. We identified 10 new cyt b haplotypes, which generated a total dataset of 28 fat dormouse haplotypes. The phylogenetic reconstruction clustered the new haplotypes into the Iranian lineage which hold a sister position against all other fat dormouse haplotypes from Europe and Asia Minor. The divergence between these lineages suggests a fragmentation event of an ancestral population at 5.76 mya (95% HPD = 3.21–8.92). This early evolutionary divergence was possibly triggered in the Middle East by dramatically divergent environmental conditions at the Messinian Salinity Crisis. The divergence clearly exceeds the intraspecific divergence, and is well within the range between congeneric rodent species. We suggest a long-term persistence of the Iranian lineage in the Hyrcanian refugium which is consitent with a high number of endemics along the southern Caspian coastal areas.     

New data on Longidorus aetnaeus Roca, Lamberti, Agostinelli & Vinciguerra, 1986 (Nematoda: Longidoridae) from Iran and Ajaria (Georgia)

Longidorus aetnaeus Roca, Lamberti, Agostinelli & Vinciguerra, 1986 is reported for the first time from Iran and Ajaria (Georgia). Morphological and morphometric data are provided for two Iranian and one Ajarian populations. The D2–D3 region of 28S rDNA for both Iranian populations was sequenced for the first time and the data reported. A detailed study of juveniles of L. aetnaeus from Iran, Georgia and Bulgaria demonstrated that this species develops through three juvenile stages. Furthermore, phylogenetic studies inferred from sequences for the D2–D3 region of 28S rRNA gene revealed that L. aetnaeus is most closely related to L. leptocephalus.     

Calculation of Fluctuating Asymmetry of the biggest Caspian whipsnake population in Hungary compared to a common snake species

The Fluctuating Asymmetry (FA) is a special type of asymmetry in the populations of bilaterally symmetrical creatures. The FA gives us numerical data on the developmental instability of the populations and refers to potential genetical and environmental stressors affecting the populations. Here we give the first data on the FA of the protected Caspian whipsnake (Hierophis caspius) from Hungary. The FA indices of the biggest population from Villány Mts were compared to the FA indices of two differently stressed Dice snake (Natrix tessellata) populations [stressed (Mád) and seminatural (Lake Balaton)]. Based on the values of the multiple and the simple indices derived from sublabial scales, we can say that the status quo of the highly protected Caspian whipsnake population does not represent significant deviation from the near-natural dice snake population from the Lake Balaton.     

Reassessment of the taxonomic position of Iranocypris typhlops Bruun & Kaiser, 1944 (Actinopterygii,Cyprinidae)

The Iranian cave barb (Iranocypris typhlops Bruun & Kaiser, 1944) is a rare and endemic species of the family Cyprinidae known from a single locality in the Zagros Mountains, western Iran. This species is “Vulnerable” according to the IUCN Red List and is one of the top four threatened freshwater fish species in Iran. Yet, the taxonomic position of I. typhlops is uncertain. We examined phylogenetic relationships of this species with other species of the family Cyprinidae based on the mitochondrial cytochrome b gene. Our results show that I. typhlops is monophyletic and is sister taxon of a cluster formed by Garra rufa (Heckel, 1843) and Garra barreimiae (Fowler & Steinitz, 1956) within a clade that includes other species of the genus Garra. Based on previous molecular and morphological studies, as well as our new results, we recommend that I. typhlops should be transferred to the genus Garra Hamilton, 1822.     

Phylogeography of the Ibero-Maghrebian red-eyed grass snake (<Emphasis Type="Italic">Natrix astreptophora</Emphasis>)

We examined phylogeographic differentiation of the red-eyed grass snake (Natrix astreptophora) using 1984 bp of mtDNA and 13 microsatellite loci from specimens collected across its distribution range in southwestern Europe and northwestern Africa. Based on phylogenetic analyses of mtDNA, European N. astreptophora constituted the sister clade to a weakly supported North African clade comprised of two deeply divergent and well-supported clades, one corresponding to Moroccan snakes and the other to snakes from Algeria and Tunisia. This tripartite differentiation was confirmed by analyses of microsatellite loci. According to a fossil-calibrated molecular clock, European and North African N. astreptophora diverged 5.44 million years ago (mya), and the two Maghrebian clades split 4.64 mya. These dates suggest that the radiation of the three clades was initiated by the environmental changes related to the Messinian Salinity Crisis and the reflooding of the Mediterranean Basin. The differentiation of N. astreptophora, with distinct clades in the Iberian Peninsula and in the western and eastern Maghreb, corresponds to a general phylogeographic paradigm and resembles the differentiation found in another co-distributed Natrix species, the viperine snake (N. maura). Despite both species being good swimmers, the Strait of Gibraltar constitutes a significant biogeographic barrier for them. The discovery that North Africa harbours two endemic lineages of N. astreptophora necessitates more conservation efforts for these imperilled snakes.     

