Nuclear Ribosomal DNA ITS Research Articles

Antifungal Potential of Cladosporium sp. (Endophytic fungi) Associated with Olea europaea L. Leaves

In the leaves of Olea europaea L. Olive trees an endophytic fungus was discovered. Cladosporium sp. was identified to be the fungus based on its morphological characteristics and nuclear ribosomal DNA ITS sequence analysis and was registered in NCBI as the Cladosporium genus has been registered under the number (0P939922.1) The species was not specified, and it was considered of unknown species after comparing it to global isolates. In comparison to olive leaf extract, Cladosporium sp. including total flavonoid, total phenolic, total terpenoid, and total saponins, Which were 121.9%, 198.1%, 89.13%, and 29.87 % respectively compared to its content in olive leaf extract, which was 61.54 %, 67.88 % , 17.1%, and 20.19% respectively. The Cladosporium sp. extract inhibited the growth of 27 isolates belonging to different species of candida which were Candida albicans , C. lypolitica , C. tropicalis , C. sphaerica , C. krusei , C. guilliermondii , C. parapsilosis , C. norvegicus , C. glabrata , and C. kefyr , the inhibition effects increased with increasing concentration to reach the highest level to suppress fungal growth when concentrated 30 mg/ml. This proves the antifungal potential of endophytic fungi in the future.

Taxonomy and phylogeny of some geophilic Umbelliferae - Apioideae genera based on anatomical, morphological and molecular data

The paper provides an overview of the concordance between molecular (nuclear ribosomal DNA ITS) and morphological data in the group of geophilic Umbelliferae of Middle Asia, which includes the genera Elwendia, Elaeosticta, Hyalolaena, Galagania, Oedibasis, Mogoltavia and Gongylotaxis. In general, a good consistency of data of different types can be observed. At the same time, at some levels of the taxonomic hierarchy, the consistency between morphological and molecular data looks much higher than at others. The identification of the group of geophilic Umbelliferae agrees much better with molecular data than the boundaries between genera within it and the proposed intrageneric groupings. Some observed cases of inconsistency, such as the position of Hyalolaena melanorrhiza among the species of the genus Elaeosticta and the polyphyly of the genera Hyalolaena and Oedibasis, are rather difficult to explain from a morphological point of view, only from a biogeography.

Isolation and Characterization of Novel Huperzine-Producing Endophytic Fungi from Lycopodiaceae Species.

Huperzine A (HupA) is an important drug for treating Alzheimer's disease (AD) and is primarily extracted from the Huperzia serrata (Lycopodiaceae). Failures in the chemical synthesis of Hup and in vitro culture have put H. serrata in danger of extinction, and there is a need for an extensive investigation of Hup from alternative perspectives. The aim of this study is to identify endophytic fungi that produce high Hup or simultaneously produce many types of Hup and have high genetic stability derived from other Lycopodiaceae species as a source of materials for natural Hup production. In this work, Hup-producing endophytic fungi were isolated from three species: Lycopodium clavatum, Phlegmariurus squarrosus, and P. phlegmaria. Of these, L. clavatum and P. squarrosus were confirmed as novel sources of Hup-producing fungi. Based on morphological characteristics and nuclear ribosomal DNA ITS sequences, four endophytic fungi Colletotrichum siamense THG1-17, Epicoccum sorghinum THG01-18, Phoma sp. TKH3-2, and Phyllosticta sp. THG2-27 were firstly isolated from these Lycopodiaceae plants, which were capable of simultaneously producing both HupA and HupB, as evidenced by high-performance liquid chromatography analysis. The four strains showed stability in Hup yield over 50 generations of culture with an in vitro storage period of 3 months. These isolated fungi will provide a new source of materials for further research to develop drugs containing HupA as well as HupB for AD treatment in the future.

Ganoderma gibbosum newly recorded from Pakistan

Ganoderma gibbosum is a new record for Pakistan, identified by morphological and molecular data. DNA sequence analysis of ribosomal 5.8S rRNA gene including the flanking nuclear ribosomal DNA-ITS regions confirmed the Pakistani specimens as G. gibbosum. Morphologically, the specimens are characterized by their non-laccate sessile basidiomata, truncate, short fine minute inter walled pillars, elongated basidiospores (7.5– 10.2 × 4.6–5.9 μm), and greyish to greyish-brown dull pileal surface.

