Nuclear Internal Transcribed Spacer Research Articles

Phylogenetic evidence of a possible Trichuris globulosa species complex in Arabian camels from Kuwait.

During a 1-year study, Trichuris adults were obtained after necropsy of Arabian camels (Camelus dromedarius) from a slaughterhouse in Kuwait. Morphological and molecular identification was performed to confirm the identity of the Trichuris specimens obtained from C. dromedarius. Fifteen male Trichuris specimens were selected, and molecular identification was performed using mitochondrial cytochrome c oxidase subunit I, 12S ribosomal RNA, 16S ribosomal RNA genes and the nuclear internal transcribed spacer 2 (ITS2) region. Through phylogenetic analysis, 2 distinct groups were obtained using the mitochondrial genes, where group 1 showed a close relationship to Trichuris globulosa while group 2 showed a close relationship to Trichuris ovis, providing molecular evidence of a possible T. globulosa species complex. Additionally, the nuclear ITS2 region did not provide enough resolution to distinguish between the 2 groups of Trichuris specimens. Observation of morphological characters revealed variations in the shape of the male spicule sheath, where specimens present either a globular posteriorly truncated swelling or the absence of posteriorly truncated swelling. Moreover, the variations in male spicule sheath does not corroborate with the results of molecular data, suggesting the limited use of this character for identification of T. globulosa. In conclusion, molecular analysis suggests a possible species complex in T. globulosa, with the mitochondrial genetic markers successfully differentiating between the 2 groups. The limited use of the male spicule sheath as a diagnostic character for identification of T. globulosa is suggested.

Morphology and phylogenetic relationships of two Antarctic strains within the genera Carolibrandtia and Chlorella (Chlorellaceae, Trebouxiophyceae)

The genera Carolibrandtia and Chlorella have been described as small green algae with spherical cell shapes that inhabit various environments. Species of these genera are often difficult to identify because of their simple morphology and high phenotypic plasticity. We investigated two small coccoid strains from Antarctica based on morphology, molecular phylogeny by two alignment methods which have been applied to previous phylogenetic studies of the genus Chlorella, and comparison of the secondary structures of nuclear small subunit (SSU) and internal transcribed spacer (ITS) rDNA sequences. Light microscopy of two strains revealed spherical cells containing chloroplasts with pyrenoids, and the morphological characteristics of the strains were nearly identical to those of other Chlorella species. However, based on the phylogenetic analyses of nuclear SSU and ITS rDNA sequences, it was determined that the Antarctic microalgal strains belonged to two genera, as the Chlorella and Carolibrandtia. In addition, the secondary structures of the SSU and ITS2 sequences were analyzed to detect compensatory base changes (CBCs) that were used to identify and describe the two strains. A unique CBC in the SSU rDNA gene was decisive for distinguishing strain CCAP 211/45. The ITS2 rDNA sequences for each strain were compared to those obtained previously from other closely related species. Following the comparison of morphological and molecular characteristics, we propose KSF0092 as a new species, Chlorella terrestris sp. nov., and the reassignment of the strain Chlorella antarctica CCAP 211/45 into Carolibrandtia antarctica comb. nov.

Applying an internal transcribed spacer as a single molecular marker to differentiate between Tetraselmis and Chlorella species.

In the realm of applied phycology, algal physiology, and biochemistry publications, the absence of proper identification and documentation of microalgae is a common concern. This poses a significant challenge for non-specialists who struggle to identify numerous eukaryotic microalgae. However, a promising solution lies in employing an appropriate DNA barcoding technique and establishing comprehensive databases of reference sequences. To address this issue, we conducted a study focusing on the molecular characterization and strain identification of Tetraselmis and Chlorella species, utilizing the internal transcribed spacer (ITS) barcode approach. By analyzing the full nuclear ITS region through the Sanger sequencing approach, we obtained ITS barcodes that were subsequently compared with other ITS sequences of various Tetraselmis and Chlorella species. To ensure the reliability of our identification procedure, we conducted a meticulous comparison of the DNA alignment, constructed a phylogenetic tree, and determined the percentage of identical nucleotides. The findings of our study reveal the significant value of the ITS genomic region as a tool for distinguishing and identifying morphologically similar chlorophyta. Moreover, our results demonstrate that both the ITS1 and ITS2 regions are capable of effectively discriminating isolates from one another; however, ITS2 is preferred due to its greater intraspecific variation. These results underscore the indispensability of employing ITS barcoding in microalgae identification, highlighting the limitations of relying solely on morphological characterization.

