Structure Of ITS2 Research Articles

DNA Barcoding and ITS2 Secondary Structure Predictions in Taro (Colocasia esculenta L. Schott) from the North Eastern Hill Region of India.

Taro (Colocasia esculenta L. Schott, Araceae), an ancient root and tuber crop, is highly polygenic, polyphyletic, and polygeographic in nature, which leads to its rapid genetic erosion. To prevent the perceived loss of taro diversity, species discrimination and genetic conservation of promising taro genotypes need special attention. Reports on genetic discrimination of taro at its center of origin are still untapped. We performed DNA barcoding of twenty promising genotypes of taro indigenous to the northeastern hill region of India, deploying two chloroplast-plastid genes, matK and rbcL, and the ribosomal nuclear gene ITS2. The secondary structure of ITS2 was determined and molecular phylogeny was performed to assess genetic discrimination among the taro genotypes. The matK and rbcL genes were highly efficient (>90%) in amplification and sequencing. However, the ITS2 barcode region achieved significant discrimination among the tested taro genotypes. All the taro genotypes displayed most similar sequences at the conserved matK and rbcL loci. However, distinct sequence lengths were observed in the ITS2 barcode region, revealing accurate discriminations among the genotypes. Multiple barcode markers are unrelated to one another and change independently, providing different estimations of heritable traits and genetic lineages; thus, they are advantageous over a single locus in genetic discrimination studies. A dynamic programming algorithm that used base-pairing interactions within a single nucleic acid polymer or between two polymers transformed the secondary structures into the symbol code data to predict seven different minimum free energy secondary structures. Our analysis strengthens the potential of the ITS2 gene as a potent DNA barcode candidate in the prediction of a valuable secondary structure that would help in genetic discrimination between the genotypes while augmenting future breeding strategies in taro.

Organization and expression analysis of 5S and 45S ribosomal DNA clusters in autotetraploid Carassius auratus

BackgroundAutotetraploid Carassius auratus (4n = 200, RRRR) (abbreviated as 4nRR) is derived from whole genome duplication of Carassius auratus red var. (2n = 100, RR) (abbreviated as RCC). Ribosome DNA (rDNA) is often used to study molecular evolution of repeated sequences because it has high copy number and special conserved coding regions in genomes. In this study, we analysed the sequences (5S, ITS1-5.8S-ITS2 region), structure, methylation level (NTS and IGS), and expression level (5S and 18S) of 5S and 45S ribosomal RNA (rRNA) genes in 4nRR and RCC in order to elucidate the effects of autotetraploidization on rDNA in fish.ResultsResults showed that there was high sequence similarity of 5S, 5.8S and ITS1 region between 4nRR and RCC. This study also identified two different types of ITS2 region in 4nRR and predicted the secondary structure of ITS2. It turns out that both secondary structures are functional. Compared with RCC, there was no significant difference in NTS (5S rRNA) methylation level, but the expression level of 5S rRNA was lower in 4nRR, indicating that methylation had little effect on the expression level in 4nRR. IGS (45S rRNA) was hypermethylated in 4nRR compared to RCC, but the expression of 18S rRNA gene was no significantly different from that in RCC, indicating that methylation regulation affected gene expression in 4nRR.ConclusionThe above studies initially revealed the effects of autotetraploidization on the structure and function of 5S and 45S rRNA in Carassius auratus, and provided a theoretical support for the systematic study of the evolution pattern and characteristics of rDNA in vertebrates.

Identification and molecular study of medicinal Plectranthus species (Lamiaceae) from Saudi Arabia using plastid DNA regions and ITS2 of the nrDNA gene

