Phylogenetic Relationships of <i>Termitomyces aurantiacus</i>Inferred from Internal Transcribed Spacers DNA Sequences

Fungus-growing termites cultivate species of the mutualistic basidiomycete genus Termitomyces on a substrate called the fungal comb. Identification of fungal species based on morphological features is complicated, tedious, and prone to errors. As an alternative, nuclear ribosomal DNA sequences consisting of the internal transcribed spacers (ITS1 and ITS2) and 5.8S rDNA were used to identify Malaysian isolates of Termitomyces sp. The morphological characteristics and molecular data indicate that Malaysian Termitomyces isolated is clearly monophyletic and belongs to the Tricholomataceae family. The Malaysian isolates analyzed in this study represent the termite fungus species called T. aurantiacus.

Characterization of Fusarium Oxysporum Isolates Obtained from Wax Gourd and Chieh-qua in China by Pathogenicity, RAMs and Sequence Analysis of the rDNA Internal Transcribed Spacers (ITS1 and ITS2)

Wax gourd (Benincasa hispida Thumb. Cogn) is called white gourd, winter melon, Chinese preserving melon, Chinese squash, and don kwa. It has been cultivated in China for over 2 300 years. It probably

Phylogeny of Ptychostomum (Bryaceae,Musci) inferred from sequences of nuclear ribosomal DNA internal transcribed spacer (ITS) and chloroplast rps4

The phylogeny of Ptychostomum was first spacer （ITS） region of the nuclear ribosomal （nr） DNA DNA rps4 sequences. Maximum parsimony, maximum undertaken based on analysis of the internal transcribed and by combining data from nrDNA ITS and chloroplast likelihood, and Bayesian analyses all support the conclusion that the reinstated genus Ptychostomum is not monophyletic. Ptychostomum funkii （Schwagr.） J. R. Spence （≡ Bryum funkii Schwaigr.） is placed within a clade containing the type species of Bryum, B. argenteum Hedw. The remaining members of Ptychostomum investigated in the present study constitute another well-supported clade. The results are congruent with previous molecular analyses. On the basis of phylogenetic evidence, we agree with transferring B. amblyodon Mull. Hal. （≡ B. inclinatum （Brid.） Turton≡ Bryum archangelicum Bruch ＆ Schimp.）, Bryum lonchocaulon Mull. Hal., Bryum pallescens Schleich. ex Schwaigr., and Bryum pallens Sw. to Ptychostomum.

The homology analysis of internal transcribed spacer sequence of ribosomal DNA in common dermatophytes

In order to analyze the sequences of the internal transcribed spacer （ITS） including the 5.8 S ribosomal DNA （rDNA） of common dermatophytes, so as to obtain a rapid and accurate method to identify the species of dermatophytes and to establish the phylogenetic tree of these species to understand their relationship, 16 strains of dermatophytes were collected and preliminarily identified by morphological characteristics. General primers for fungi ITS1 and ITS4 were used to amplify the ITS rDNA of each strains with PCR. The PCR products after purification were sequenced directly and were analyzed through internet. In the results, 11 strains were identified by means of morphological features, among which 5 strains were Trichophyton, 5 strains were Microsporum and 1 was Epidermophytoa, which was consistent with the results by molecular biology. In the 5 unidentifiable strains, 1 strain was proved to be Chrysosporium by molecular biology. These strains studied could be divided into 3 different classes as indicated in the analysis of the phylogenetic tree of the sequences in ITS, which were quite different from those of morphological classification. It is evident from the above observations that the molecular method of analysis on the ITS sequences is a rapid, highly sensitive and accurate approach for the detection of dematophyte species, however, it still exhibits some limitations needing the supplementation with morphological identification.

DNA Barcoding of <i>Ricinus communis</i>from Different Geographical Origin by Using Chloroplast <i>matK</i>and Internal Transcribed Spacers

Ricinus communis have attracted considerable attention because of its specific industrial and pharmacological activities. DNA barcodes can be used as reliable tools to facilitate the identification of medicinal plants for the safe use, quality control and forensic investigation. In this study, the differential identification of eight accessions of R. com-munis was investigated through DNA sequence analysis of two candidate DNA barcodes. The nucleotide sequence of internal transcribed spacers (ITS2) and chloroplast maturase gene (matK) have been determined to construct the phylogenetic tree. The phylogenetic relationships of accessions based on the nrITS2 region and partial matK region showed that all accessions in this study were related to three geographical origins. Based on sequence align-ment and phylogenetic analyses we concluded that the ITS2 sequences can distinguish R. communis accessions from different geographical distributions.