Plastome evolution in the genus Sium (Apiaceae, Oenantheae) inferred from phylogenomic and comparative analyses

BackgroundSium L. (Apiaceae) is a small genus distributed primarily in Eurasia, with one species also occurring in North America. Recently, its circumscription has been revised to include 10 species, however, the phylogenetic relationships within its two inclusive clades were poorly supported or collapsed in previous studies based on nuclear ribosomal DNA ITS or cpDNA sequences. To identify molecular markers suitable for future intraspecific phylogeographic and population genetic studies, and to evaluate the efficacy of plastome in resolving the phylogenetic relationships of the genus, the complete chloroplast (cp) genomes of six Sium species were sequenced.ResultsThe Sium plastomes exhibited typical quadripartite structures of Apiaceae and most other higher plant plastid DNAs, and were relatively conserved in their size (153,029–155,006 bp), gene arrangement and content (with 114 unique genes). A total of 61–67 SSRs, along with 12 highly divergent regions (trnQ, trnG-atpA, trnE-trnT, rps4-trnT, accD-psbI, rpl16, ycf1-ndhF, ndhF-rpl32, rpl32-trnL, ndhE-ndhG, ycf1a and ycf1b) were discovered in the plastomes. No significant IR length variation was detected showing that plastome evolution was conserved within this genus. Phylogenomic analysis based on whole chloroplast genome sequences produced a highly resolved phylogenetic tree, in which the monophyly of Sium, as well as the sister relationship of its two inclusive clades were strongly supported.ConclusionsThe plastome sequences could greatly improve phylogenetic resolution, and will provide genomic resources and potential markers useful for future studies of the genus.

The Occurrence of the Strongylid Nematodes, Kalicephalus viparae viparae (Nematoda: Diaphanocephalidae), in Viper Snakes, Macrovipera lebetina (Reptilia: Viperidae), Southwestern Iran.

ancylostomatid Kalicephalus spp. is the common parasitic intestinal nematode of reptiles. West-Asian blunt-nosed viper is a venomous snake found in extensive areas of Iran. From June to September 2017, two dead viper snakes were referred to a parasitology laboratory and examined for intestinal parasites. Several white elongated roundworms were collected and fixed to identify under light and scanning electron microscopes (SEM) based on morphological and molecular characteristics. For the molecular survey, some parts of the identified worms were extracted and the ITS of nuclear ribosomal DNA (rDNA) was amplified by polymerase chain reaction (PCR). Five roundworms were found in one snake and three worms with similar morphological characteristics in another one. All the collected female hookworms were taxonomically identified as Kalicephalus viperae viperae. The SEM findings showed the head was small and had three dorsal, ventral, and middle circumoral papillae with a spike-like process on the median papilla of K. viperae. Moreover, the buccal capsule was bivalvular and included two lateral valves consisting of several chitonid pieces. The tail of the female worm was slim and long with a blunt end and had a terminal spike at its end. In the molecular survey, the ITS of rDNA amplified at about 850 bp was identified as K. viperae. The ITS gene rDNA phylogeny analysis of the K. viperae sequence showed that the isolated species had high similarity to Ancylostoma species from around the world and is close to Ancylostoma braziliense with 88% discrepancies in the phylogenetic tree. The morphological characteristics and a large part of K. viperea viperea rDNA nucleotide sequence were reported in viper snakes for the first time in the world and in Iran.

A Molecular Systematics and Taxonomy Research on Trechispora (Hydnodontaceae, Trechisporales): Concentrating on Three New Trechispora Species from East Asia.