Plectranthus is a genus of the Lamiaceae family that includes many species of medicinal and agricultural importance. However, this genus has been the subject of taxonomic debate and contains species that are difficult to distinguish. The present study focused on six Plectranthus species commonly found in Saudi Arabia: P. arabicus, P. tenuiflorus, P. barbatus, P. pseudomarrubioides, P. asirensis, and P. hijazensis. P. hijazensis is endemic to Saudi Arabia. The capacities of five different plastid DNA barcodes (matK, rbcL, trnH-psbA, and ITS1 and ITS2 regions of the nrDNA gene) to identify and distinguish between Plectranthus species were evaluated. The following analytical methods were used to evaluate the efficiencies of the selected markers: BLAST, inter- and intraspecific distance, barcode gap, secondary structure of ITS2, and maximum likelihood (ML) phylogenetic trees. The results demonstrated that the nuclear ITS2 region can be successfully amplified and sequenced (100%), leading to a strong ability to discriminate between species and a clear barcode gap. Furthermore, there were significant differences in the ITS2 secondary structure among Plectranthus spp. Samples of Plectranthus formed monophyletic groups according to species in the ML tree, with high supported values. Our results establish that all Plectranthus species in Saudi Arabia can be classified into two groups within the Coleus clade. To our knowledge, this is the first time that local and endemic Plectranthus spp. have been identified and compared with Plectranthus samples of different geographical origins. Our results confirm the diversity of Plectranthus species growing naturally in southwestern Saudi Arabia. In addition, P. hijazensis, which is endemic to Saudi Arabia, was determined to be genetically distinct from other Plectranthus species and should, therefore, be the focus of future research, in addition to the preservation of the natural environment of these species.

Two New and Nontoxigenic Pseudo-nitzschia species (Bacillariophyceae) from Chinese Southeast Coastal Waters.

To explore the species diversity and toxin profile of Pseudo-nitzschia, monoclonal strains were established from Chinese southeast coastal waters. The morphology was examined under light and transmission electron microscopy. The internal transcribed spacer region of ribosomal DNA was sequenced for phylogenetic analyses, and the secondary structure of ITS2 was predicted and compared among allied taxa. A combination of morphological and molecular data showed the presence of two new species, Pseudo-nitzschia hainanensis sp. nov. and Pseudo-nitzschia taiwanensis sp. nov. Pseudo-nitzschiahainanensis was characterized by a dumpy-lanceolate valve with slightly blunt apicesand a central nodule, as well as striae comprising two rows of poroids. Pseudo-nitzschiataiwanensis was characterized by a slender-lanceolate valve, and striae comprising one row of split poroids. The poroid structure mainly comprised two sectors. Both taxa constituted their own monophyletic lineage in the phylogenetic analyses inferred from ITS2 rDNA and were well differentiated from other Pseudo-nitzschia species. Morphologically, P.hainanensis and P.taiwanensis could be assigned to the Pseudo-nitzschia delicatissima and the Pseudo-nitzschia pseudodelicatissima complex, respectively. Particulate domoic acid was measured using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), but no detectable pDA was found. With the description of the two new species, the species diversity of genus Pseudo-nitzschia reaches 58 worldwide, among which 31 have been recorded from Chinese coastal waters.

Structure and characterization of the ribosomal transcription units of small liver flukes, Opisthorchis viverrini, O. Felineus and Clonorchis sinensis

Opisthorchiasis is a zoonotic parasitic infection caused by small liver fluke species, Opisthorchis viverrini,O. felineus and Clonorchis sinensis, in the family Opisthorchiidae. Vietnam has both species, of which C.sinensis is distributed in the northern and O. viverrini in the central provinces. In addition to the mitochondrialgenomes, the ribosomal DNA sequences (rDNA) of these species are highly needed to obtain for providingmolecular markers in species identification, classification, phylogeny and evolutionary studies. In this study,the near/complete nucleotide sequences of ribosomal transcription units (rTU) from O. viverrini (Vietnamesesample), O. felineus (Russian sample) and C. sinensis (Vietnamese sample) were analyzed. All rTUs for threespecies were determined, which is 7,839 bp for O. viverrini, 6,948 bp for O. felineus and 7,296 bp for C.sinensis containing structures of 18S, ITS1, 5,8S, ITS2 and 28S. The IGS region was not obtained for all threespecies. In all three species, sequence analysis revealed 2 tandem repetitive elements of 47-48 bp/each in ITS1but not in ITS2. The nucleotide sequences of 18S, ITS1, ITS2 and 28S are valuable ribosomal markers that thisstudy provides for diagnosis, identification, taxonomic classification and population genetics. In conclusion,the rTU sequences for the three species of the family Opisthorchiidae have been identified and providesmolecular markers for the use of phylogenetic analysis for species/family classification in the superfamilyOpisthorchioidea and the class Trematoda.