Astragalicola ephedrae sp. nov., isolated from the stem of Ephedra gerardiana in Ladakh, India.

An endophytic fungus designated as EIT4T (MCC 9756T) was isolated from the asymptomatic stem tissue of Ephedra gerardiana collected from the Kargil district of Ladakh Union territory, India. Phylogenetic analysis based on concatenated nuclear ribosomal ITS (internal transcribed spacer) and LSU (large ribosomal subunit) sequence datasets revealed its placement within the genus Astragalicola. However, it formed a separate clade exhibiting strong bootstrap support value (80%). The highest nrITS sequence similarity between EIT4T and species of Astragalicola was 95.19% (A. vasilyevae) and 94.26% (A. amorpha), while nrLSU sequence similarity was 99.27% (A. amorpha). Morphologically, EIT4T differs from the other species of Astragalicola in having larger sub-globose to pyriform conidiomata, smaller and mostly unbranched conidiophores, and polymorphic translucent conidia with two terminal guttules. Based on combined cultural, micromorphological, molecular, and phylogenetic analyses, EIT4T represents a novel species in the genus Astragalicola proposed here as Astragalicola ephedrae sp. nov. Detailed description and illustrations of the novel species are provided. The type strain is EIT4T (= MCC 9756T = MN29T).

Plastome-based phylogeny and biogeography of Lactuca L. (Asteraceae) support revised lettuce gene pool categories.

This study generated and analyzed complete plastome and internal transcribed spacer (ITS) data of 46 Lactuca species, 13 African endemic (AE) Lactuca species, and 15 species from eight related genera in Lactucinae. The new plastome and nuclear ITS sequences were then used to reconstruct the phylogenetic relationships of Lactuca species. The whole-plastome data were used to estimate divergence time and ancestral area reconstruction of the identified major Lactuca lineages. The results showed that Lactuca species are generally similar in plastome size, Guanine and Cytosine (GC) content, gene structure, and categories, although crop lettuce (Lactuca sativa L.) and its gene pool relatives were found to have one unique pseudogene (ψ ndhF), and accD, atpF, cemA, clpP, and rpl22 showed signs of positive selection. Our phylogenomic analysis demonstrated that Lactuca is monophyletic after excluding Lactuca alatipes Collett and Hemsl and AE Lactuca species. AE Lactuca species are morphologically distinct from core Lactuca lineage and need to be excluded from Lactua. The core Lactuca species most likely originated from Asia-Temperate W ~6.82 Mya and then dispersed globally and formed nine clades. Finally, the lettuce gene pool concept was amended according to the phylogenetic and historical biogeographic analyses. This study revised the circumscription of Lactuca, revealed robust phylogenetic relationships within the genus, and provided insights into Lactucinae phylogeny. The lettuce gene pool species could be used as potential genetic resources for lettuce breeding.

Population genetics and demography of the coral-killing cyanobacteriosponge, Terpios hoshinota, in the Indo-West Pacific.

