ITS2 Secondary Structure Research Articles

Dunaliella viridis TAV01: A Halotolerant, Protein-Rich Microalga from the Algarve Coast

Tolerance to harsh environmental conditions, high growth rates and an amino acid profile adequate for human consumption are beneficial features observed in Dunaliella viridis TAV01, a novel strain isolated from a salt pond in the Algarve, Portugal. TAV01 was identified down to the species level by maximum likelihood and Bayesian phylogenetic analyses of the ribosomal internal transcribed spacers one and two regions (ITS1 and ITS-2) and was supported by ITS2 secondary structure analysis. The biochemical profile revealed high protein (35.7 g 100 g−1 DW; 65% higher than the minimum recommended by the World Health Organization) and lipid contents (21.3 g 100 g−1 DW), a relatively higher proportion of the polyunsaturated fatty acids (PUFAs), α-linolenic (26.3% of total fatty acids (TFA)) and linoleic acids (22.8% of TFA), compared to those of other Dunaliella strains, and a balanced essential amino acids profile containing significant levels of leucine, phenylalanine, valine, and threonine. The major carotenoid was lutein, making up over 85% of total carotenoids. The presence of high-quality natural products in D. viridis TAV01 offers the possibility of using this new strain as a valuable biological resource for novel feed or food products as ingredients or supplements.

Insights into the phylogenetic inconsistencies of the genus Amazonocrinis and description of epilithic Amazonocrinis malviyae sp. nov. (Cyanobacteria, Nostocales) from Jammu and Kashmir, India.

A dark-coloured thin film of cyanobacteria growing on the bottom of a submerged stone was isolated from Basantgarh village in Udhampur district, Jammu and Kashmir, India. The isolated strain (designated 19C-PST) was characterized using a polyphasic approach. The strain exhibited typical Nostoc-like morphology with a characteristic feature of having heterocytes in series. The 16S rRNA gene phylogeny placed the strain at a well-supported and distinct node. Notably, the recently described genus, Amazonocrinis, on the addition of more 16S rRNA gene sequences, reflected a critical split, which proved to be stable and well supported in all phylogenetic analyses of the 16S rRNA gene. Interestingly, Amazonocrinis nigriterrae CENA67T (type species of the genus) clustered together with our strain 19C-PST in the 16S rRNA gene phylogenetic analysis while the rest of the members of the genus Amazonocrinis were placed at a separate and distant node. This clearly indicated that strain 19C-PST is a member of Amazonocrinis sensu stricto. However, the results of phylogenetic analysis of ITS sequences only, in strains purported to belong to Amazonocrinis did not agree with the 16S rRNA gene results and placed our strain 19C-PST in a sister clade to three strains that have not yet been speciated, UHCC 0702, NIES-4103 and SA22, with A. nigriterrae falling into a separate clade. Further, folded secondary structures of the D1-D1', V2, BoxB and V3 helices of strain 19C-PST were found to be significantly different from those of all the phylogenetically related taxa. The study revealed an interesting case where low taxon sampling and phylogenomic interpretations came across as points of attention in cyanobacterial taxonomy. Based on the morphological, phylogenetic, 16S-23S ITS secondary structure analyses, we describe our strain as Amazonocrinis malviyae sp. nov. in accordance with the International Code of Nomenclature for algae, fungi and plants. This work also illuminates the need for further research to resolve the taxonomic discrepancies among Amazonocrinis strains.

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.

Morphology, molecular phylogeny, and species delimitation within microalgal genera Eubrownia, Spongiococcum, and Chlorococcum (Chlorophyceae, Chlorophyta)

• Our knowledge of their taxonomic and phylogenetic relationships is still deficient. • Some genus and species from Moewusinia clade differ specific feature combinations. • The ITS2 of Eubrownia and Chlorococcum genera is unique within the chlorophytes. Despite that the diversity of chlorophycean microalgae has been studied for a long time, our knowledge of their taxonomic and phylogenetic relationships is still deficient. This study represents one of the first efforts to examine the congruence of the morphological identification of microalgal species from genera Eubrownia, Spongiococcum , and Chlorococcum with DNA-based species delimitation methods. We found unique combinations of morphological, molecular, and ecological features, distinguishing some members of the Moewusinia clade at the genus and species levels. Most genetic groups recovered by GMYC, ABGD, and PTP analyses of 18S rRNA and ITS2 were consistent with their morphological identification. The ITS2 region of genera Eubrownia and Chlorococcum is among the longest within the chlorophytes theretofore studied and has a unique secondary structure with branched helix III and uncommonly long helix IV. Moreover, the molecular signatures were found in the basal stem region of helix I, distinguishing two genera. The CBC analysis is able to effectively discriminate between E. isobilateralis, E. aggregata , and all Chlorococcum species, except for C. infusionum and C. echinozygotum . Thus, we confirmed tentative hypotheses of species, obtained by DNA-based delimitation methods, with morphological data, molecular signatures, ITS2 secondary structure, and CBC criterion.