Investigation of the genetic diversity of domestic Capra hircus breeds reared within an early goat domestication area in Iran

Background

Iran is an area of particular interest for investigating goat diversity. Archaeological remains indicate early goat domestication (about 10 000 years ago) in the Iranian Zagros Mountains as well as in the high Euphrates valley and southeastern Anatolia. In addition, mitochondrial DNA data of domestic goats and wild ancestors (C. aegagrusor bezoar) suggest a pre-domestication management of wild populations in southern Zagros and central Iranian Plateau. In this study genetic diversity was assessed in seven Iranian native goat breeds, namely Markhoz, Najdi, Taleshi, Khalkhali, Naini, native Abadeh and Turki-Ghashghaei. A total of 317 animals were characterized using 14 microsatellite loci. Two Pakistani goat populations, Pahari and Teddy, were genotyped for comparison.

Results

Iranian goats possess a remarkable genetic diversity (average expected heterozygosity of 0.671 across loci, 10.7 alleles per locus) mainly accounted for by the within-breed component (G_ST = 5.9%). Positive and highly significant F_IS values in the Naini, Turki-Ghashghaei, Abadeh and Markhoz breeds indicate some level of inbreeding in these populations. Multivariate analyses cluster Iranian goats into northern, central and western groups, with the western breeds relatively distinct from the others. Pakistani breeds show some relationship with Iranian populations, even if their position is not consistent across analyses. Gene flow was higher within regions (west, north, central) compared to between regions but particularly low between the western and the other two regions, probably due to the isolating topography of the Zagros mountain range. The Turki-Ghashghaei, Najdi and Abadeh breeds are reared in geographic areas where mtDNA provided evidence of early domestication. These breeds are highly variable, located on basal short branches in the neighbor-joining tree, close to the origin of the principal component analysis plot and, although highly admixed, they are quite distinct from those reared on the western side of the Zagros mountain range.

Conclusions

These observations call for further investigation of the nuclear DNA diversity of these breeds within a much wider geographic context to confirm or re-discuss the current hypothesis (based on maternal lineage data) of an almost exclusive contribution of the eastern Anatolian bezoar to the domestic goat gene pool.     

Phylogenetic relationships,molecular taxonomy,biogeography of <Emphasis Type="Italic">Betula</Emphasis>, with emphasis on phylogenetic position of Iranian populations

Birch trees inhabiting the high-altitude Hyrcanian forest (the southern shores of the Caspian Sea of Iran and Azerbaijan) are classified in the EN (endangered) category of the International Union for Conservation of Nature (IUCN). Taxonomic status and phylogenetic relationship of Iranian populations, molecular taxonomy, and biogeography of the genus Betula in the world have been considered. Four remnant populations of Betula were selected from north and northwestern parts of Iran. The internal ITS and trnH-psbA intergenic spacer regions were sequenced. Based on the trnH-psbA and ITS, Iranian birch and white birch were placed in a clade, but based on trnH-psbA divided into two subclades. Phylogenetic trees based on ITS and trnH-psbA data did not completely support the morphological classification. Network analysis confirms a close relationship of B. pendula with B. platyphylla and B. papyrifera with B. humilis, and B. ermanii were in a group with the other tetraploid species of the subgenus Neurobetula. Divergence time analysis showed that about 75 Ma ago the Betula genus separated from the other genus of Betulaceae and then divided into two main clades in Oligocene. Our divergence analysis supports that two subgenera of Betulenta and Betulaster are the oldest subgenera in the genus Betula and they date back to Eocene. The ancestral reconstruction suggests that ancestors of the genus Betula originated from Southeast Asia.     

Phylogeography and conservation genetics of the Columbia spotted frog (Rana luteiventris; Amphibia, Ranidae)

The Columbia spotted frog (Rana luteiventris) has a widespread distribution in western Canada and the western US, although the southern reach of its range is highly fragmented into several isolated populations. Threats from various factors have raised concerns regarding the long-term survival of many small, isolated populations. Here, we report a study designed to determine the phylogeographic and conservation genetic parameters of R. luteiventris in the western US. Mitochondrial DNA (mtDNA) sequences were examined for phylogeographic structuring using phylogenetic reconstruction methods, coupled with networking and nested clade analyses. These methods permitted a distinction to be made between historic and demographic forces acting to generate geographical patterning of genetic variation. Phylogenetic analysis revealed four geographically correlated monophyletic clades. Three of these clades correspond to well-defined, nonoverlapping geographical locations in the fragmented portion of the range. The other is comprised of all samples collected from the contiguous range and includes one isolate from northern Wyoming. Networking and nested clade analyses confirmed these results and revealed that historical processes, such as range expansion and vicariance, rather than recurrent gene flow are likely responsible for observed patterns of genetic variation. A measure of genetic variation (theta = 4N(e)mu) revealed that R. luteiventris populations in Utah have a relatively low amount of genetic variation compared with populations in the continuous portion of the range.     