The first occurrence of the cyanobacteriosponge Terpios hoshinota was reported from coral reefs in Guam in 1973, but was only formally described in 1993. Since then, the invasive behavior of this encrusting, coral-killing sponge has been observed in many coral reefs in the West Pacific. From 2015, its occurrence has expanded westward to the Indian Ocean. Although many studies have investigated the morphology, ecology, and symbiotic cyanobacteria of this sponge, little is known of its population genetics and demography. In this study, a mitochondrial cytochrome oxidase I (COI) fragment and nuclear ribosomal internal transcribed spacer 2 (ITS2) were sequenced to reveal the genetic variation of T. hoshinota collected from 11 marine ecoregions throughout the Indo-West Pacific. Both of the statistical parsimony networks based on the COI and nuclear ITS2 were dominated by a common haplotype. Pairwise FST and Isolation-by-distance by Mantel test of ITS2 showed moderate gene flow existed among most populations in the marine ecoregions of West Pacific, Coral Triangle, and Eastern Indian Ocean, but with a restricted gene flow between these regions and Maldives in the Central Indian Ocean. Demographic analyses of most T. hoshinota populations were consistent with the mutation-drift equilibrium, except for the Sulawesi Sea and Maldives, which showed bottlenecks following recent expansion. Our results suggest that while long-range dispersal might explain the capability of T. hoshinota to spread in the IWP, stable population demography might account for the long-term persistence of T. hoshinota outbreaks on local reefs.

Rapid identification of Amanita citrinoannulata poisoning using colorimetric and real-time fluorescence and loop-mediated isothermal amplification (LAMP) based on the nuclear ITS region

Health concerns and financial losses caused by mushroom poisoning have been reported worldwide. Amanita citrinoannulata, a poisonous mushroom commonly found in China, results in a toxic reaction in humans after mistaken ingestion. To reduce the mistaken ingestion of poisonous mushrooms and to improve clinical diagnosis of mushroom poisoning, a rapid mushroom species identification method is required. Such identification methods could be advantageous in the identification of other poisonous mushroom species. This study developed two rapid and sensitive methods for the detection of A. citrinoannulata utilizing colorimetric and real-time loop-mediated isothermal amplification (LAMP) technology and specifically designed primers for the internal transcribed spacer (ITS) genes of A. citrinoannulata. The methods demonstrated high sensitivity as 0.2 ng of A. citrinoannulata DNA could be detected, with no cross-reaction with 41 non-target mushroom species. The entire detection process could be completed within 40 min without requiring complex instruments and can be observed by the naked eye. Therefore, these novel methods can be used for the identification of fresh and cooked mushroom samples and vomit samples, which contain only 1% A. citrinoannulata. Furthermore, these methods facilitate the detection of mushroom poisoning, and thus, have potential to reduce the number of mushroom poisoning-related deaths worldwide.

Pragmatic Applications and Universality of DNA Barcoding for Substantial Organisms at Species Level: A Review to Explore a Way Forward.

DNA barcodes are regarded as hereditary succession codes that serve as a recognition marker to address several queries relating to the identification, classification, community ecology, and evolution of certain functional traits in organisms. The mitochondrial cytochrome c oxidase 1 (CO1) gene as a DNA barcode is highly efficient for discriminating vertebrate and invertebrate animal species. Similarly, different specific markers are used for other organisms, including ribulose bisphosphate carboxylase (rbcL), maturase kinase (matK), transfer RNA-H and photosystem II D1-ApbsArabidopsis thaliana (trnH-psbA), and internal transcribed spacer (ITS) for plant species; 16S ribosomal RNA (16S rRNA), elongation factor Tu gene (Tuf gene), and chaperonin for bacterial strains; and nuclear ITS for fungal strains. Nevertheless, the taxon coverage of reference sequences is far from complete for genus or species-level identification. Applying the next-generation sequencing approach to the parallel acquisition of DNA barcode sequences could greatly expand the potential for library preparation or accurate identification in biodiversity research. Overall, this review articulates on the DNA barcoding technology as applied to different organisms, its universality, applicability, and innovative approach to handling DNA-based species identification.

Hybrid origin of the invasive Spartina anglica inferred from chloroplast and nuclear ITS phylogenies