Neocylindrospermum variakineticum gen. & sp. nov. (Nostocales, Cyanobacteria), a novel genus separated from Cylindrospermum using a polyphasic method

ABSTRACT The large genus Cylindrospermum is polyphyletic as currently circumscribed, and its cryptic diversity requires detailed taxonomic evaluation. Two novel cyanobacterial strains were isolated by scraping the surface of wet wood above the water level, in the stream below Mae Phun Waterfall in the northern part of Thailand, and investigated with a polyphasic study combining morphological and molecular analyses. The morphological features of these two novel strains fitted the original description of the genus Cylindrospermum but they differed from known species in cell size, exospore features and akinete formation. Phylogenetic analyses based on 16S rRNA, 16S–23S rRNA ITS region, rpoC1 and nifH genes indicated that the novel strains formed a unique clade with strongly supported values from BI and ML methods. This unique clade was distantly separated from the Cylindrospermum sensu stricto clade and other Cylindrospermum-like clades. Molecular analyses showed that the two novel strains had <96.3% 16S rRNA gene sequence similarity and <83.2% 16S–23S rRNA ITS region sequence similarity to known cyanobacterial genera and other related taxa. Furthermore, ITS secondary structures between the 16S–23S rRNA gene in both strains also exhibited singular folding patterns of D1–D1', Box-B and V3 helix, thereby distinguishing them from the closely related taxa. The establishment of Neocylindrospermum gen. nov. with the type species as N. variakineticum sp. nov. is proposed according to the International Code of Nomenclature for Algae, Fungi, and Plants.

Zarconia navalis gen. nov., sp. nov., Romeriopsis navalis gen. nov., sp. nov. and Romeriopsis marina sp. nov., isolated from inter- and subtidal environments from northern Portugal.

The morphology, 16S rRNA gene phylogeny and 16S-23S rRNA gene ITS secondary structures of three strains of marine Cyanobacteria, isolated from inter- and subtidal environments from north Portugal were studied, resulting in the description of Zarconia navalis gen. nov., sp. nov. (Oscillatoriales incertae sedis), Romeriopsis navalis gen. nov., sp. nov. (Leptolyngbyaceae) and Romeriopsis marina sp. nov., named under the International Code of Nomenclature for algae, fungi, and plants. No diacritical morphological characters were found for the new genera and species. The 16S rRNA gene maximum-likelihood and Bayesian phylogenies supported that the genus Zarconia is a member of the Oscillatoriales, morphologically similar to the genera Microcoleus and Phormidium, but distant from them. The genus Romeriopsis is positioned within the Leptolyngbyaceae (Synechococcales) and is closely related to Alkalinema. The secondary structures of the D1-D1', Box B, V2 and V3 helices corroborate the phylogenetic results. Furthermore, our study supports previous observations of polyphyletic Oscillatoriales families and reinforces the need for their taxonomic revision.

Phylogeny and fatty acid profiles of Aliinostoc vietnamicum sp. nov. (cyanobacteria) from the soils of Vietnam.

A new cyanobacterial species of Aliinostoc, A. vietnamicum sp. nov., is recorded in the tropical forest soil from the Cát Tiên National Park, Vietnam. The analysis is based on morphological characters, 16S rDNA phylogeny, ITS secondary structure, and fatty acid composition analysis. Aliinostoc vietnamicum differed from the other species of the genus by the size and shape of vegetative cells, size of akinetes and heterocytes, and presence of granular polyphosphate inclusions in vegetative cells. The evolutionary distance matrix based on the 16S rRNA gene shared 96.2-98.2% similarities with other Aliinostoc sequences. The phylogeny inferred by maximum likelihood and Bayesian inference placed A. vietnamicum in the Aliinostoc clade, within the Nostocaceae. For the first time, fatty acid composition analysis was obtained for a member of the genus Aliinostoc with cultivation time experiments. α-linolenic (27.54-37.75%), palmitic (13.87-22.65%), and stearic (10.08-20.27%) acids were the dominant fatty acids when cultured during the exponential growth phase, as well as during stationary. This is the first finding of a strain with such a high content of stearic acid among cyanobacteria with Nostoc-like morphology.