Trechispora are an important genus of wood-inhabiting fungi that have the ability to decompose rotten wood in the forest ecosystem. In this study, we reported three new species of Trechispora: T. murina, T. odontioidea, T. olivacea from a subtropical region of Yunnan Province, China. Species descriptions were based on a combination of morphological features and phylogenetic analyses of the ITS and LSU region of nuclear ribosomal DNA. Trechispora murina is characterized by the resupinate basidiomata, grandinioid hymenial surface with a greyish tint, monomitic hyphal system and ellipsoid, thick-walled, ornamented basidiospores; T. odontioidea has an odontioid hymenial surface with cylindrical to conical, blunt aculei and subglobose to globose, colorless, slightly thick-walled, ornamented basidiospores; T. olivacea has a farinaceous hymenial surface with olivaceous tint, basidia clavate and thick-walled, ornamented, broadly ellipsoid to globose basidiospores. Sequences of the ITS and nLSU rDNA markers of the studied samples were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony, and Bayesian inference methods. After a series of phylogenetic analyses, the 5.8S+nLSU dataset was constructed to test the phylogenetic relationship of Trechispora with other genera of Hydnodontaceae. The ITS dataset was used to evaluate the phylogenetic relationship of the three new species with other species of Trechispora. Using ITS phylogeny, the new species T. murina was retrieved as a sister to T. bambusicola with moderate supports; T. odontioidea formed a single lineage and then grouped with T. fimbriata and T. nivea; while T. olivacea formed a monophyletic lineage with T. farinacea, T. hondurensis, and T. mollis.

Phylogenetic analysis of some fungi species in West Kazakhstan based on nuclear ribosomal DNA ITS sequences

In this study, phylogenetic analysis of some fungal species distributed in West Kazakhstan was performed based on ITS sequences. All of the mushroom samples were collected from different regions of West Kazakhstan and brought to the laboratory. Total genomic DNA was extracted using a GeneMark commercial kit. ITS1 and ITS4 primers were used for the amplification of the ITS region in PCR analyses. The resulting DNA sequences were then edited using BioEdit and FinchTV. For phylogenetic analysis used MEGA 6.0 program. As a result of the study, ITS sequences ranged from 532 to 715 nucleotides, while the divergence values of the sequences differed between 0.000 and 0.468. The maximum likelihood tree constructed using ITS sequences consists of two clades. According to the phylogenetic analysis results obtained using other fungal species, ITS results were found to be a good indicator for the differentiation of fungal genera.

Phylogenomic insight into dysploidy, speciation, and plastome evolution of a small Mediterranean genus Reichardia (Cichorieae; Asteraceae)

Reichardia Roth is a small Mediterranean genus comprising ten homogeneous species with basic chromosome numbers of 7, 8, and 9. To assess the plastid genome evolution and differentiation of Reichardia species, we assembled the complete plastome sequences of seven Reichardia and two Launaea species and conducted various phylogenomic analyses comparatively with nuclear ribosomal DNA ITS sequences. Reichardia and Launaea plastomes were highly conserved in gene content and order, containing 130 genes. Plastid phylogenomic reconstruction strongly suggested that Reichardia was a sister to Launaea, and its common ancestor initially diverged into two major lineages: the first containing species with n = 8 chromosomes exclusively, and the other with n = 9, 8, and 7 chromosomes. Although the ancestral Reichardia karyotype was suggested to most likely be n = 9 from ancestral chromosome number reconstruction, the pattern of descending dysploidy indicated by the phylogenetic trees based on nuclear ribosomal DNA ITS was less evident in the trees based on the plastome. Possible reasons for these findings are discussed.

DNA Barcoding and Phylogeny of Acari Species Based on ITS and COI Markers

Acari harbor numerous minute species of agricultural economic importance, mainly Tetranychidae and Phytoseiidae. Great efforts have been established by means of recovering morphological, molecular, and phylogenetic traits for species identification. Traditional identification still relies on external diagnostic characters, which are limited and usually exhibit large phenotypic plasticity within the species, rendering them useless for species delimitation and identification. We decided to increase the number of sequences of the Acari mitochondrial COI (Cytochrome C oxidase I) marker and ITS nuclear ribosomal DNA region for species identification in Tetranychidae and Phytoseiidae. The molecular data allow us to establish species boundaries and phylogenetic relationships among several clades of Acari, mainly Tetranychidae and Phytoseiidae. Sequence comparisons between complete COI and the Acari mitochondrial COI, ITS1-5,8S-ITS2, and ITS2 among all Acari sequences have demonstrated that the selected regions, even small, gave enough informative positions for both species’ identification and phylogenetic studies. Analyses of both DNA regions have unveiled their use as species identification characters, with special emphasis on Acari mitochondrial COI for Tetranychidae and Phytoseiidae species in comparison with the Folmer fragment, which has been universally used as a barcode marker. We demonstrated that the Acari mitochondrial COI region is also a suitable marker to establish a barcode dataset for Acari identification. Our phylogenetic analyses are congruent with other recent works, showing that Acari is a monophyletic group, of which Astigmata, Ixodida, Mesostigmata, Oribatida, and Prostigmata are also monophyletic.