Spartina anglica originated from interspecific hybridization between S. alterniflora and S. maritima which produced a sterile hybrid S. × townsendii. Subsequent whole genome duplication of the hybrid, gave rise to a vigorous allopolyploid S. anglica. In earlier studies, both parental nuclear sequences were detected in S. anglica. However, to date, parental copies of nuclear ribosomal DNA (nrDNA) have not been detected in a phylogenetic context. In the present study, we used the nuclear internal transcribed spacer (ITS) region in conjunction with chloroplast DNA (trnL-trnF, matK, and rbcL) to infer the hybrid origin of S. anglica invading Korean coasts. Our results revealed that the chloroplast DNA sequences of S. anglica were similar to those of S. alterniflora, which confirms its maternal origin. However, in the nuclear rDNA ITS region, we detected two highly divergent groups of ITS sequences that were divided between the two parent lineages. The maternal copies were indistinguishable from S. alterniflora, while the paternal repeats were highly differentiated from S. maritima and exhibited characteristics of ITS pseudogenes. This indicates that S. anglica has undergone rapid changes at the rDNA loci after the allopolyploid formation. Our study demonstrated the two divergent copies of ITS sequences, thus resolving the phylogenetic hybrid origin of S. anglica. This study offers further insights into the evolutionary history of S. anglica.

Molecular identification and phylogenetic analysis of Aloe shadensis from Saudi Arabia based on matK, rbcL and ITS DNA barcode sequence

The Kingdom of Saudi Arabia thrives with great plant diversity, including rare plants of the family Asphodelaceae that have multiple benefits and are still being studied. Aloe shadensis is one of these plants that must be preserved and documented in its natural environment. The most appropriate molecular approach currently approved for documentation is the sequencing of some genomic markers. The current study is the first to use genomic markers to record this rare plant. In this study, the plastid genes matK (Maturase K), rbcL (Ribulose-bisphosphate carboxylase/oxygenase large subunit), and the nuclear region ITS (Internal transcribed spacer) were used to reveal their efficiency in identifying the plant under study. This study is the first to deal with this plant and document it using these genetic markers. The study showed a promising result concerning identifying the sequence of the matK gene and ITS region, while the rbcL gene did not give a good indicator through the used primers. The obtained sequences of the matK gene and the ITS region were determined through two different sets of primers in each case then deposited in GenBank. The evolutionary relatedness of Aloe shadensis was established with the different species of Aloe. The study showed that the closest species is Aloe vera with a similarity of more than 99 %. The study concludes with the possibility of using these genes to correctly identify, distinguish and document the species of Aloe shadensis.

Homogeneity and Possible Replacement of Populations of the Dengue Vectors Aedes aegypti and Aedes albopictus in Indonesia.

Currently, Aedes aegypti, the principal vector of dengue virus in Indonesia, has spread throughout the archipelago. Aedes albopictus is also present. Invasion and high adaptability of the Aedes mosquitoes to all of these areas are closely related to their ecology and biology. Between June 2016 and July 2017, larval and adult mosquito collections were conducted in 43 locations in 25 provinces of Indonesia using standardized sampling methods for dengue vector surveillance. The samples collected were analyzed for polymorphism and phylogenetic relationship using the mitochondrial cox1 gene and the nuclear ribosomal internal transcribed spacer 2 (ITS2). Almost all Ae. aegypti samples collected in this study (89%) belonged to the same haplotype. A similar situation is observed with the nuclear ITS2 marker. Populations of Ae. aegypti characterized few years ago were genetically different. A closely related observation was made with Aedes albopictus for which the current populations are different from those described earlier. Ae. aegypti populations were found to be highly homogenous all over Indonesia with all samples belonging to the same maternal lineage. Although difficult to demonstrate formally, there is a possibility of population replacement. Although to a lower extent, a similar conclusion was reached with Ae. albopictus.

Phylogenetic origin of two Japanese Torreya taxa found in two regions with strongly contrasting snow depth.