Molecular Phylogeny, DNA Barcoding, and ITS2 Secondary Structure Predictions in the Medicinally Important Eryngium Genotypes of East Coast Region of India.

Commercial interest in the culinary herb, Eryngium foetidum L., has increased worldwide due to its typical pungency, similar to coriander or cilantro, with immense pharmaceutical components. The molecular delimitation and taxonomic classification of this lesser-known medicinal plant are restricted to conventional phenotyping and DNA-based marker evaluation, which hinders accurate identification, genetic conservation, and safe utilization. This study focused on species discrimination using DNA sequencing with chloroplast–plastid genes (matK, Kim matK, and rbcL) and the nuclear ITS2 gene in two Eryngium genotypes collected from the east coast region of India. The results revealed that matK discriminated between two genotypes, however, Kim matK, rbcL, and ITS2 identified these genotypes as E. foetidum. The ribosomal nuclear ITS2 region exhibited significant inter- and intra-specific divergence, depicted in the DNA barcodes and the secondary structures derived based on the minimum free energy. Although the efficiency of matK genes is better in species discrimination, ITS2 demonstrated polyphyletic phylogeny, and could be used as a reliable marker for genetic divergence studies understanding the mechanisms of RNA molecules. The results of this study provide insights into the scientific basis of species identification, genetic conservation, and safe utilization of this important medicinal plant species.

Diagnosis and characterization of the ribosomal DNA-ITS of potato rot nematode (Ditylenchus destructor) populations from Chinese medicinal herbs

The potato rot nematode (Ditylenchus destructor) is a very economically important nematode in agronomic and horticultural plants worldwide. In this study, 43 populations of D. destructor were collected from different hosts across China, including 37 populations from Chinese herbal medicine plants. Obtained sequences of ITS-rDNA and D2–D3 of 28S-rDNA genes of D. destructor were compared and analyzed. Nine types of significant length variations in ITS sequences were observed among all populations. The differences in ITS1 length were mainly caused by the presence of repetitive elements with substantial base substitutions. Reconstructions of ITS1 secondary structures showed that the minisatellites formed a stem structure. Ten haplotypes were observed in all populations based on mutations and variations of helix H9. Among them, 3 known haplotypes (A–C) were found in 7 populations isolated from potato, sweet potato, and Codonopsis pilosula, and 7 unique haplotypes were found in other 36 populations collected from C. pilosula and Angelica sinensis compared with 7 haplotypes (A–G) according to Subbotin’ system. These unique haplotypes were different from haplotypes A–G, and we named them as haplotypes H–N. The present results showed that a total of 14 haplotypes (A–N) of ITS-rDNA have been found in D. destructor. Phylogenetic analyses of ITS-rDNA and D2–D3 showed that all populations of D. destructor were clustered into two major clades: one clade only containing haplotype A from sweet potato and the other containing haplotypes B–N from other plants. For further verification, PCR-ITS-RFLP profiles were conducted on 7 new haplotypes. Collectively, our study suggests that D. destructor populations on Chinese medicinal materials are very different from those on other hosts and this work provides a paradigm for relevant researches.

Semicryptic Diversity around Chaetoceros elegans (Bacillariophyta, Mediophyceae), and the Description of Two New Species

The globally distributed Chaetoceros elegans belongs to the Chaetoceros lorenzianus (C. lorenzianus) complex and is characterized by having tear-shaped setae poroids. Several strains of C. elegans were established from Chinese coastal waters. The vegetative cells and the resting spores were observed using light and electron microscopy. Phylogenetic analyses of two nuclear ribosomal RNA genes (SSU and the D1–D3 region of LSU) and the internal transcribed spacer (ITS) revealed that the C. elegans strains clustered into three clades, corresponding to different morphotypes. Based on the type material, the delineation of C. elegans was amended, and two new taxa, (Chaetoceros macroelegans) C. macroelegans sp. nov. and (Chaetoceros densoelegans) C.densoelegans sp. nov., were described. The two new taxa are featured by the presence of two types of setae poroids, tear-shaped and round-oval setae poroids, whereas only tear-shaped setae poroids are seen in C. elegans. The setae base is distinct in C. elegans, but absent or short in the two new taxa. In C. macroelegans, the tear-shaped poroids on the intercalary setae are larger and less densely spaced than in the other two species. The round-oval setae poroids are more densely spaced in C.densoelegans than in C. macroelegans, although they have more or less the same size. Resting spores characterize the two new taxa, but are unknown in the amended C. elegans. When comparing the ITS2 secondary structure, two and four compensatory base changes (CBCs) distinguish C. elegans from C. macroelegans and C.densoelegans, respectively. Between the two new taxa, no CBC but five hemi-CBCs (HCBCs) are present. The shape, size and density of the setae poroids, as well as the morphology of the resting spores, are important characteristics for species identification among the presently nine known species within the C. lorenzianus complex.

