Ribosomal DNA-internal Transcribed Spacer Region Research Articles

Molecular prevalence of Enterocytozoon bieneusi in stray cats of İzmir, Türkiye

The phylum Microsporidia contains obligate single celled parasites that can infect many vertebrate hosts including humans. Enterocytozoon bieneusi is considered as the most diagnosed species in humans. E. bieneusi has also been detected in many animals such as cats, dogs and cattle. Among these animals, cats are carriers of type D and IV which are the most common human pathogenic genotypes of E. bieneusi. In Türkiye, the prevalence of E. bieneusi in stray cats is not well known. Therefore, in this study, the molecular prevalence of E. bieneusi in stray cats (n = 339) was determined by Real-Time PCR targeting ribosomal DNA ITS (internal transcribed spacer) region of E. bieneusi. Initially, the analytical sensitivity of Real-Time PCR was determined by a plasmid control and then E. bieneusi DNA was investigated in fecal samples of stray cats. The results showed that the analytical sensitivity of Real-Time PCR targeting ITS region of E. bieneusi was ≤1 copy plasmid/reaction. Analysis of fecal samples revealed that the molecular prevalence of E. bieneusi was 50.15% (170/339). Overall, these results showed that the Real-Time PCR successfully detected E. bieneusi in cat’s fecal samples and stray cats can be an important source for transmission of E. bieneusi to humans and other animals.

Morphological and molecular characterization of Aspergillus elegans from small indigenous dry fish ‘Jat Puti’ Puntius sophore (Hamilton 1822) in Bangladesh

Small indigenous species (SIS) of fishes are available source of animal protein, vitamin and minerals, not commonly available in other foods in Bangladesh. These fishes are eaten both in fresh and dried condition. Puntious sophore was collected from different regions of Baikka Beel at Sreemangal Upazila, Moulvibazar district, Bangladesh to investigate mycoflora association with dried condition. Fungus was isolated from dried fish using tissue planting methods for classical and molecular characterization. Aspergillus elegans was identified through classical taxonomy and molecular approach based on ITS region of ribosomal DNA (rDNA) of fungi. For analyzing ITS4 and ITS5 were used. Mycelium of the identified fungus septated, hyaline, abundant and branched. Long, straight or flexous conidiophore were present. The molecular phylogeny in morphologically identified dry fish fungi of P. sophore such as A. elegans was studied based on their internal transcribed spacer (ITS). The sequences of ITS 600 bp region of A. elegans had 5.8S of rDNA sequences were identical. The highest mycelial growth was recorded in potato dextrose agar (PDA) medium for the growth and development of A. elegans. Temperature 30°C was optimum, this fungus grew well in pH 7. So, tested fungi grew well in neutral condition.
 Jahangirnagar University J. Biol. Sci. 10(1 & 2): 39-47, 2021 (June & December)

A Survey of Trunk Disease Pathogens within Citrus Trees in Iran.

Citrus trees with cankers and dieback symptoms were observed in Bushehr (Bushehr province, Iran). Isolations were made from diseased cankers and branches. Recovered fungal isolates were identified using cultural and morphological characteristics, as well as comparisons of DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region, translation elongation factor 1α, β-tubulin, and actin gene regions. Dothiorella viticola, Lasiodiplodia theobromae, Neoscytalidium hyalinum, Phaeoacremonium (P.) parasiticum, P. italicum, P. iranianum, P. rubrigenum, P. minimum, P. croatiense, P. fraxinopensylvanicum, Phaeoacremonium sp., Cadophora luteo-olivacea, Biscogniauxia (B.) mediterranea, Colletotrichum gloeosporioides, C. boninense, Peyronellaea (Pa.) pinodella, Stilbocrea (S.) walteri, and several isolates of Phoma, Pestalotiopsis, and Fusarium species were obtained from diseased trees. The pathogenicity tests were conducted by artificial inoculation of excised shoots of healthy acid lime trees (Citrus aurantifolia) under controlled conditions. Lasiodiplodia theobromae was the most virulent and caused the longest lesions within 40 days of inoculation. According to literature reviews, this is the first report of L. theobromae and N. hyalinum on citrus in Iran. Additionally, we report several Phaeoacremonium species, S. walteri, Pa. pinodella and C. luteo-olivacea on citrus trees for the first time in the world.