The Japanese archipelago exhibits a notable difference in snow depth in winter, deep snow on the Sea of Japan side and low snow cover on the Pacific Ocean side. This contrasting pattern has shaped the distribution of infraspecific taxon pairs in a range of woody plants, with taxa found on the Sea of Japan side typically exhibiting a stunted shrub form with multiple decumbent stems. The phylogenetic origin of these taxon pairs is unknown, i.e., whether the two taxa diverged from the same species or if they have different origins. This study aimed to reveal the phylogenetic origin of two varieties of Torreya nucifera (Taxaceae); var. nucifera is a tree found on the Pacific Ocean side, whereas var. radicans is a shrub found on the Sea of Japan side. We examined the phylogenetic relationships of the two varieties and worldwide Torreya taxa using whole chloroplast genomes, chloroplast DNA fragments, and the nuclear ribosomal internal transcribed spacer (ITS). The whole chloroplast genome phylogeny indicated that T. nucifera var. radicans was a sister taxon to Chinese T. grandis, rather than to var. nucifera. In contrast, the nuclear ITS phylogeny indicated that while several haplotypes of T. nucifera var. radicans were closely related to T. grandis, most haplotypes of T. nucifera var. radicans formed a single clade with those of var. nucifera. This implies that the homogenization of the ITS has occurred between the two taxa, while taxon-specific chloroplast DNA haplotypes were retained. These discordant phylogenies suggested that the two taxa have different phylogenetic origins, but have an intricate evolutionary history, involving inter-taxa hybridization and gene flow, possibly when their distributions were confined to sympatric refugia. Given the genetic evidence and distinct difference in growth form, we propose that T. nucifera var. radicans should be taxonomically treated as a distinct species, T. fruticosa.

The use of DNA barcoding to avoid adulteration in olive plant leaf products

Abstract. Partovi R, Iranbakhsh A, Sheidai M, Ebadi M. 2020. The use of DNA barcoding to avoid adulteration in olive plant leaf products. Asian J For 5: 42-47. The leaves of olive plant species Olea europeae, and O. europeae var. cuspidata have been used for medicinal perfused in Iran. The first species leaves have been used to control the blood pressure, while the leaves of wild olive have been used for abortion by locals. Our preliminary inspection of the medicinal plant market revealed that the leaves of these two olive species are sold mistakenly to the consumers and their health might be at risk. Therefore, we permed this investigation to produce DNA barcodes for correct identification of these two olive species and also identify the potential adulteration in our local market. We used Internal transcribed spacer (ITS), as well as plastid genome trnL-F intergenic spacer and ribosomal protein L16 (rpL16) sequences. These sequences after alignment and curation produced DNA barcodes that can differentiate the two olive species from each other. The phylogenetic trees constructed also separated the samples of these olive species and confirmed the potential use of these short DNA sequences for olive barcoding. The present study revealed that some of the local shops mistakenly sell the wild olive leaves instead of the cultivated olive leaf to be used for blood pressure. This mistake endangers the health of pregnant women consumers if they carry a child. We suggest using a combination of nuclear ITS and plastid intergenic spacer (trnL-F and rpL16) regions for DNA barcoding of olive plants to avoid leaf product adulteration.

<p><strong><em>Urocystis piptatheri</em></strong><strong> sp. nov. (Urocystaceae), a new species of smut fungus on <em>Piptatherum laterale</em> (Poaceae) from Pakistan</strong></p>

A new species of smut fungi, Urocystis piptatheri is described from Kumrat Valley, Pakistan based on morphological characters and molecular phylogenetic analysis of nuclear ribosomal DNA comprising the internal transcribed spacer (ITS) regions along with large subunit (LSU). The new species is characterized by the large size of its spore balls, size and number of central spores, spore wall thickness, sori in leaves and by the host plant species (Piptatherum laterale). Phylogenetic analysis of nuclear ribosomal ITS and LSU nucleotide sequences confirmed the novelty of Urocystis piptatheri. A key for the identification of Urocystis species on Pipthatherum spp. is provided.

First report of the isolation and phylogenetic characterization of equine Setaria digitata from India based on mitochondrial COI, 12S rDNA, and nuclear ITS2 sequence data.