Siamcapillus rubidus gen. et sp. nov. (Oculatellaceae), a novel filamentous cyanobacterium from Thailand based on molecular and morphological analyses

Cyanobacterial diversity has been extensively studied; however, scant knowledge exists concerning genetic data and higher accuracy for tropical cyanobacterial diversity. In particular, the existence of unnamed monophyletic genera within the Oculatellaceae requires further investigation. Here, two novel strains (NUACC13 and NUACC14) were isolated from a cyanobacterial mat covering border soil of a small freshwater pond in Phitsanulok Province, Thailand. A polyphasic approach combining genetic (16S rDNA and rpoC1 gene phylogenies, evolutionary distance and ITS secondary structures) and morphological information was applied to evaluate the taxonomic status of these novel strains. All phylogenetic analyses of 16S rDNA and rpoC1 gene sequences placed our strains within the Oculatellaceae, strongly supporting a novel monophyletic clade that did not belong to other known Oculatellaceae genera. The 16S rDNA sequence similarity (&lt;96.2%) values among the closest described genera also supported the novelty of our strains. The hypothetical secondary structures (D1–D1′, Box-B, V2 and V3 helices) of the ITS region confirmed the uniqueness of our strains, indicating differences from the related genera. Morphological features of the novel strains differed from the phylogenetically closest taxa by size and color of filament and vegetative cell and the presence of a frayed sheath. Considering all the results, we herein described our strains as the novel genus Siamcapillus gen. nov. in the Oculatellaceae with the type species S. rubidus sp. nov.

Taxonomic study of a novel terrestrial alga, Spongiosarcinopsisqinghaiensis sp. nov. (Protosiphonaceae, Chlorophyta), from the Qinghai-Tibet Plateau.

There is only one species of Spongiosarcinopsis in the literature currently. It was found in gray soil in Russia for the first time. According to molecular data analysis results, the isolated algal strain is most closely related to Spongiosarcinopsisterrestris. Unlike Spongiosarcinopsisterrestris, the isolated strain was found on soil surfaces at high altitudes, the young vegetative cell is spherical, vegetative cells are relatively large, and pyrenoids are generally fewer. In view of such morphological differences, phylogenetic analysis results, and comparison of ITS2 secondary structure and ultrastructure, the strain isolated in the present study was proposed to be a novel species.

Roholtiella volcanica sp. nov., a New Species of Cyanobacteria from Kamchatkan Volcanic Soils

During a study of biodiversity of cyanobacteria in Gorely volcano soils (Kamchatka Peninsula), a strain of heterocytous, a false branching cyanobacterium with gradually tapered filaments, was isolated. Prominent features of the strain were purplish-grey trichomes and firm, distinct multilayered sheaths. Based on the results obtained from the morphological, ecological, and phylogenetic analysis using the 16S rRNA and 16S–23S ITS region, 16S–23S ITS secondary structure analysis, comparison of flanking regions of BoxB and V3 helices, and the p-distance between the 16S–23S ITS region, we describe our strain K7 as a novel species of the genus Roholtiella with the name Roholtiella volcanica sp. nov., in accordance with the International Code of Nomenclature for algae, fungi, and plants. This work continues the rapid expansion of the description of new taxa of cyanobacteria, and particularly demonstrates a coming phase in cyanobacterial taxonomy in which the discovery of new species in recently described genera rapidly increases our understanding of the diversity in this phylum.