Phylogeny, classification and biogeography of

Philotheca sect. Erionema includes 14 species from eastern Australia and one from south-western Australia. We conducted a phylogenetic analysis of the section, including samples of all species, using sequences of the ITS and ETS regions of nuclear ribosomal DNA. Results were broadly congruent with a previous analysis based on morphological and flavonoid data. The analysis is consistent with the monophyly of the section and supports the monophyly of six species represented by multiple samples. Philotheca verrucosa (A. Rich.) Paul G. Wilson was resolved as paraphyletic with respect to P. freyciana Rozefelds but with poor support. Philotheca glasshousiensis, P. myoporoides and P. myoporoides subsp. myoporoides were clearly polyphyletic, including separate geographic clades and the classification of each of these taxa requires revision. In particular, disjunct northern populations of P. glasshousiensis probably represent a distinct species, the five subspecies of P. myoporoides could be treated as separate species and at least two other distinct groups that are currently included under the circumscription of subsp. myoporoides could be treated as species. The phylogeny revealed deeply divergent, geographically overlapping clades in eastern Australia and substantial distances (up to 900 km) between sister taxa. We infer that biogeography of the group has been shaped largely by vicariant differentiation of taxa.

A note on systematic position of Indian species of Cyathocline (Compositae)

The Genus Cyathocline has been merged with Blumea following molecular phylogenetic evidence (Plastid trnL-F and nuclear ribosomal DNA ITS sequences data). While the type species, C. purpurea has been transferred to Blumea, other species have not been transferred. In light of this, new combinations are made here for C. lutea & C. manilaliana. C. purpurea var. alba and C. purpurea var. bicolor are synonymized under Blumea purpurea. B. manilaliana is reported here the first time from Maharashtra state.

Five novel and highly efficient endophytic fungi isolated from Huperzia serrata expressing huperzine A for the treatment of Alzheimer’s disease

Huperzine A (Hup A) is an important drug for treating Alzheimer's disease (AD) and mainly extracted from the Huperzia serrata (Thunb.) Trevis. (Lycopodiaceae) (HS). Nevertheless, the content of Hup A in HS is very low of 0.007% with growing circle of 8 to 10 years, and the chemical synthesis of Hup A still has some insurmountable limitations in the industrialized production. So, the available resources of Hup A for clinical treatment of AD are scarce. The purpose of this work was to construct a biosynthesis platform based on the endophytic fungi from HS. In this work, five endophytic fungi Mucor racemosus NSH-D, Mucor fragilis NSY-1, Fusarium verticillioides NSH-5, Fusarium oxysporum NSG-1, and Trichoderma harzianum NSW-V were firstly found and isolated from the Chinese folk medicine HS, which were identified according to their morphological characteristics and nuclear ribosomal DNA ITS sequences. The highest efficient fungus could effectively biosynthesize Hup A in a liquid culture of 319.8 ± 0.17 mg/L which were 112 times higher than that of other reported conventional endophytic fungi. Moreover, these fungi with higher hereditary stability could possess the initial expressing ability of Hup A after 40 generations, and the expressed Hup A from these biosynthesis systems has prior physicochemical properties, a better inhibition activity of acetylcholinesterase and a lower cytotoxicity compared with the listed active pharmaceutical ingredients (APIs) of Hup A. These results provide promising alternative resources for producing Hup A at an industrial scale by biosynthesis, and it may also shed light on millions of AD patients. KEY POINTS: • Five novel endophytic fungi with high stability could highly express prior Hup A Graphical abstract.