Application of the first internal transcribed spacer(ITS-1)of ribosomal DNA as a molecular marker to population analysis in farrer's scallop Chlamys farreri

Sequence variation of the first internal transcribed spacer of ribosomal DNA （ ITS - 1 ） was examined and its application to the study of genetic variation was explored in four populations of farter＇ s scallop Chlamys farreri. ITS - 1 fragments, with a length of about 300 bp,of 78 individuals collected from Dalian, Qingdao, Yantai in China and Korea respectively were amplified via PCR, cloned and sequenced. Intra-genomic variation was examined by sequencing several clones of single individuals. Alignment and polymorphism analysis detected 44 haplotypes and 50 polymorphic sites which consist of 30 substitutions and 20 indels, indicating a high level of polymorphisms. Sequence analysis also showed a very low level of intra-individual variation. All these features validated the feasibility of application of ITS - 1 fragment to population analysis. Polymorphism analysis showed that the Korea sample has the richest genetic variation, followed by Yantai and Qingdao samples. AMOVA （analysis of molecular variance） showed that the majority （96.26%） of genetic variation was distributed within populations and 3.74% resulted from among populations, but with P 〈 0.05 （ = 0.042）, indicating that the populations in this study have significant divergence. This output was basically concordant with the result arising from RAPD data and different from that from mitochondrial 16S rDNA sequence data. Discussion on this inconsistency was made accordingly.

Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer(ITS)region of peritrich ciliates in environmental samples

Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifi cations of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specifi c PCR primers were newly designed to amplify a fragment including the internal transcribed spacer(ITS) region of ribosomal rDNA from environmental samples. The primers showed high specifi city in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28 S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18 S rDNA in peritrichs. The newly designed specifi c primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in dif ferent systems.

Molecular phylogeny of Pneumocystis based on 5.8S rRNA gene and the internal transcribed spacers of rRNA gene sequences

To clarify the phylogenetic relationships and species status of Pneumocystis, the 5.8S rRNA gene and the internal transcribed spacers (ITS, 1 and 2) of Pneumocystis rRNA derived from rat, gerbil and human were amplified, cloned and sequenced. The genetic distance matrix of six Pneumocystis species compared with other fungi like Taphrina and Saccharomyces indicated that the Pneumocystis genus contained multiple species including Pneumocystis from gerbil. The phylogenetic tree also showed that Pneumocystis from human and monkey formed one group and four rodent Pneumocystis formed another group. Among the four members, Pneumocystis wakefieldiae was most closely related to Pneumocystis murina and Pneumocystis carinii, and was least related to gerbil Pneumocystis.

A preliminary analysis of phylogenetic relationships of Arundinaria and related genera based on nucleotide sequences of nrDNA （ITS region） and cpDNA （trnL-F intergenic spacer）

Phylogenetic relationships of Arundinaria and related genera (Pleioblastus, Pseudosasa, Oligostachyum, Bashania, Clavinodum, etc.) were assessed by analyzing the sequences of the nrDNA internal transcribed spacer (ITS) and the cpDNA trnL-F intergenic spacer (IGS). Comparison with trnL-F IGS sequence, the ITS region provided the higher number of parsimony informative characters, and the interspecific variation of the ITS sequence was higher than that of the trnL-F IGS sequence.The tree obtained by combining both sets of data showed that the species sampled in Arundinaria and the related genera were monophyletic and divided into two clades. The relationships and positioning of all the taxa surveryed (including A. oleosa, A. hsienchuensis, A. chino, A. amara, A. yixingensis, A. amabilis, A. fortunei, A. pygmaea, A. gramineus, A. fargesii, A. faberi, A. hupehense, Pseudosasa japonica cv. Tsutsumiana, P. japonica and Brachystachyum densiflorum) were also discussed. The results from the sequences were broadly consistent with morphological characters, appearing all these taxa sampled belong to the genus of Arundinaria. The topologies of the trees generated from individual data and the combined data were similar.