Identification of morphologically similar species of Tribulus (Zygophyllaceae) by employing DNA barcodes and rRNA secondary structures

Abstract Tribulus terrestris is one of the essential constituents in traditional Indian systems of medicine and commonly used in several herbal preparations. It is adulterated with morphologically similar species, viz., T. subramanyamii, T. lanuginosus and T. alatus. T. terrestris is often misidentified for T. subramanyamii and T. lanuginosus. Hence, the error-free recognition and collection of this medicinal herb are important to ensure the drug’s effectiveness and biosafety. In the present study, DNA barcoding technique was employed to identify and to differentiate T. terrestris from its closely related species. Phylogenetic analysis was carried out for the three species of Tribulus using barcode candidates, such as, nuclear ribosomal DNA region ITS, chloroplast plastid gene psbA-trnH and rbcL. Sequence alignment revealed 7.5% polymorphic sites in ITS, with different specific regions showing the distribution of polymorphic sites as follows: ITS1 1.7%, ITS2 3.6% and the plastid intergenic spacer psbA-trnH 2.33%. The region ITS2 possesses a higher rate of transition/transversion ratio, percentage of variation and mean distance as compared to ITS1 and psbA-trnH. In addition, ITS2 has unique polymorphic loci which aid to differentiate T. terrestris from other species of Tribulus. Additionally we predicted the structure of ITS2 and 5.8S rRNA which distinguishes three phenotypically alike species based on the helical structure. The present study opens up new avenue for the identification of closely related species based on ITS2 region and 5.8S rRNA structure which could be employed for diversity studies

Nucleotide variation and secondary structures of nuclear ribosomal ITS2 in phylogeny relationships of some wheat species

This research aimed to estimate the nucleotide polymorphism and point mutation/substitutions for ITS2 region in 12 different Triticum and Aegilops species, and to demonstrate their phylogenetic relationships. The aligned ITS2 dataset showed a total of 67 SNPs and transitions (75%) predominant over transversions (25%). The C/T transitions were observed at highest level in the studied species. The highest nucleotide substitutions occurred in ITS2 sequences of T. aestivum, T. turgidum, and A. speltoides that showed similar substitutions on the base sites 76, 193, 215, 230 and 241 (TCCAG changed to ATTTA). T. boeticum and T. urartu showed similar substitutions on the base sites 85, 201, 231, 234 and 252 (GTGCC changed to AGATA). Remarkably, the nucleotide of base site 146 was C in all Triticum species, but T in all Aegilops species. The substitution rate in A. neglecta, and A. umbellulata was zero, it was concluded that their ITS2 is conserved. The secondary structures of ITS2 in the studied species was similar to that of the other flowering plants with four helices and the variation in helices length. The helix III had the longest length compared to the other helices. The length ranges of helices I, II, IV varied from 9 (T. turgidum, and T. urartu) to 10, 11 (A. cylindrica) 13 and 6 (A. speltoides) and 10 (T. boeticum) paired bases, respectively. Using pairwise ITS2 nucleotide comparisons, the closeness of A. caudata with A. cylindrica, T. boeticum with T. urartu, A. neglecta with A. umbellulata was observed. Phylogeny trees classified 12 species into four main clades. The highest point mutations occurred in ITS2 sequences related to the species grouped in the first clade.

Phylogeographical patterns of Campanula gr. arvatica, an endemic group of the Cantabrian mountains (NW Iberian Peninsula), based on plastid and nuclear DNA polymorphisms

Endemicity is a key consideration for conservation, and taxa that are endemic to a single country are vaulable conservation targets. In this study, we investigate endemic species of the genus Campanula in northern Iberian Peninsula based on sequences of nrDNA (internal transcribed spacer, ITS) and plastid regions (rbcL, trnL-F and trnS-G) and additionally the secondary structure of ITS2. The main aim of this work was to evaluate the evolutionary history of Campanula gr. arvatica from the Cantabrian Range (northern Iberian Peninsula) and establish phylogeographical patterns. Our results showed that molecular data from nuclear DNA and plastid regions should be applied for management and conservation strategies to monitor this endemic group from northern Spain. Furthermore, molecular evidence indicates significant differences among the three taxa analysed and shows the importance of the Cantabrian Mountains as a refuge for endemic Iberian flora in which processes of neo-speciation and/or crypto-speciation are under way.