On the identity of Goodyera rosulacea (Orchidaceae: Orchidoideae: Cranichideae: Goodyerinae)

Morphological studies based on the protologues, observations of herbarium specimens and field surveys suggest that Goodyera rosulacea is a synonym of G. brachystegia. According to genetic studies, accessions of G. rosulacea and G. brachystegia form a single clade in the phylogenetic trees obtained from analyses of the nuclear ribosomal DNA ITS region and four plastid regions (accD-psaI, matK, trnK-rps16 and trnL-F). This phylogenetic evidence further underpins the conclusion that the two names refer to one and the same species.

An Empirical Analysis Rejects the Hybrid Speciation Hypothesis of a Crucial Kiwifruit Species, Despite Genomic Evidence of Frequent Interspecific Gene Flow in the Genus.

Hybrid speciation is an important way to generate species diversity. In general, however, interspecific hybridization is easily confused with the formation of hybrid species. Using the genomic resequencing data of the kiwifruit genus (Actinidia), at least ten species were documented recently as homoploid hybrid species, and thus a two-layer mode of species diversification has been proposed. As a crucial piece of evidence, Actinidia fulvicoma was identified as a hybrid derivative of Actinidia eriantha × Actinidia cylindrica, representing a rare case of hybrid species in kiwifruit that won the competition of ecological niches with one of its putative parental species, A. cylindrica. However, the hypothesized hybrid origin of A. fulvicoma is inconsistent with our specimen observations. Here, we present multiple lines of evidence to reject the hybrid speciation hypothesis for this species, despite genomic evidence for frequent interspecific gene flow. We collected the samples of A. fulvicoma in type locality and neighboring regions to contrast them with type specimen, and sequenced nuclear ribosomal DNA ITS, chloroplast trnL-trnF and mitochondrial nad2-i3, as well as four single-copy nuclear genes explored from kiwifruit genomes, to infer phylogenetic relationships among A. fulvicoma, its putative parental species, and their relatives. Our data definitely reveal that A. fulvicoma occupies an independent backbone lineage and it is not a hybrid. This study suggests that correct evolutionary applications on extensive surveys of the putative hybrid and its possible parents with strict criteria are necessary in the documentation of hybrid speciation to advance our understanding of the genomic basis of hybrid species.

Laserocarpum, a new genus of Apiaceae endemic to Greece

<em>Laserpitium pseudomeum</em> is an endemic umbellifer of Greece occurring in the mountains of Sterea Ellas and northern Peloponnese. Molecular data indicate that it is not related to its putative congeners, but instead constitutes an isolated lineage. The nuclear ribosomal DNA ITS tree places it as a weakly supported sister group to <em>Portenschlagiella ramosissima</em>, the sole species in its genus, whereas the tree inferred from three noncoding cpDNA loci does not confirm this relationship. <em>Portenschlagiella ramosissima</em> is sometimes placed in <em>Athamanta</em>, an affinity supported neither by molecular data nor by fruit morphology. <em>Laserpitium pseudomeum</em> notably differs from <em>P. ramosissima</em> in vegetative, flower, and fruit characteristics, and no obvious morphological synapomorphies indicate their close relationship. Therefore, we place <em>L. pseudomeum</em> in the newly described genus <em>Laserocarpum</em> and lectotypify the species with the specimen <em>Orphanides 2019</em> (G00766460) at G-Boiss.

Dissimilar molecular and morphological patterns in an introgressed peripheral population of a sand dune species (Armeria pungens, Plumbaginaceae).

Introgression is a poorly understood evolutionary outcome of hybridisation because it may remain largely undetected whenever it involves the transfer of small parts of the genome from one species to another. Aiming to understand the early stages of this process, a putative case from the southernmost border of the Armeria pungens range from its congener A.macrophylla is revisited following the discovery of a subpopulation that does not show phenotypic signs of introgression and resembles typical A.pungens. We analysed morphometrics, nuclear ribosomal DNA ITS and plastid DNA (trnL-trnF) sequences, genome size, 45S and 5S rDNA loci-FISH data and nrDNA IGS sequences. Within the study site, most individuals match morphologies of either of the two hybridising species, particularly the new subpopulation, with intermediate phenotypes being scarce. This pattern does not fully fit molecular evidence revealing two ITS ribotypes co-occurring intragenomically in most plants from the study site and one single plastid haplotype. Genome size and structural features of the IGS sequences both indicate that A.pungens from the study site is genetically more similar to its sympatric congener than to the remainder of its conspecifics. Introgression of A.macrophylla into A.pungens and plastid capture explain all the evidence analysed. However, important features to understand the origin and fate of the introgressed population, such as the degree and direction of introgression, which are important for understanding early stages of hybridisation in plants with low reproductive barriers, should be addressed with new data.