A New Deep-Water Epilithic Green Alga, Ulvella lacustris, from an Alpine Brackish Lake in Qinghai–Tibet Plateau

Ulvella species are widely distributed in ocean and freshwater habitats and from high latitudes to the tropics. However, no species of this genus have been found in the Qinghai–Tibetan Plateau. In the present study, five rock specimens were collected from the bottom of Qinghai Lake at a depth of about 15–28 m, from which three unialgal strains were isolated. These strains were characterized by prostrate pseudoparenchyma, irregular or radial branch filaments, and mono- or polystromatic discs or rosettes formed by consecutive filaments, with thalli that can grow up to 180 um in length, suggesting that they may be Ulvella species. Phylogenetic analysis based on 18S rDNA and tufA cpDNA sequences revealed that the isolates formed a strong branch among other Ulvella species and were closely related to Ulvella leptochaete and Ulvellawaernii. This result was also confirmed by neighbor-joining tree inference based on ITS2 secondary structure. Unlike other members of Ulvella, the isolated strain was characterized by its unique habitat, field morphology of thallus, size of vegetative cells, and number of pyrenoids. Based on these morphological differences, the phylogenetic analysis, and the comparison of ITS2 secondary structure with relative species, the strain isolated in this study was proposed to be a novel Ulvella sp. The alga was found on the bottom of brackish lakes on the Qinghai–Tibetan Plateau, which meant that our sampling range needed to be expanded further.

Description of Desmonostoc caucasicum sp. nov. (Cyanobacteria) using an integrative taxonomic approach

ABSTRACT A new cyanobacterial species of the genus Desmonostoc is described according to the International Code of Nomenclature for algae, fungi, and plants. The new species Desmonostoc caucasicum sp. nov. is recorded in the mountain meadow subalpine soil from the Greater Caucasus, Russia. The analysis is based on morphological characters, the 16S rRNA gene phylogenetic analysis, and ITS secondary structure. Desmonostoc caucasicum differs from the other species of the genus in colony morphology, size of vegetative cells and heterocytes, and habitat type. The 16S rRNA gene sequence of the new strain displayed 95.3%–97.9% similarities to other species of the genus Desmonostoc. The phylogeny inferred by maximum likelihood and Bayesian inference placed D. caucasicum in the Desmonostoc clade, within the Nostocaceae. The novel strain formed an independent lineage within the clade. The D1–D1ʹ, Box–B and V3 helices obtained from the 16S–23S ITS region didn’t fit those of any described species of Desmonostoc. Amplification of a fragment of the mcy gene involved in microcystin biosynthesis from D. caucasicum confirmed that it has this genetic determinant. An integrative taxonomic approach, based on morphological, 16S rRNA and mcy genes molecular analyses, ITS secondary structure, along with ecological data, is used to delimit this new species.

Hidden diversity of Chlorococcum (Chlorophyta) in a shallow temporary freshwater lake: description of Chlorococcum szentendrense sp. nov

ABSTRACT Chlorococcum is a coccoid green algal genus, which contains almost 50 species. The genus is polyphyletic, but in a traditional sense, the cells are coccoid and non-motile with a cup-shape chloroplast in the vegetative stage, while the spores have chlamydomonad appearance. They are distributed worldwide mainly in terrestrial habitats, and the biotechnological potential of several strains has been reported. In this study, three new green algal strains from a shallow, temporary freshwater lake in Hungary are characterized using microscopic (light and transmission electron microscopy) and DNA-based methods (phylogenetic analysis of the ribosomal ITS region, the 18S ribosomal RNA and rbcL genes, and ITS secondary structure comparison). Based on the obtained results, one of the three new isolates is considered to represent a new species, which is described here as Chlorococcum szentendrense sp. nov.

Description of Limnomonas gen. nov., L. gaiensis sp. nov. and L. spitsbergensis sp. nov. (Chlamydomonadales, Chlorophyta)

Two novel Chlamydomonas-like species, belonging to the Moewusii clade, have been described. The first species inhabits eutrophic and neutral to basic pH waters in Sweden and England. It is easily recognizable under a light microscope due to its morphology (a small green prolate spheroidal shape with a large and truncated papilla at its anterior end, two equal flagella, a single lateral eyespot, a basal nucleus, and a well-defined pyrenoid) and to its peculiar whole-body pendulum movement while resting on surfaces or attached to floating particles. The species occurs as free-living individuals and is able to gather temporarily into groups of individual cells. No particular binding structures or palmelloid cells were observed in cultures. The second species, previously assigned to Chlamydomonas cf. proboscigera, was collected from persistent snow in Svalbard, Norway. Its morphology is revised herein. Using SSU rDNA sequence analyses, these two species formed a well-supported clade. Moreover, ITS-2 secondary structure analyses confirmed sexual incompatibility between these biological species. Considering these results, a new genus Limnomonas and its type species L. gaiensis and L. spitsbergensis are proposed.