Widespread inspection and comparative analysis of ITS secondary structure conservation and covariation of Coccinellidae

ITS1 and ITS2 of ribosomal DNA (rDNA) of six subfamilies i.e. Epilachaninae, Chilocorinae, Coccinellinae, Coccidulinae, Scymininae Ortaliinae and Sticholotidinae were examined, aligned and conserved motifs in folding patterns of stable consensus secondary structures were assessed. RNAalifold and RNA fold algorithms applied by RNAz program to compute consensus secondary structures and MFE efficiently and derive a number of statistical scores i.e. SCI, Z-score and MPI. Assembled subsets in ITS1 (three sets) and ITS2 (ten sets) were significantly conserved. In this study, analysis of these consensus secondary structures shows that a motif sequence is remain conserved in all the species. Helices in subsets of ITS1 showed compensatory base involvement to maintain the stability and conservation of secondary structures during natural selection. Secondary structures of ITS2 found more conserved than ITS1. MP, BI and ML methods used to establish the evolutionary relationship, compared the topology significance on the basis of SCI and MPI in defined sets. Coccinellinae, Epilachaninae and Scymninae (all strains of species) were monophyletic whereas Coccidulinae, and Chilocorinae had inadequate sequence information. Of Ortaliinae and Sticholotidinae, yet no ITS sequences have been submitted. Thus, analysis of the structural folding patterns of ITS regions provided further prime information of species relatedness and inter-intra-specific variations.

<p><strong>A taxonomic reassessment of <em>Chlamydomonas meslinii</em> (Volvocales, Chlorophyceae) with a description of <em>Paludistella</em> gen.nov.</strong></p>

Chlamydomonas (Volvocales, Chlorophyceae) is a large polyphyletic genus that includes numerous species that should be classified into independent genera. The present study aimed to examine the authentic strain of Chlamydomonas meslinii and related strains based on morphological and molecular data. All the strains possessed an asteroid chloroplast with a central pyrenoid and hemispherical papilla; however, they were different based on cell and stigmata shapes. Molecular phylogenetic analyses based on 18S rDNA, atpB, and psaB indicated that the strains represented a distinct subclade in the clade Chloromonadinia. The secondary structure of ITS-2 supported the separation of the strains into four species. Based on the results, we propose a novel genus, Paludistella, including P. meslinii comb. nov., P. chlorostelatta comb. nov., P. asymmetrica sp. nov., and P. trianguloculus sp. nov.

Genetic diversity of Paramecium species on the basis of multiple loci analysis and ITS secondary structure models.

Among ciliates, Paramecium has become a privileged model for the study of "species problem" particularly in the case of the "Paramecium aurelia complex" that has been intensely investigated. Despite extensive studies, the taxonomy of Paramecium is still challenging. The major problem is an uneven sampling of Paramecium with relatively few representatives of each species. To investigate species from the less discovered region (Pakistan), 10 isolates of Paramecium species including a standing-alone FT8 strain previously isolated by some of us were subjected to molecular characterization. Fragments of 18S recombinant DNA (rDNA), ITS1-5.8S-ITS2-5'LSU rDNA, cytochrome c oxidase subunit II, and hsp70 genes were used as molecular markers for phylogenetic analysis of particular isolates. The nucleotide sequences of polymerase chain reaction products of all markers were compared with the available sequences of relevant markers of other Paramecium species from GenBank. Phylogenetic trees based on all molecular markers showed that all the nine strains had a very close relationship with Paramecium primaurelia except for the FT8 strain. FT8 consistently showed its unique position in comparison to all other species in the phylogenetic trees. Available sequences of internal transcribed spacer 1 (ITS1) and ITS2 and some other ciliate sequences from GenBank were used for the construction of secondary models. Two highly conserved helices supported by compensatory base changes among all ciliates of ITS2 secondary structures were found similar to other eukaryotes. Therefore, the most conserved 120 to 180 base pairs regions were identified for their comparative studies. We found that out of the three helices in ITS1 structure, helix B was more conserved in Paramecium species. Despite various substitutions in the primary sequence, it was observed that secondary structures of ITS1 and ITS2 could be helpful in interpreting the phylogenetic relationships both at species as well as at generic level.