Infrageneric and Sectional Relationships in the Genus Rhododendron (Ericaceae) Inferred from ITS Sequence Data

The internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (including 5.8S rRNA) of 15 Rhododendron, species, representing most sections of the genus, one Ledum species and Cassiope fastigiata were sequenced. Together with the ITS sequences of 13 selected Rhododendron species and Bejaria racemosa downloaded from GenBank, we explored the infrageneric and sectional relationships of this important North Temperate genus by employing maximum-parsimony analysis using PAUP software. C. fastigiata and B. racemosa were designated as outgroups. The ITS-based tree inferred that: (1) Rhododendron was a well-supported monophyletic group, while subg. Therorhodion was basal to the rest of the genus; (2) Ledum was a member of Rhododendron, and its close relationship with the lepidote rhododendron was confirmed; (3) the lepidote rhododendron plus Ledum formed a strongly-supported monophyletic clade which was sister to the rest of the elepidote rhododendron; (4) the elepidote rhododendron formed a weakly-supported clade within which the monophyly of subg. Hynwrianthes and subg. Tsutsusi were strongly supported, while subg. Pentanthera and subg. Azaleastrum were polyphyletic; and (5) the monophyly of sect. Choniastnini, (subg. Azaleastrum) was strongly-supported, while subg. Tsutsusi could be sister to a weakly-supported clade composed of two sampled species of sect. Azaleastrum (subg. Azaleastrum) together with R. sentibarbatum, of subg. Mumeazalea.

The Phylogenetic Relationships of an Endemic Genus Sinadoxa in the Qinghai-Xizang Plateau: Evidence from ITS Sequence Analysis

对青藏高原特有的濒危植物华福花 (SinadoxacorydalifoliaC .Y .Wu ,Z .L .WuetR .F .Huang)的核糖体DNA中的内转录间隔区 (ITS)序列及 5 .8SrRNA基因的序列进行了测定。同川续断目和五加目有关类群序列的比较及分支分析表明华福花属与五福花属 (AdoxaL .)近缘 ,不支持它可能与五加目或败酱科有亲缘关系的假设。尽管形态上它与五福花属分化十分明显 ,但ITS碱基分异却较小。引起其系统位置发生争论的外部形态为什么进化得如此之快 ,是值得进一步深入探讨的课题。

Systematic positions of Lamiophlomis and Paraphlomis(Lamiaceae) based on nuclear and chloroplast sequences

Genera Lamiophlomis and Paraphlomis were originally separated from genus Phlomis s.1. on the basis of particular morphological characteristics. However, their relationship was highly contentious, as evidenced by the literature. In the present paper, the systematic positions of Lamiophlomis, Paraphlomis, and their related genera were assessed based on nuclear internal transcribed spacer （ITS） and chloroplast rpl16 and trnL-F sequence data using maximum parsimony （MP） and Bayesian methods. and outgroup were sampled. Analyses of both separate In total, 24 species representing six genera of the ingroup and combined sequence data were conducted to resolve the systematic relationships of these genera. The results reveal that Lamiophlomis is nested within Phlomis sect. Phlomoides and its generic status is not supported. With the inclusion ofLamiophlomis rotata in sect. Phlomoides, sections Phlomis and Phlomoides of Phlomis were resolved as monophyletic. Paraphlomis was supported as an independent genus. However, the resolution of its monophyly conflicted between MP and Bayesian analyses, suggesting the need for expended sampling and further evidence.

Complete nuclear ribosomal DNA sequence amplification and molecular analyses of Bangia (Bangiales, Rhodophyta) from China

Filamentous Bangia,which are distributed extensively throughout the world,have simple and similar morphological characteristics.Scientists can classify these organisms using molecular markers in combination with morphology.We successfully sequenced the complete nuclear ribosomal DNA.approximately 13 kb in length,from a marine Bangia population.We further analyzed the small subunit ribosomal DNA gene(nrSSU) and the internal transcribed spacer(ITS) sequence regions along with nine other marine,and two freshwater Bangia samples from China.Pairwise distances of the nrSSU and 5.8S ribosomal DNA gene sequences show the marine samples grouping together with low divergences(0-0.003;0-0.006,respectively) from each other,but high divergences(0.123-0.126;0.198,respectively) from freshwater samples.An exception is the marine sample collected from Weihai,which shows high divergence from both other marine samples(0.063-0.065;0.129,respectively) and the freshwater samples(0.097;0.120,respectively).A maximum likelihood phylogenetic tree based on a combined SSU-ITS dataset with maximum likelihood method shows the samples divided into three clades,with the two marine sample clades containing Bangia spp.from North America,Europe,Asia,and Australia;and one freshwater clade,containing Bangia atropurpurea from North America and China.