Occurrence and Diversity of Black-Foot Disease Fungi in Symptomless Grapevine Nursery Stock in Spain.

In this study, 3,426 grafted grapevines ready to be planted from 15 grapevine nursery fields in Northern Spain were inspected from 2016 to 2018 for black-foot causing pathogens. In all, 1,427 isolates of black-foot pathogens were collected from the asymptomatic inner tissues of surface sterilized secondary roots and characterized based on morphological features and DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region, histone H3, translation elongation factor 1-alpha and β-tubulin genes. Eleven species belonging to the genera Dactylonectria, Ilyonectria, Neonectria, and Thelonectria were identified, including Dactylonectria alcacerensis, D. macrodidyma, D. novozelandica, D. pauciseptata, D. torresensis, Ilyonectria liriodendri, I. pseudodestructans, I. robusta, Neonectria quercicola, Neonectria sp. 1, and Thelonectria olida. In addition, two species are newly described, namely D. riojana and I. vivaria. Twenty-four isolates representing 13 black-foot species were inoculated onto grapevine seedlings cultivar 'Tempranillo'. The pathogenicity tests detected diversity in virulence among fungal species and between isolates within each species. The most virulent species was D. novozelandica isolate BV-0760, followed by D. alcacerensis isolate BV-1240 and I. vivaria sp. nov. isolate BV-2305. This study improves our knowledge on the etiology and virulence of black-foot disease pathogens, and opens up new perspectives in the study of the endophytic phase of these pathogens in grapevines.

Assessment of genetic diversity among different sugarcane genotypes using internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA)

Internal transcribed spacer or ITS region of nuclear ribosomal DNA (rDNA) has been used to evaluate genetic assortment and phylogenetic relationship in nine sugarcane genotypes including Saccharum species and another related genus as Erianthus, Narenga and hybrid. DNA was extracted from selected genotypes and ITS (ITS-1 and ITS-2) regions were amplified using specific primers. The sequence lengths ITS-1 showed 205- 207 bp, while ITS2 was ranged from 211- 218 bp. However, G+C content (%) 65.2% - 67% in ITS-1 and in ITS-2 68.4% - 99.7%. The sequence lengths of fragment and GC content of ITS-1 and ITS-2 regions showed variable. To evaluate the phylogenetic association of both the region of ITS (ITS-1 and ITS-2) neighbor-joining (NJ) method was employed. The cluster A of ITS-1 and cluster B for ITS-2 and cluster C combined between ITS1+ ITS2 sequences gave two distinct groups A and B. The group A represented the ITS1 sequences which showed two subgroups I and II. The A-I subgroup consisted of wild species of sugarcane; Erianthus, Narenga and S. robustum, whereas the A-II subgroup consisted of the Saccharum species and hybrid. The ITS2 sequences in the group B showed better correlation amongst each other. The sequences ITS-1 & ITS-2 combined and compared with some selected sequences from NCBI database using NJ method. The results have confirmed that ITS region can be used for evaluating the genetic assortment in Saccharum and its closely related genes.

Three new species and the molecular phylogeny of Antipathozoanthus from the Indo-Pacific Ocean (Anthozoa, Hexacorallia, Zoantharia).

In this study, three new species of macrocnemic zoantharians (Hexacorallia, Zoantharia) are described from localities in the Indo-Pacific Ocean including the Red Sea, the Maldives, Palau, and southern Japan: Antipathozoanthus obscurus sp. n., A. remengesaui sp. n., and A. cavernus sp. n. Although the genus Antipathozoanthus is currently restricted to species living on antipatharians, A. obscurus sp. n. is not associated with any living substrate and instead is found on coral reef carbonate substrate within narrow caves or cracks. The two new species that have association with antipatharians, A. remengesaui sp. n. and A. cavernus sp. n., can be distinguished by their relative coenenchyme development and the antipatharian species that each uses as substrate. Additionally, all new species described in this study have unique nuclear internal transcribed spacer region of ribosomal DNA (ITS-rDNA) sequences. Our results indicate that more phylogenetic studies focusing on increasing the numbers of species examined within each of the genera of Parazoanthidae are required in order to better understand the evolutionary history of substrate specificity within the family Parazoanthidae.