Application of ITS2 secondary structure phylogenetic information in DNA barcode identification of Chrysanthemum indicum and its related plants

By exploring additional phylogenetic information hidden in ITS2 secondary structure,the possibility of identifying Chrysanthemum indicum and its related species with DNA barcode of ITS2 nucleic acid sequence and its structure information were discussed.The genomic DNA was extracted from 12 samples. The ITS2 fragments were amplified by PCR and sequenced bidirectionally to obtain ITS2 sequence information. 28 sequences of related species for Ch. indicum were downloaded from Gen Bank. Until all 40 ITS2 sequences were aligned,ITS2 secondary structure prediction and structure comparison were finished. Then ITS2 secondary structure information was coded. After comparing ITS2 structure information and nucleic acid information,MP phylogenetic trees were built. The results showed that the secondary structures of ITS2 shared the same structure model--a four-fingered hand. They not only have the common characteristics of ITS2 secondary structures in plants,but also have many other conservative sequences,and their overall conservativeness is high. Among all species used in this study,their ITS2 secondary structures had obvious difference. In addition,the number of mutation sites in the joint matrix compared with the nucleic acid sequences increased by nearly 90%,which greatly enriched the number of mutation sites. This method of information analysis distinguished Ch. indicum from its related species. At the same time,the support rate of the branches of evolutionary trees and the identification rate of species were significantly improved. Although there was no distinction between Ch. zawadskii and Ch. morifolium,it effectively distinguished the three species,namely,Ch. hypargyrum,Ch.oreastrum,and Ch. dichrum. Therefore,the authors suggest that the ITS2 sequence combined with its structural data information should be applied to the identification of Ch. indicum and its related species,and be widely applied to DNA barcode research.

Characterization of Clinostomum metacercariae using microscopic and molecular approaches

One of the fundamental aspects in understanding the biology, diversity and epidemiology of a parasite lies in its proper identification. In the present study, morphological and molecular characterization of Clinostomum metacercariae recovered from an ornamental fish, Trichogaster fasciata, was carried out in order to ascertain its identity. To serve the purpose, scanning electron micrographs and gene sequences for two commonly used molecular markers, i.e., nuclear ribosomal internal transcribed spacer 2 (rDNA-ITS2) and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) were obtained. The sequences were further used for generating similarity index matrix as well as inferring phylogenies. Light and electron microscopic observations on metacercariae of the parasite revealed that it belongs to the genus Clinostomum. Identification of the same up to the level of species was made possible through sequence and phylogenetic analyses. The ITS2 sequence analyses of our species (KX758630) showed similarity to unidentified Clinostomum sp. reported from Nigeria (KY865625) and China (KP110579), and C. tilapiae recorded from South Africa (KX034048) and Nigeria (KY649353). However, the CO1 gene analyses suggested it to be highly identical to C. philippinense and the same was also corroborated in the phylogenetic analysis. Thus, morphological and molecular characterization revealed that the recovered metacercariae belong to the species C. philippinense. Additionally, a brief description of secondary structures of ITS2 of various species of Clinostomum has also been discussed.

Usual alga from unusual habitats: Biodiversity of Klebsormidium (Klebsormidiophyceae, Streptophyta) from the phylogenetic superclade G isolated from biological soil crusts

Seven new species and two varieties of Klebsormidium were described using an integrative approach on the base of 28 strains from the poorly studied phylogenetic superclade G. These strains originated from the unusual and exotic habitats (semi-deserts, semi-arid shrublands, Mediterranean shrub and deciduous vegetation, temperate Araucaria forests, peat bogs, dumps after coal mining, maritime sand dunes etc.) of four continents (Africa, South and North America, and Europe). Molecular phylogenies based on ITS-1,2, rbcL gene and concatenated dataset of ITS-1,2–rbcL, secondary structure of ITS-2, morphology, ecology and biogeography, micrographs and drawings of the investigated strains were assessed. Additionally, phylogeny and morphology of 18 Klebsormidium strains from other lineages isolated from the same localities (different vegetation types of Chile and maritime sand dunes of Germany) were investigated for the comparison with representatives of clade G. Clade G Klebsormidium is characterized by distant phylogenetic position from the other Klebsormidium lineages and prominent morphology: four-lobed chloroplasts and mostly short swollen cells in young culture, compact small pyrenoids, curved or disintegrated filaments, unusual elongation of cells in old culture, formation of specific cluster- and knot-like colonies on agar surface, especially prominent in strains isolated from desert regions, from which the group probably originated. Comparison of Klebsormidium diversity from different biogeographic regions showed that the representatives of clade G are common algae in regions of the southern hemisphere (South Africa and Chile) and rare representatives in terrestrial ecosystems of the northern hemisphere. Further investigation of mostly unstudied territories of the southern hemisphere could bring many surprises and discoveries, leading to a change of the present concept that Klebsormidium is cosmopolitan in distribution.