Morphological and molecular characterization of two populations of <Emphasis Type="Italic">Aphelenchus avenae</Emphasis> (Nematoda: Aphelenchidae) from Iran and their phylogenetic relationships

Soil samples were collected from the rhizosphere of faba bean (Vicia faba L.) in Khuzestan province, south-western Iran. Many of the localities were positive for the presence of Aphelenchus avenae. The morphological and morphometric characters of the two Iranian populations are discussed and illustrated based on morphological and morphometrics data. Additionally, sequences of the D2-D3 expansion segments of 28S rRNA gene for two populations were used for molecular phylogenetic analysis. One population was without male and formed a separate branch in the phylogenetic tree. Results indicate high genetic variation among A. avenae populations from different geographic regions.     

Occurrence and Genome Analysis of Cucurbit chlorotic yellows virus in Iran

In 2011 and 2012, several cucurbit‐growing regions of Iran were surveyed and samples with symptoms similar to those induced by Cucurbit chlorotic yellows virus (CCYV) were collected. The pathogen was transmitted to cucumber and melon under greenhouse conditions by whiteflies (Bemisia tabaci). RT‐PCR using designed CCYV‐specific primer pair (CCYV‐F/CCYV‐R) resulted in amplification of the predicted size DNA fragment (870 bp) for the coat protein (CP) gene in samples collected from Boushehr, Eyvanakay and Varamin. Nucleotide sequences of the CP of the three Iranian CCYV isolates were compared with five CCYV isolates obtained from GenBank and analysed. Phylogenetically, all CCYV isolates clustered in two groups; Group I is composed of five non‐Iranian isolates from China, Lebanon, Japan, Sudan and Taiwan, and the three Iranian isolates formed Group 2. Among Iranian isolates, the Eyvanakay isolate clustered in a distinct clade with the Boushehr and Varamin isolates. A phylogenetic tree based on amino acid identity of CP showed that CCYV was closely related to Lettuce chlorosis virus (LCV), Bean yellow disorder virus (BnYDV) and Cucurbit yellow stunting disorder virus (CYSDV). This is the first report of CCYV in Iran.     

Cryptic diversity in a Eurasian water snake (Natrix tessellata, Serpentes: Colubridae): Evidence from mitochondrial sequence data and nuclear ISSR-PCR fingerprinting

The dice snake, Natrix tessellata (Laurenti, 1768), is a suitable study organism to address questions of Eurasian phylogeography due to its wide Palearctic distribution. We analysed complete mitochondrial cytochrome b sequences and nuclear ISSR-PCR fingerprints of more than 300 specimens representing nearly the entire geographic range. Nine major mitochondrial lineages were discovered based on mtDNA sequences. The three most basal lineages comprised populations from Iran, Jordan–Egypt, and Greece, respectively. Other lineages were associated with samples from the Turkish peninsula, the Caucasus, the Aral Sea, and eastern Kazakhstan. A sister-group relationship was found between two lineages from Crete and the European mainland. Assuming an evolutionary rate of 1.35% sequence divergence per million years, among-lineage p-distances of 1.7–8.4% suggest that intraspecific differentiation might date back as far as the Miocene/Pliocene transition 5–6 million years ago. The pattern of genetic differentiation in mitochondrial phylogeny with regard to Asia Minor and the region of the Aral Sea was not congruent with the results of the nuclear ISSR-PCR analyses, and suggests admixing within some mtDNA clades at contact zones. The taxonomic implications of the high intraspecific variation in the dice snake are discussed.     

Characterization of spirolide producing Alexandrium ostenfeldii (Dinophyceae) from the western Arctic

Toxin producing dinoflagellates of the genus Alexandrium Halim represent a risk to Arctic environments and economies. This study provides the first record and a characterization of Alexandrium ostenfeldii in the western Arctic. During a cruise along the coasts of western and southern Greenland 36 isolates of the species were established in August 2012. Plankton samples taken at three different stations from the upper water layer at water temperatures of approx. 4–7 °C, contained low amounts of A. ostenfeldii. Sequencing of SSU and ITS-LSU rDNA and subsequent phylogenetic analyses identified all Greenland strains as members of a NW Atlantic spirolide producing phylogenetic clade. Molecular results were confirmed by morphological features typical for this group (=Group 5 of a recent ITS-LSU phylogeny of A. ostenfeldii). The Greenland isolates did not contain either Paralytic Shellfish Poisoning toxins or gymnodimines, but produced several spirolides. Altogether 12 different analogs were detected, of which only SPX-1, C, 20-meG and H have been described earlier. The remaining 8 spirolides have not been identified so far. Some of them were found to dominate the toxin profiles of a number of isolates. Among the 36 investigated strains spirolide composition varied considerably, particularly isolates from western Greenland (Station 516) exhibited a high diversity of analogs, with different profiles in nearly all 22 isolates. All of the 34 tested Greenland strains showed considerable lytic capacity when exposed to Rhodomonas salina.     