Dominant Root Associated Fungi (RAF) from Drynaria quercifolia L. either Induce or Retard Growth of PSB Rc10 Rice (Oryza sativa L.) in Gibberellic Acid-Inhibited Medium

The testing and use of microorganisms for in vitro growth promotion of agriculturally significant crops such as rice has increased but remains underexploited. The current study aims to explore growth-enhancing mechanisms of dominant root-associated fungi (RAF) isolates from Drynaria quercifolia and test their effects on rice. The most abundant RAF on five tree-collection sites were cultured in vitro. Genomic DNA of the RAF were extracted and the ITS (internal transcribed spacer) region of the 18S ribosomal DNA (rDNA) were sequenced and molecularly identified. Two RAF isolates significantly increased the plant shoot/total length (Meyerozyma guilliermondii: 10.29±4.18/13.46±4.18 cm; Trichoderma simmonsii: 10.33±1.38/13.23+1.58 cm), shoot/total fresh weight (Meyerozyma guilliermondii: 57.33±15.76/71.00±16.10 mg; Trichoderma simmonsii: 63.22±12.23/76.00±10.67 mg) and shoot/total dry weight (Meyerozyma guilliermondii: 16.99±6.74/22.78±7.41 mg; Trichoderma simmonsii: 16.89±3.33/23.11±5.30 mg) weight comparedto the negative control. These results possibly show the ability of the two isolates to produce the hormone gibberellic acid. On the other hand, three of the RAF isolates did not significantly increase seedling growth and biomass. The Trichoderma yunnanense (shoot: 0.36±0.16 cm; total: 0.53±0.20 cm), unidentified Mucoromycotina isolate F5P1RAF16 (shoot: 1.87±0.59 cm; total: 2.12±0.58 cm) and the unidentified Mucoromycotinaisolate F9P2RAF21 (shoot: 3.26±1.56 cm; total: 5.19±2.00 cm) approximated the growth of rice seedlings inoculated with broth and water negative control (shoot: 4.40+2.27 cm; total: 6.38+2.28 cm). This possibly indicates the inability of these isolates to produce GA or their potential ability to produce growth-retarding metabolites. Preliminary data from this study reveal potential growth-promoting capacity of RAF isolates on rice.

Rare zooxanthellate Nanipora octocoral (Helioporacea) in the Gulf of Thailand

The octocoral Nanipora kamurai is the fifth species of octocoral described with an aragonite skeleton. So far, it has only been known from a single location in Okinawa, Japan. Here, we report on the second recorded location of the genus Nanipora, in the Gulf of Thailand, where its colonies were found in relatively high densities. A total of 143 separate Nanipora colonies were counted in transects on a coral reef at Koh Tao, Thailand. They were found living on dead coral skeletons and on the shell of a living Tridacna squamosa clam. Phylogenetic analyses of cytochrome oxidase subunit 1 (COI) and mitochondrial mismatch repair protein (mtMutS) sequences showed that they belong to N. kamurai or to a close relative of this species. Additionally, a symbiotic relationship with Symbiodinium was observed for the first time in Nanipora, confirmed by sequences of the internal transcribed spacer region of ribosomal DNA (ITS-rDNA) of the dinoflagellate.

Characterization and Pathogenicity of Botryosphaeriaceae Species Isolated from Almond Trees on the Island of Mallorca (Spain).

In this study, 31 almond orchards with trees showing severe decline symptoms were surveyed from 2009 to 2014 on the island of Mallorca (Spain). In all, 45 Botryosphaeriaceae isolates were collected and characterized based on phenotypical features and comparisons of DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region and elongation factor 1-α gene. Five species were identified as Diplodia olivarum, D. seriata, Neofusicoccum luteum, N. mediterraneum, and N. parvum. Pathogenicity tests were performed on four cultivars ('Pons', 'Vivot', 'Jordi', and 'Ferragnes') under field conditions for two consecutive years (2013 to 2014), and confirmed that all five species cause canker and dieback of almond, with Neofusicoccum spp. more virulent than Diplodia spp. in both years. Jordi was less sensitive to fungal infection in 2013. First reports from almond in Spain include N. mediterraneum and N. luteum.