Phylogenetic relationships within the genus Aspidisca(Protozoa,Ciliophora,Euplotida) revealed by ITS1-5.8S-ITS2 region sequences

Abstract The internal transcribed spacer regions （ITS1 and ITS2） and 5.8S rRNA genes were sequenced in six populations of four Aspidisca species, namely A. leptaspis, A. orthopogon, A. rnagna and A. aculeata. Phylogenetic trees were constructed by means of Bayesian inference （BI）, Maximum Parsimony （MP）, Neighbor-Joining （NJ）, and Maximum Likelihood （ML） to assess the inter- and intra-species relationships within the genus Aspidisca. All trees show similar topologies with stable supports and indicate that： （1） four well known groups, i.e., Oligotrichia, Stichotrichia, Choreotrichia and Hypotrichia, are distinctly outlined within the class Spirotrichea, and all are monophyletic other than Hypotrichia; （2） members of Aspidisca can be distinguished well, based on the ITSI-5.SS-ITS2 region sequences, and A. leptaspis and A. magna shared a closer relationship than other species; （3） Aspidisca and Euplotes branch early in the subclass Hypotrichia. To compare the phylogenetic relationships based on different genes, SSU rRNA trees were also constructed with nearly the same species inclusion, which revealed different topologies of inter-species, inter-genera and inter-subclasses.

Molecular phylogenetic analyses based on the complete plastid genomes and nuclear sequences reveal Daphne(Thymelaeaceae)to be non-monophyletic as current circumscription

The diverse members of the genus Daphne are prized for their fragrant flowers.Despite being promising ornamental plants in many countries,genetic information of Daphne is scarce.In this study,the plastomes of four species and one variety of Daphne were sequenced and analyzed.The plastomes were typical and contained a pair of inverted repeat(IR)regions that separated the large single-copy(LSC)region from the small single-copy(SSC)region.With a length ranging from 132,869 bp(D.genkwa)to 174,773 bp(D.championii),106 to 141 genes were predicted.Comparative plastome analysis of the newly sequenced plastomes with four publicly available Daphne plastomes identified an expansion of the IRs,sequence variations,and mutational hotspots.Phylogenetic analyses indicated that the genus Daphne in its current circumscription is polyphyletic.Daphne genkwa was nested within the genus Wikstroemia,while D.championii was well resolved as sister to Edgeworthia.These findings concurred with results from our study that used nuclear ribosomal internal transcribed spacer sequence data.The conflicts on the molecular placement of D.championii and D.genkwa and the present taxonomic classification in Daphne suggest that a new intergeneric classification system of Daphneae warrants consideration.

Phylogeography of Batrachospermum arcuatum in North China based on ITS sequence data

Batrachospermum arcuatum Kylin is typically dioecious,although some monoecious specimens have been collected from four locations in North China.In this study,B.arcuatum populations,including monoecious and dioecious thalli,were collected from seven stream segments.The nuclear DNA internal transcribed spacer(ITS)region was sequenced for the seven populations,and a phylogenetic tree was constructed using Bayesian inference to assess intraspecifi c relationships.A haplotype network was also created.The ITS region in B.arcuatum from North China comprised 822–853 bp,with 10 haplotypes detected from the seven locations.The results of this study support the inclusion of monoecious individuals in the species B.arcuatum.

Nucleotide Sequence Variations in a Medicinal Relative of Asparagus, <i>Asparagus cochinchinensis</i>(Lour.) Merrill (Asparagaceae)

To determine the eVolutionary history of Asparagus cochinchinensis (Asparagaceae), we investigated the geographic pattern of its nucleotide sequence variations in Japan, Taiwan, South Korea and China. We found 21 polymorphic nucleotide sites by sequencing the internal transcribed spacer (ITS) region, which gave rise to a total of 15 haplotypes, labeled A to O. The A-type was found only in inland China (Guizhou and Sichuan), and the other haplotypes in China extended to several lineages;therefore, A. cochinchinensis may have its origin in the interior of China. The I-type has large distribution area and it also experienced a quick expansion in relative recent years. Haplotype differentiation was observed between the eastern and western side of the Central Mountain Ridge in Taiwan. Two lineages were found in Japan, one in the Yaeyama Islands and the other in remaining areas of Japan, implying that A. cochinchinensis independently colonized in Japan at least twice.