Equine ocular setariasis arising mainly from ectopic infestation of Setaria digitata is a common vision impairing ophthalmic disease in India, and the identification of this filarial nematode is based solely on morphology. However, morphological characters alone are inadequate to detect and differentiate S. digitata from its congeners. The present communication reports the first phylogenetic characterization of equine S. digitata from India based on sequences derived from the mitochondrial cytochrome c oxidase subunit 1 (COI), the mitochondrial small subunit ribosomal DNA (12S rDNA), and the nuclear internal transcribed spacer 2 (ITS2). Three isolates were characterized for each gene, and respective sequences were submitted to NCBI database (MN078131, MN078132, and MN095798). The sequences were also compared with the other related sequences available from PubMed around the globe, and phylogenetic analysis was carried out in conjunction with nucleotide homologies. There was no intraspecific variation among the Indian isolates. The phylogenetic analysis of S. digitata, inferred from these genes, showed that the isolate sequences obtained from different host species created a separate monophyletic clade within the genus Setaria with minor sequence variations revealing similar molecular characteristics of S. digitata isolates throughout the globe. In addition, the studied Indian isolates were found closer to Sri Lankan isolates. The S. digitata and S. labiatopapillosa appeared as sister species.

Genetic and morphological characterization of Ixodes apronophorus from Western Siberia, Russia

Genetic variability of I. apronophorus from Western Siberia, Russia was examined using the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial 16S rRNA and cytochrome c oxidase subunit 1 (cox1) genes and compared to those of Ixodes persulcatus and Ixodes trianguliceps from the same site. The I. apronophorus sequences demonstrated the highest nucleotide and haplotype diversity for both mitochondrial genes, whereas I. persulcatus was more variable in the nuclear ITS2. Phylogenetic analysis of the molecular sequence data showed that I. apronophorus differed from other Ixodes species, including Romanian I. apronophorus. The level of identity between 16S rRNA gene sequences of Siberian and Romanian I. apronophorus was only 91%; these sequences did not form a monophyletic group, indicating that I. apronophorus from Siberia and Romania could be different tick species. The analysis of morphological features of the Siberian I. apronophorus confirmed their consistency with those for the previously described I. apronophorus species. Based on the 16S rRNA and ITS2 sequences, Siberian I. apronophorus clustered together with Ixodes kazakstani and Ixodes scapularis, which are the recognized members of the Ixodes ricinus-I. persulcatus species complex within the subgenus Ixodes, and can be assigned to this complex.

Description of a new species and understanding the genetic diversity of Saccocoelioides Szidat, 1954 (Haploporidae) in Middle America using mitochondrial and nuclear DNA sequences

Members of the genus Saccocoelioides Szidat, 1954, include endoparasites from freshwater and brackish fishes from the Americas. Adult specimens were collected from the intestines of Poecilia catemaconis Miller, 1975, a poeciliid fish endemic to Catemaco Lake, and the white mullet Mugil curema Valenciennes, 1836, from Alvarado Lagoon, Veracruz, Mexico. The specimens were sequenced for three molecular markers, internal transcribed spacer 2 (ITS2) and domains D1-D3 from the large subunit (LSU) of nuclear ribosomal DNA and cytochrome c oxidase subunit 1 (cox 1) from mitochondrial DNA. The newly sequenced specimens were aligned with other sequences downloaded from GenBank. Maximum likelihood and Bayesian inference analyses were inferred with three data sets (a combination of nuclear DNA ITS2 + LSU, cox 1 alone and the concatenated cox 1+ ITS2 + LSU). The phylogenetic analyses inferred with the combined data set of the two nuclear molecular markers (ITS2 + LSU) revealed that Saccocoelioides is monophyletic and formed 11 independent lineages representing 11 valid species previously recognized plus the new lineage that is herein described as a new species named Saccocoelioides macrospinosus n. sp., however, the new species was placed in a basal polytomy in the tree. Therefore, the addition of a mitochondrial gene with a fast rate of substitution was fundamental to clarify the phylogenetic relationships of the new species. The genetic divergences estimated with the cox 1 gene were high, ranging from 8.3 to 15.5% among Saccocoelioides macrospinosus n. sp. and sister taxa. The new species has a slightly elongated body measuring 440–850 μm long and was classified in the diminutive morphotype. In addition, seven adult specimens recovered from the intestines of the banded tetra fish Astyanax aeneus Günther, 1860 from Nicaragua and Costa Rica formed a monophyletic clade with other specimens identified previously as Saccocoelioides tkachi, expanding its distribution range in other areas of Middle America.