Phenotypic and molecular characterization of the causal agent of chafer beetle mortality in the wheat fields of the Kurdistan province, Iran

AbstractWe report the first case of chafer beetle [Anisoplia austriaca(Herbst 1783)] mortality caused byActinomucor elegansvar.elegansin wheat fields of the Kurdistan province, Iran. For three years, dead larvae ofAnisoplia austriacawere collected from wheat fields of the Kurdistan province. Similar isolates of a fast-growing fungus were recovered from all samples. The fungal isolates were identified asA. elegansvar.elegansbased on morphological and cultural characteristics. The identity of the species was further confirmed using sequence data of the ITS (Internal Transcribed Spacer) region of ribosomal DNA. Koch’s postulates were fulfilled by the inoculation of the larvae ofA. austriacaandGalleria mellonella(Linnaeus, 1758) (as the model insect) using the spore suspension ofA. elegansvar.elegans. The viability of sporangiospores was evaluated using a spore dilution technique on germination medium. The results on the pathogenicity (100% mortality inA. austriacalarvae) and viability tests (germination: 95.45%) demonstrated thatA. elegansvar.eleganscan be considered as a potential biocontrol agent against the chafer beetle. Field experiments are still required to evaluate the capacity ofA. elegansas a biological control agent.

First Record of Alternaria simsimi Causing Leaf Spot on Sesame (Sesamum indicum L.) in Korea

Leaf spot disease was observed in sesame (Sesamum indicum L.) during 2009 and 2010 in Korea. The pathogen was identified as Alternaria simsimi based on morphological and cultural characteristics. The morphological identification was well supported by phylogenetic analysis of the ribosomal DNA-internal transcribed spacer region. A. simsimi isolates caused spot symptoms on leaves and stems of sesame plants 2 wk after artificial inoculation, which were similar to those observed in the field. This is the first record of leaf spot disease in Korea caused by A. simsimi.

Genetic Variation of Rice Populations Estimated Using nrDNA ITS Region Sequence

The rice belonging to Oryza sativa is not only has significant economic importance, for it is the major source of nutrition for about 3 billion all around the world. But also plays a vital role as a model organism, because it has a number of advantages to be a model plant, such as efficient transformation system and small genome size. Many methods and techniques have been conducted to attempt to distinguish different Oryza sativa species, such as amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and so on. However, studies using sequence analysis of internal transcribed spacer (ITS), a region of ribosomal RNA has not been reported until now. This study was undertaken with an aim to understand the phylogenetic relationships among sixteen isolates of Oryza sativa collected from abroad and fifteen isolates collected from Korea, using ribosomal RNA (rRNA) internal transcribed spacer (ITS) sequences to compare the phylogeny relationships among different Oryza sativa species. The size variation obtained among sequenced nuclear ribosomal DNA (nrDNA) ITS region ranged from 515bp to 1000bp. The highest interspecific genetic distance (GD) was found between Sfejare 45 (FR12) and Anapuruna (FR15). Taebong isolate showed the least dissimilarity of the ITS region sequence with other thirty isolates. This consequence will help us further understanding molecular diversification in intra-species population and their phylogenetic analysis.

Molecular analyses of shallow-water zooxanthellate zoanthids (Cnidaria: Hexacorallia) from Taiwan and their Symbiodinium spp

Although we previously reported on surveys of shallow-water zoanthid (Anthozoa: Hexacorallia: Zoantharia) species diversity from localities in Taiwan, no study has yet examined the molecular phylogeny of the majority of specimens, nor has any study examined zoanthid zooxanthellae (Symbiodinium spp.) diversity in Taiwan. As many phylogenetic results have been reported from Japan, a critical step in testing DNA identification of zoanthids and their symbionts is the application of these techniques to specimens from neighboring regions such as Taiwan. We utilized three DNA markers (mitochondrial cytochrome oxidase subunit I (COI), mitochondrial 16S ribosomal DNA (mt 16S rDNA), and the nuclear internal transcribed spacer region of ribosomal DNA (ITS-rDNA)) to examine the molecular phylogeny of 110 zooxanthellate specimens collected from 10 localities in Keelung, Kenting, and Green Island. Additionally, we report on the types of Symbiodinium spp. (zooxanthellae) from the three regions above plus the Penghu Islands, utilizing sequences of ITS2-rDNA. Most specimens in the suborder Brachycnemina belonged to previously reported species. However, it appears that there are still some undescribed Palythoa species groups in the northwestern Pacific that are cryptic in having small colony sizes (<5 polyps) and occurring in low numbers. With regards to Symbiodinium spp. diversity, Palythoa tuberculosa and Palythoa mutuki associated with subclade C1 and related types, Zoanthus sansibaricus associated with subclade C3-derived types, and Zoanthus kuroshio that harbored C15/C91-related Symbiodinium. Notably, 1/2 of the P. tuberculosa specimens from the Penghu Islands harbored a previously unreported clade C symbiont, and two Zoanthus. vietnamensis specimens from Keelung also had a unique symbiont type within clade C. It appears that undescribed Palythoa species in the northwestern Pacific may be widely distributed yet low in numbers, often cryptic in their habitat, and with a small colony size. As well, it is clear that much work remains to understand zoanthid-symbiont associations in Taiwan.