Increased frequency of scale anomalies and loss of genetic variation in serially bottlenecked populations of the dice snake, Natrix tessellata

Documented demographic bottlenecks resultingfrom introductions of the dice snake to severallakes in Switzerland provide a rare opportunityto study the effect of serial bottlenecks onthe genetic properties of Natrixtessellata populations. We investigated twointroduced populations using informationderived from eight microsatellite markers. Bothintroduced populations had significantlyreduced levels of allelic diversity relative tonon-bottlenecked populations. The severity ofthe bottlenecks was underlined by thesignificant reduction in observed and expectedheterozygosity. The loss of allelic diversityand observed heterozygosity was stronger in theserially bottlenecked population than in thepopulation that was bottlenecked only once.From previous studies, scale anomalies wereknown to be more common in introducedpopulations relative to native populations. Weinvestigated whether the higher occurrence ofscale anomalies in introduced populations isassociated with individual heterozygosity andmean genomic diversity d ². We founda significant relationship between theoccurrence of scale anomalies and individualheterozygosity but no significant relationshipbetween scale anomalies and the microsatellitespecific measurement, d ², was found.Because of their known history, introducedpopulations in Switzerland may serve as a modelto demonstrate the effect of severe populationbottlenecks on genetic variability anddevelopmental stability in N. tessellata.The results therefore help to device strategiesfor the management and protection of endangerednatural N. tessellata populations.     

DNA taxonomy of the potamid freshwater crabs from Northern Africa (Decapoda,Potamidae)

The Mediterranean area, from southern Balkans to western Maghreb, is inhabited by the Potamon subgenus Euthelphusa, with three currently recognised species. The Maghrebian species P. (E.) algeriense is isolated from other Potamon species by the Mediterranean Sea and the Sahara Desert, and nearly no molecular data are currently available for this taxon. Present study investigated the mtDNA and nuDNA diversity in Potamon algeriense s.l. with the aims of exploring its molecular diversity pattern throughout its known distribution range, and testing the possible presence of cryptic taxa currently lumped under this binomen. The phylogenetic and DNA taxonomy analyses showed the presence of two well-supported clades of species rank within P. algeriense s.l, with an eastern clade including the populations from Tunisia and Numidia, and a western, highly structured clade including the populations from central Algeria and Morocco. In agreement with a typical Maghrebian biogeographic pattern, the distribution of these two species shows a clear east–west divide, with a disjunction zone located in Kabylia, and a strong link between molecular diversity and segregation within different hydrographical basins is evident. The Maghreb thus proved to host an unexpectedly high genetic diversity of, and to constitute a biodiversity hot-spot for, the Potamon subgenus Euthelphusa. In the light of the existence of two well-characterised species currently lumped under P. algeriense s.l., and of their noteworthy molecular structuring, the status of Maghrebian Potamon populations should be re-assessed for both the species present in the area, which are to be considered as independent management units.     

Molecular characterization of Plasmodium vivax clinical isolates in Pakistan and Iran using pvmsp-1, pvmsp-3α and pvcsp genes as molecular markers

In this study, the diversity of Plasmodium vivax populations circulating in Pakistan and Iran has been investigated by using circumsporozoite protein (csp) and merozoite surface proteins 1 and 3α (msp-1 and msp-3α) genes as genetic markers. Infected P. vivax blood samples were collected from Pakistan (n = 187) and Iran (n = 150) during April to October 2008, and were analyzed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 (variable block 5) revealed the presence of type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant, in both study areas. The sequence analysis of 33 P. vivax isolates from Pakistan and 30 from Iran identified 16 distinct alleles each, with one allele (R-8) from Iran which was not reported previously. Genotyping pvcsp gene also showed that VK210 type is predominant in both countries. Moreover, based on the size of amplified fragment of pvmsp-3α, three major types: type A (1800 bp), type B (1500 bp) and type C (1200 bp), were distinguished among the examined isolates that type A was predominant among Pakistani (72.7%) and Iranian (77.3%) parasites. PCR/RFLP products of pvmsp-3α with HhaI and AluI have detected 40 and 39 distinct variants among Pakistani and Iranian examined isolates, respectively. Based on these three studied genes, the rate of combined multiple genotypes were 30% and 24.6% for Pakistani and Iranian P. vivax isolates, respectively. These results indicate an extensive diversity in the P. vivax populations in both studies.