New insight into genetic variation and haplotype diversity of Fasciola hepatica from Algeria.

The liver fluke Fasciola hepatica is the main cause of fasciolosis in North Africa leading to significant economic losses and public health problems. In this study, the ribosomal internal transcribed spacer (ITS), cytochrome c oxidase I (COI), the mitochondrial region spanning the COI-trnT-rrnL, and the NADH dehydrogenase subunit I (NADI) markers were used to characterize Fasciola flukes from Algeria. Fasciola appeared widespread from the east to the west of Algeria. Among 1701 sampled cattle from 8 Algerian provinces, 5% were infected. Using morphological and morphometric analysis, one morphotype of Fasciola was observed. Nuclear ITS marker indicated that all collected flukes belong to F. hepatica. Multiple alignments of ITS dataset revealed two haplotypes, one described here for the first time. Analysis of molecular variance (AMOVA) of mitochondrial markers revealed weak population structure in Algeria. Mismatch distributions, neutrality tests, and median-joining network analysis all were compatible with a recent expansion of Algerian F. hepatica population. Fasciolosis appeared common in Algerian cattle, it seems that the absence of control strategy coupled to the favorable Mediterranean climate may lead to a reconstruction and dispersion of its populations. This study provides important results concerning the genetic characterization and variability of F. hepatica in Algeria as well as the significant role of cattle importation in shaping its dispersal route worldwide.

Phenotypic and molecular analysis of Mexican Metarhizium anisopliae strains

Metarhizium anisopliae is an anamorphic fungus widely studied due to its use as a biological control agent. In Mexico, it is mainly used as an integrated management strategy to control populations of spittlebugs (Hemiptera: Cercopidae) that affect sugarcane fields and cattle pastures. In this study, 13 strains isolated from Aeneolamia spp. (Hemiptera: Cercopidae) from the main areas of sugarcane production of Mexico and 11 reference strains of Metarhizium from different insect hosts and geographic origins were evaluated based on their conidial dimensions, thermotolerance at 15, 25, 30, and 35 oC, random amplification of polymorphic DNA (RAPD) patterns using a combined analysis with 14 different oligonucleotides and diversity estimators, and a phylogenetic analysis with the ribosomal RNA internal transcribed spacer (ITS) region. All strains used in this study showed typical morphological characteristics corresponding to M. anisopliae and were able to grow at 25 and 30 oC with restricted growth at low (15 oC) and high temperatures (35 oC). Polymorphism analysis clustered all strains from Aeneolamia sp. in one welldefined group with low variability among them. Phylogenetic relationships based on nuclear ribosomal ITS region sequences recovered a subclade within M. anisopliae formed by 10 of the Mexican strains.

The genetic relationship between R. microplus and R. decoloratus ticks in South Africa and their population structure

Rhipicephalus microplus and R. decoloratus are one-host ticks that preferentially feed on cattle. They are capable of transmitting various tick-borne pathogens which may be detrimental to the agricultural and livestock industry in South Africa. Previous studies have shown that R. microplus forms five lineages in the R. microplus complex, segregating into different geographical areas based on mitochondrial markers. This study examined the phylogenetic relationship within and between R. microplus and R. decoloratus using the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome oxidase subunit I (COI) genes. The results showed that the nuclear ITS2 marker is informative for interspecific variation but lacks the resolution for intraspecific variation. Analysis of the mitochondrial COI gene revealed that R. microplus ticks from South Africa grouped into a clade comprised of ticks from Asia and South America. The population structure of these two tick species was also investigated using novel microsatellite markers. Population structure analyses revealed that both the R. microplus and R. decoloratus populations presented with two genetic clusters. Rhipicephalus microplus ticks from the Kwa-Zulu Natal (KZN) province belonged to cluster 1, and those from the Eastern Cape (EC) province predominantly grouped into cluster 2. No observable population structure was noted for R. decoloratus. The overlap of genetic clusters in both species could be attributed to inbreeding between the regions by unrestricted movement of cattle across provinces. Such movement promotes tick mobility, gene flow and the homogenisation of tick populations.