First report of black scurf on carrot caused by binucleate Rhizoctonia AG-U

Black scurf on carrot roots was found in Hokkaido, Japan, in 2010. An isolate of a binucleate Rhizoctonia was obtained from sclerotia on the root surface. This isolate was identified as anastomosis group (AG)-U based on cultural characteristics, hyphal fusions and the sequence of ribosomal DNA-internal transcribed spacer region. The AG-U isolate caused black scurf symptoms on carrot roots in an inoculation test. The reference isolate of Rhizoctoniasolani AG-2-2 IV, which causes carrot root rot, a disease with symptoms that differ from the black scurf symptom. This is the first report of carrot black scurf caused by binucleate Rhizoctonia AG-U.

Coiling dimorphism within a genetic type of the planktonic foraminifer Globorotalia truncatulinoides

Abstract The coiling direction—sinistral (left-coiled) or dextral (right-coiled)—of planktonic foraminiferal shells is a classical proxy used to assess past environmental changes and to understand their evolutionary patterns. Globorotalia truncatulinoides is composed of five different genetic types (I to V), each with a specific biogeographic distribution. So far, type II is the only type within which both coiling types have been frequently found. Here we examine the coiling direction as a dimorphic variation in a single genetic type, and show that the two forms with different coiling directions differ in ecology. The studied 140 left- and 137 right-coiled specimens from eight depth layers at four stations in the Sargasso Sea all belonged to Type II, based on the phylogenetic analyses of the ITS (internal transcribed spacer) region of ribosomal DNA. The distributions of left- and right-coiled Type II (Type II-L and II-R) differed strongly: Type II-L dominated between 400 and 200 m depth in the central water mass, whereas Type II-R was common in the top 200 m of the subtropical gyre. The left- and right-coiled forms are thus associated with different water masses. However, the vertical distribution along a temperature gradient indicates that their coiling direction is not determined by temperature, and might have some genetic basis. Our molecular phylogenetic analyses showed that the two forms probably share the same gene pool through their dispersals between different water masses. Moreover, the lineage of G. truncatulinoides is young (

Analysis of variability among the isolates of Peronosclerospora sorghi from sorghum and cornbased on restriction fragment length polymorphismof ITS region of ribosomal DNA

Downy mildew, caused by Peronosclerospora sorghi [Weston and Uppal (Shaw)], is one of the most serious diseases of sorghum and corn worldwide. Pathogenic variability among various isolates of P. sorghi has been reported. However, the molecular relationships between the isolates from sorghum and corn are unknown. In the present study DNA was extracted from P. sorghi isolates from sorghum and corn collected from different locations of Tamil Nadu, India, and their genetic variability was investigated using restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA. PCR amplification of the ITS regions of the P. sorghi isolates from sorghum and corn gave products of approximately 550 base pairs in length with slight variations in a few isolates. Restriction fragment length polymorphism analysis of the ITS regions of nuclear rDNA of P. sorghi with HhaI, EcoRI and MspI confirmed the heterogeneity among the P. sorghi isolates from sorghum and corn.