Polyphasic Identification and Genomic Insights of Leptothermofonsia sichuanensis gen. sp. nov., a Novel Thermophilic Cyanobacteria Within Leptolyngbyaceae

Thermal environments are an important reservoir of thermophiles with significant ecological and biotechnological potentials. However, thermophilic isolates remain largely unrecovered from their habitats and are rarely systematically identified. In this study, we characterized using polyphasic approaches a thermophilic strain, PKUAC-SCTAE412 (E412 hereafter), recovered from Lotus Lake hot spring based in Ganzi prefecture, China. The results of 16S rRNA/16S-23S ITS phylogenies, secondary structure, and morphology comparison strongly supported that strain E412 represent a novel genus within Leptolyngbyaceae. This delineation was further confirmed by genome-based analyses [phylogenomic inference, average nucleotide/amino-acid identity, and the percentages of conserved proteins (POCP)]. Based on the botanical code, the isolate is herein delineated as Leptothermofonsia sichuanensis gen. sp. nov, a genus adjacent to recently delineated Kovacikia and Stenomitos. In addition, we successfully obtained the first complete genome of this new genus. Genomic analysis revealed its adaptations to the adverse hot spring environment and extensive molecular components related to mobile genetic elements, photosynthesis, and nitrogen metabolism. Moreover, the strain was capable of modifying the composition of its light-harvesting apparatus depending on the wavelength and photoperiod, showing chromatic adaptation capacity characteristic for T1 and T2 pigmentation types. Other physiological studies showed the strain’s ability to utilize sodium bicarbonate and various sulfur compounds. The strain was also shown to be diazotrophic. Interestingly, 24.6% of annotated protein-coding genes in the E412 genome were identified as putatively acquired, hypothesizing that a large number of genes acquired through HGT might contribute to the genome expansion and habitat adaptation of those thermophilic strains. Most the HGT candidates (69.4%) were categorized as metabolic functions as suggested by the KEGG analysis. Overall, the complete genome of strain E412 provides the first insight into the genomic feature of the genus Leptothermofonsia and lays the foundation for future global ecogenomic and geogenomic studies.

Morphological, histopathological and secondary structure analysis of second internal transcribed spacer (ITS-2) region of Gigantocotyle explanatum (Trematoda: Paramphistomidae) in buffaloes of Pakistan

Liver infections due to amphistomes in buffaloes cause significant economic losses in livestock sector. The present study investigated the classical morphological features of adult amphistomes combined with histopathology and molecular identity in slaughtered buffaloes from Khyber Pakhtunkhwa, Pakistan. Adult amphistome were collected and morphologically characterized as Gigantocotyle explanatum. Morphometric measurement (n=50) were obtained with 12.05±1.68 mm in length and 5.77±0.81 mm in width, and the values of sagittal section was 6.35±1.03 x 2.98±0.50 mm in size. Severe bile duct pathology was observed with fibrosis of the duct wall with an irregular epithelial border, hyperplasia and inflammatory response with numerous neutrophils and eosinophils. The molecular identity of G. explanatum within Paramphistomidae was confirmed by ITS-2 rDNA sequences phylogenetic analysis based on maximum likelihood method. The genetic data based on ITS-2 secondary structure of G. explanatum consisted of four helix, Helix I, II and IV were conserved as compared with other closely related reference taxa of family Paramphistomidae and Gastrothylacidae. Helix III expressed some variations. The study concluded that rDNA ITS-2 and secondary structure information provides a guide for other researchers to determine the molecular taxonomic position of Paramphistomidae trematodes, data will support future clinical studies and control measures to reduce the amphistomiasis in buffaloes.

Characterization and taxonomic utility of ITS2 in Dolerus Panzer, 1801 (Hymenoptera: Tenthredinidae)

The widespread use of ITS2 as a potential marker in insects has accelerated species-level phylogenetic studies. Reliable and quality data can be obtained thanks to the features such as rapid evolution and secondary structure of this marker. This paper presents the phylogenetic relationship among 36 individuals of Dolerus Panzer, 1801 (Hymenoptera: Tenthredinidae) collected between 2002 and 2018 to obtain the first data on ITS2 secondary structure. Aligned of ITS2 data were analyzed by application of maximum likelihood method to reveal phylogenetic relationship among the specimens. Also, the structural properties, length variation and presence of compensatory base changes make the ITS2 useful marker in determination of species boundaries of closely related species. Four species ( Dolerus triplicatus (Klug, 1818), Dolerus germanicus (Fabricius, 1775), Dolerus puncticollis Thomson, 1871 and Dolerus nigratus (Müller, 1776) and two putative species ( Dolerus spp. 1 and 2) were determined from 36 individuals belonging to Dolerus .