New Taxa of Streptophyte Algae (Streptophyta) from Terrestrial Habitats Revealed Using an Integrative Approach

Two new genera (Streptosarcina and Streptofilum) and three new species (Streptosarcina arenaria, S. costaricana and Streptofilum capillatum) of streptophyte algae were detected in cultures isolated from terrestrial habitats of Europe and Central America and described using an integrative approach. Additionally, a strain isolated from soil in North America was identified as Hormidiella parvula and proposed as an epitype of this species. The molecular phylogeny based on 18S rRNA and rbcL genes, secondary structure of ITS-2, as well as the morphology of vegetative and reproductive stages, cell ultrastructure, ecology and distribution of the investigated strains were assessed. The new genus Streptosarcina forms a sister lineage to the genus Hormidiella (Klebsormidiophyceae). Streptosarcina is characterized by packet-like (sarcinoid) and filamentous thalli with true branching and a cell organization typical for Klebsormidiophyceae. Streptofilum forms a separate lineage within Streptophyta. This genus represents an easily disintegrating filamentous alga which exhibits a cell coverage of unique structure: layers of submicroscopic scales of piliform shape covering the plasmalemma and exfoliate inside the mucilage envelope surrounding cells. The implications of the discovery of the new taxa for understanding evolutionary tendencies in the Streptophyta, a group of great evolutionary interest, are discussed.

Phylogenetic analysis of Uncaria species based on internal transcribed spacer (ITS) region and ITS2 secondary structure

Context: The plant genus Uncaria (Rubiaceae), also known as Gouteng, is the source of an important traditional Chinese medicine. Misidentification and adulteration of Gouteng affect the safety and efficacy of the medication. Phylogenetic relationships among the species of this genus are unknown.Objective: The present study sought to detect the phylogenetic relationships based on internal transcribed spacer (ITS) region of all 12 species of Uncaria recorded in the Flora of China.Materials and methods: Accession of seven species of Uncaria served as reference samples. ITS region was used for polymerase chain reaction (PCR) amplification of the reference samples representing 39 specimens. Distance analysis, species discrimination, and secondary structure of ITS2 were used to assess the ability of ITS sequence in authenticating. The phylogenetic relationships were detected using three methods: Bayesian inference (BI), maximum likelihood (ML), and neighbor joining (NJ).Results: Five species of traditional Chinese medicine Gouteng were well resolved in molecular phylogenetic tree. Besides, Uncaria lancifolia Hutch. was closer to U. rhynchophylloides F.C. How and U. sessilifructus Roxb. was closer to U. laevigata Wall. within the tree. Further, we also found that ITS2 secondary structure can be a candidate tool in distinguishing two closely related species U. yunnanensis K.C.Hsia and U. lanosa Wall. For accurate identification of different species of Uncaria based on species-specific nucleotide sites, a consensus sequences database with all 12 species is established.Discussions and conclusions: The results are able to discriminate Uncaria species and illustrate the phylogenetic relationships, which are essential for the investigation of adulterants and misidentifications of Uncaria.

Phylogenic Diversity and Taxonomic Problems of the Dictyosphaerium Morphotype within the Parachlorella Clade (Chlorellaceae, Trebouxiophyceae).

The Parachlorella clade was put forward as a group within the family Chlorellaceae in 2004. Recent molecular analyses have revealed that Dictyosphaerium morphotype algae form several independent lineages within the Parachlorella clade, and new genera and species have been established. In this study, we focus on the diversity of Dictyosphaerium morphotype algae within the Parachlorella clade, based on 42 strains from China. We used combined analyses of morphology and molecular data based on SSU and internal transcribed spacer region (ITS) rDNA sequences to characterize these algae. In addition, the secondary structure of ITS2 was compared to delineate new lineages. Our results revealed high phylogenic diversity of Dictyosphaerium morphotype algae, and we describe five distinct lineages. We examined the morphological features of these five lineages, and morphological differences are difficult to find compared with other Dictyosphaerium morphotype algae. The five distinct lineages were not described as new genera currently. We lastly discuss the taxonomic problems regarding the Dictyosphaerium morphotype within the Parachlorella clade, and possible solutions are considered.