Identification of Rhizoctonia solani isolates from sugar beet roots by analyzing the ITS region of ribosomal DNA

Rhizoctonia solani (K?hn) is one of the most important sugar beet pathogens Rhizoctonia solani anastomosis groups (AGs) 2-2 and 4 are proven to be the most common pathogenic strains on sugar beet. AG 2-2 (intraspecific groups IIIB and IV) can cause root and crown rot while damping-off of seedlings is most frequently attributed to AG 4. Four isolates of R. solani from sugar beet roots showing characteristic crown and root rot symptoms, collected from different localities in Vojvodina Province, were chosen and compared to the well-characterized R. solani isolate R9, AG 2-2 IV, from the USA. All Vojvodinian isolates showed medium level of pathogenicity and were able to cause crown and root rot symptoms on inoculated sugar beet roots. Based on anastomosis reaction, isolates from Vojvodina did not belong to the AG 2-2 group. Sequencing of the ITS (internal transcribed spacer) region of ribosomal DNA was performed on the Vojvodinian isolates from R9 in order to determine their relatedness. Sequence analysis showed that these isolates were different than R9 and were closely related (99-100% sequence homology) to anastomosis group 4, subgroup HG II.

Phylogenetic analyses of the ITS region of ribosomal DNA reveal that Septoria passerinii from barley is closely related to the wheat pathogen Mycosphaerella graminicola

Septoria passerinii, a mitosporic ascomycete, causes speckled leaf blotch of barley. Although this fungus has been known to science for more than 120 yr, no teleomorph has been found and its phylogenetic relationships are not known. On the basis of spore morphology and growth characteristics in culture, S. passerinii appears similar to Septoria tritici, the anamorph of the wheat pathogen Mycosphaerella graminicola. To test the hypothesis that S. passerinii and M. graminicola are closely related, the internal transcribed spacer (ITS) region (ITS1, ITS2 and the intervening 5.8S rRNA gene) of both species was sequenced and compared to those of many other fungi in the Dothideales and Pleosporales of the Loculoascomycetes. Two ITS sequences were identified among seven isolates of S. passerinii. Six isolates from cultivated barley (Hordeum vulgare) shared the same 569 base-pair sequence. One isolate from the wild barley H. jubatum had a 569 base-pair sequence that differed from the first sequence by seven nucleotides. The ITS sequence of M. graminicola differed from those of both forms of S. passerinii by 10 nucleotides. These differences were large enough that all three ITS sequences could be distinguished by restriction enzyme digestion of polymerase chain reaction-amplified products. Cluster analyses revealed that both forms of S. passerinii were closely related to M. graminicola and were contained within a large, monophyletic group of Mycosphaerella species. This confirms that the teleomorph of S. passerinii, if it exists, must be a Mycosphaerella. Furthermore, the isolate from H. jubatum probably represents a new, undescribed species that is separated from S. passerinii by host specificity. Our analysis also revealed the phylogenetic relationships of the halophilic black yeasts Trimmatostroma salinum and Phaeotheca triangularis within Mycosphaerella and of Rhizopycnis vagum within the Pleosporales. The Pleosporalean genera Leptosphaeria and Phaeosphaeria were apparently polyphyletic, as were most of the anamorph genera in both the Pleosporales and Dothideales. Pathogenicity to the same host occurred in phylogenetically distinct species, indicating convergent evolution for host specificity. Large differences in size of the ITS1 region made alignment difficult; within the Dothideales and Pleosporales, the best phylogenetic analyses were obtained with ITS2 sequences.

Molecular phylogeny of Acer monspessulanum L. subspecies from Iran inferred using the ITS region of nuclear ribosomal DNA

Khademi H, Mehregan I, Assadi M, Nejadsatari T, Zarre S. 2015. Molecular phylogeny of Acer monspessulanum L. subspecies from Iran inferred using the ITS region of nuclear ribosomal DNA. Biodiversitas 17: 16-23. This study was carried out on the Acer monspessulanum complex growing wild in Iran. Internal transcribed spacer (ITS) sequences for 75 samples representing five different subspecies of Acer monspessulanum were analyzed. Beside this, 86 previously published ITS sequences from GenBank were used to test the monophyly of the complex worldwide. Phylogenetic analyses were conducted using Bayesian inference and maximum parsimony. The results indicate that most samples of A. monspessulanum species from Iran were part of a monophyletic clade with 8 samples of A. ibericum from Georgia, A. hyrcanum from Iran and one of A. sempervirens from Greece (PP= 1; BS= 79%). Our results indicate that use of morphological characteristics coupled with molecular data will be most effective.