Sequence differences of rDNA-ITS2 and species-diagnostic PCR assay of Anopheles sinensis and Anopheles anthropophagus from China

Anopheles sinensis and An. anihropophagus are two morphologically indistinguishable yet genetically and behaviorally distinct mosquito species that vary dramatically in their importance as vectors of malaria inChina. The sequence of the ribosomal DNA internal transcribed spacer 2 (ITS2) was determined for bothspecies to assess the species differentiation. The lengths of ITS2 was 468 hp for An. sinensis and 452 hp forAn. anthropophagus. Interspecies difference in sequence was 28. 8%. Intraspecies sequence divergence wasnegligible. A PCR method was developed for distinguishing the two species based on the species-specific variations of the ITS2 sequences. The method would amplify a diagnostic fragment in length of 425 hp for An.sinensis and 253 hp for An. anthropophagus. Field collections from 12 localities in 10 provinces of China weretested. In 440 mosquitoes identified by adult morphological characteristics as An. sinensis, 291 (66. 2 % ) wereidentified later as An. sinensis and 56 (12. 7 % ) as An. anthropophagus, the remaining 93 (22. 1 % ) were notamplified by PCR. The results showed that the morphological characteritics of adult was not reliable for fieldidentification. The PCR assay was a simple, fast and reliable method for species identification.

Metataxonomics of Internal Transcribed Spacer amplicons in cerebrospinal fluid for diagnosing and genotyping of cryptococcal meningitis

Background:Cryptococcal meningitis is a severe infectious disease associated with high morbidity and mortality.Rapidity and accuracy of diagnosis contribute to better prognosis,but readily available tools,such as microscopy,culture,and antigens do not perform well all the time.Our study attempted to diagnose and genotype cryptococcus in the cerebrospinal fluid(CSF)samples from patients with cryptococcal meningitis using the approach of metataxonomics of Internal Transcribed Spacer(ITS)amplicons.Methods:The CSF samples were collected from 11 clinically suspected cryptococcal meningitis patients and four non-infectious controls.Samples were recruited from the First Affiliated Hospital of Fujian Medical University Hospital,Fuzhou Fourth Hospital and the 476th Hospital of Chinese People's Liberation Army from December 2017 to December 2018.ITS1 ribosomal deoxyribonucleic acid(rDNA)genes of 15 whole samples were amplified by universal forward primer ITS1(CTTGGTCATTTAGAGGAAGTAA)and reverse primer ITS2(GCTGCGTTCTTCATCGATGC),sequenced by Illumina MiSeq Benchtop Sequencer.The results were confirmed by sanger sequencing of ITS1 region and partial CAP59 gene of microbial isolates from 11 meningitic samples.Pair-wise comparison between infectious group and control group was conducted through permutational multivariate analysis(PERMANOVA)in R software.Results:The 30,000 to 340,000 high-quality clean reads were obtained from each of the positively stained or cultured CSF samples and 8 to 60 reads from each control.The samples from 11 infected patients yielded detectable cryptococcal-specific ITS1 DNA with top abundance(from 95.90%to 99.97%),followed by many other fungal groups(each<1.41%).ITS genotype was defined in 11 CSF samples,corresponding to ITS type 1,and confirmed by Sanger sequencing.A statistically significant difference(r2=0.65869,P=0.0014)between infectious group and control group was observed.Conclusions:The metataxonomics of ITS amplicons facilitates the diagnosis and genotype of cryptococcus in CSF samples,which may provide a better diagnostic approach of cryptococcal infection.

Internal transcribed spacer guided multiplex PCR for species identification of Convolvulus prostratus and Evolvulus alsinoides

Shankhpushpi is a reputed drug from an Indian system of medicine for treating mental disorders and enhancing memory. Two herbs, namely Convolvulus prostratus Forssk. and Evolvulus alsinoides(L.) L., are commonly known as Shankhpushpi. Ambiguous vernacular identity can affect the scientific validity of the Shankpushpi-based herbal drug therapy. In the present investigation, a novel and sensitive multiplex PCR method based on polymorphism in the internal transcribed spacer(ITS) region was developed to establish the molecular identity of C. prostratus and E. alsinoides. DNA was isolated and the ITS region was amplified, sequenced and assembled. Sequences were aligned to identify variable nucleotides in order to develop plant-specific primers. Primers were validated in singleplex reactions and eventually a multiplex assay was developed. This assay was tested for sensitivity and validated by amplifying DNA isolated from the simulated blended powdered plant material. Primers developed for C. prostratus resulted into a 200 bp amplicon and 596 bp for E. alsinoides. The assay was found to be sensitive enough for amplification of low quantities of DNA. The method can detect 10% of the mixing of plants with each other in blended material. This PCR assay can be used for rapid botanical identification of Shankhpushpi plant materials and will improve evidence-based herbal drug therapy.