Molecular phylogeny of Botryococcus braunii strains (race A) – An integrative approach

Botryococcus braunii is a green colonial microalga which produces large quantities of liquid hydrocarbons similar to fossil petroleum. Exploring Botryococcus genus diversity entailed morphological, biochemical and phylogenetic studies. The majority of scientific literature specifies B. braunii as the only species with strains classified in three chemical races (A, B and L) which superpose the phylogenetic clusters built on 18S rDNA sequences. The cohesion of data provided by the three main research directions relies on the integrative analysis of the Botryococcus strains. The present work addresses the diversity of B. braunii race A strains by taking into account the morphology, the hydrocarbon and fatty acid profiles, and the phylogeny of 10 strains, isolated from several Romanian aquatic environments. The newly sequenced B. braunii strains increased the resolution of the analyses and yielded at least two distinct phylogenetic clades within race A cluster. This outcome was sustained by the genetic information coded by the 18S rRNA genes and both sequence- and sequence-structure analysis of ITS2. The Compensatory Base Changes occurrence within the secondary structure of ITS2 and the presence of one intron in the 18S rDNA sequences gave extra credibility to our phylogenetic results.

Application of the Ribosomal DNA ITS2 Region of Physalis (Solanaceae): DNA Barcoding and Phylogenetic Study.

Recently, commercial interest in Physalis species has grown worldwide due to their high nutritional value, edible fruit, and potential medicinal properties. However, many Physalis species have similar shapes and are easily confused, and consequently the phylogenetic relationships between Physalis species are poorly understood. This hinders their safe utilization and genetic resource conservation. In this study, the nuclear ribosomal ITS2 region was used to identify species and phylogenetically examine Physalis. Eighty-six ITS2 regions from 45 Physalis species were analyzed. The ITS2 sequences were aligned using Clustal W and genetic distances were calculated using MEGA V6.0. The results showed that ITS2 regions have significant intra- and inter-specific divergences, obvious barcoding gaps, and higher species discrimination rates (82.2% for both the BLASTA1 and nearest distance methods). In addition, the secondary structure of ITS2 provided another way to differentiate species. Cluster analysis based on ITS2 regions largely concurred with the relationships among Physalis species established by many previous molecular analyses, and showed that most sections of Physalis appear to be polyphyletic. Our results demonstrated that ITS2 can be used as an efficient and powerful marker in the identification and phylogenetic study of Physalis species. The technique provides a scientific basis for the conservation of Physalis plants and for utilization of resources.

The characterisation and taxonomic utility of ITS2 in Tenthredopsis Costa, 1859 (Tenthredinidae: Hymenoptera) with some new records from Turkey

The taxonomic value of ITS2 has not been well established in insects and the data on its secondary structural properties is limited to a few studies. Here, we present the first data on the secondary structural properties of ITS2 in sawflies and test its utility as a molecular identifier in Tenthredopsis (Tenthredinidae: Symphyta). The ITS2 regions were sequenced from 67 specimens representing 14 species and six morphotypes of Tenthredopsis and their secondary structure were predicted and characterised. A sequence-structural alignment dataset of ITS2 was analysed by application of maximum likelihood method to see relationship among the specimens. Here, a relaxed approach has been adapted to identify and to elucidate species boundaries by considering the presence of CBCs, hemi-CBCs and unique length size of ITS2 in combination with morphology. The predicted secondary structure of ITS2 was extremely branched in contrast to common core structure found in many eukaryotes. A likely protein binding side was detected in Helix III and suggested to be a synapomorphy for Tenthredinidae. Eighteen species were identified under the relaxed approach applied here, of which Tenthredopsis lactiflua (Klug, 1817), Tenthredopsis ornata (Serville, 1823), Tenthredopsis stigma (Fabricius, 1798) and Tenthredopsis coquebertii (Klug, 1817) are new records for Turkey. The structural properties and length size of ITS2 prove useful in determination of species boundaries of closely related species. However, the full account on its taxonomic utility requires more empirical evidence in Hymenoptera.