Paris variabilis (Melanthiaceae), a new species from southwestern China

Paris variabilis, a new species from the Wumengshan Mountains, southwestern China, is described and illustrated. The new species is placed in Paris section Euthyra. The new taxon was determined to be most morphologically similar to P. vietnamensis but differs in its oblong leaf blades with an acute apex, stamens 2–4 × petal number, greenish yellow filaments and an enlarged, purplish red style base. The phylogenetic placement of this species was assessed based on nuclear ribosomal ITS DNA sequences data. The results of morphological and phylogenetic analyses support the status of the taxon as a new species.

Biosynthesis of AgNPs in Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) (P-AgNPs) Using the Endophytic Fungus Fusarium solani Isolated from an Endangered Medicinal Plant Plumbago rosea and Their Anti Bacterial and Anticancer Activity on Human Breast Cancer Cells (MCF-7)

Abstract—Among the most hopeful biomaterials, metallic nanoparticles with antibacterial and anticancer properties are expected to open new avenues to fight and prevent various cancer and infectious diseases. The aim of the current study was the biosynthesis of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone-AgNPs (P-AgNPs) using the endophytic fungus Fusarium solani isolated from an endangered medicinal plant Plumbago rosea. The fungus was identified according to its morphological characteristics and nuclear ribosomal DNA ITS sequence analysis. The synthesized P-AgNPs were initially noticed through visual color change from yellow to reddish brown and further confirmed by surface plasmonic resonance (SPR) band at 450 nm using UV-visible spectroscopy. Fourier transform infrared (FTIR) analysis showed amines and amides that are responsible for the stabilization of P-AgNPs. High-resolution scanning electron microscopy (HRSEM) showed that P-AgNPs were well dispersed, spherical, and well within the range of 23.2, 33.2 and 33.7 nm. X-ray diffraction (XRD) study revealed crystalline nature of P-AgNPs. These P-AgNPs displayed good antibacterial activity in dose dependant manner. The synthesized P-AgNPs exhibited dose-depended cytotoxicity against MCF-7 human breast cancer cells, the inhibitory IC50 were found to be 14.5 μg/mL. These nanoparticles were further studied for their anticancer activity and showed high selective toxicity against the MCF-7 breast cancer cell line. The anticancer activity also executed through inhibiting the cells at the phase DNA synthesis.

Short-spored Subulicystidium (Trechisporales, Basidiomycota): high morphological diversity and only partly clear species boundaries.

Diversity of corticioid fungi (resupinate Basidiomycota), especially outside the northern temperate climatic zone, remains poorly explored. Furthermore, most of the known species are delimited by morphological concepts only and, not rarely, these concepts are too broad and need to be tested by molecular tools. For many decades, the delimitation of species in the genus Subulicystidium (Hydnodontaceae, Trechisporales) was a challenge for mycologists. The presence of numerous transitional forms as to basidiospore size and shape hindered species delimitation and almost no data on molecular diversity have been available. In this study, an extensive set of 144 Subulicystidium specimens from Paleo- and Neotropics was examined. Forty-nine sequences of ITS nuclear ribosomal DNA region and 51 sequences of 28S nuclear ribosomal DNA region from fruit bodies of Subulicystidium were obtained and analysed within the barcoding gap framework and with phylogenetic Bayesian and Maximum likelihood approaches. Eleven new species of Subulicystidium are described based on morphology and molecular analyses: Subulicystidium boidinii, S. fusisporum, S. grandisporum, S. harpagum, S. inornatum, S. oberwinkleri, S. parvisporum, S. rarocrystallinum, S. robustius, S. ryvardenii and S. tedersooi. Morphological and DNA-evidenced borders were revised for the five previously known species: S. naviculatum, S. nikau, S. obtusisporum, S. brachysporum and S. meridense. Species-level variation in basidiospore size and shape was estimated based on systematic measurements of 2840 spores from 67 sequenced specimens. An updated identification key to all known species of Subulicystidium is provided.