Accession-Specific Haplotypes of the Internal Transcribed Spacer Region in Arabidopsis thaliana--A Means for Barcoding Populations

Eukaryote genomes contain multiple copies of nuclear ribosomal DNA (nrDNA) harboring both highly conserved and variable regions. This has made nrDNA the most popular genetic marker for phylogenetic studies and the region of choice for barcoding projects. Furthermore, many scientists believe that all copies of nrDNA within one nucleus are practically identical due to concerted evolution. Here, we investigate the model plant species Arabidopsis thaliana for intragenomic variation of the internal transcribed spacer (ITS) region of nrDNA. Based on a modified deep sequencing approach, we provide a comprehensive list of ITS polymorphisms present in the two most widely used accessions of A. thaliana-Col-0 and Ler. Interestingly, we found that some polymorphisms are shared between these genetically very distinct accessions. On the other hand, the high number of accession-specific polymorphisms shows that each accession can be clearly and easily characterized by its specific ITS polymorphism patterns and haplotypes. Network analysis based on the detected haplotypes demonstrates that the study of ITS polymorphism patterns and haplotypes is an extremely powerful tool for population genetics. Using the methods proposed here, it will now be possible to extend the traditionally species-bound barcoding concept to populations.     

A Molecular Phylogeny of Lilium in the Internal Transcribed Spacer Region of Nuclear Ribosomal DNA

Phylogenetic relationships among 55 species of Lilium, Cardiocrinum giganteum, and Nomocharis saluenensis were inferred from nucleotide sequence variations in the internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA. The phylogeny derived from ITS sequences estimated using maximum-likelihood methods indicated that (1) most of the species construct their own clade according to the classification based on morphological features at the section level; (2) section Daurolirion is not independent of Sinomartagon, and it is appropriate to integrate two sections as Sinomartagon; (3) it is appropriate that L. henryi and L. bulbiferum are classified into subsection 6a and Sinomartagon–Daurolirion, respectively; (4) subsection 6b is much closer to Sinomartagon than subsection 6a and Archelirion, and it arose directly from Sinomartagon; and (5) Lilium is much closer to Nomocharis than Cardiocrinum. Phylogenetic estimation using sequences of the ITS region is suitable at the levels of genus, section, and most of subsection. Received: 18 December 1998 / Accepted: 14 March 1999     

The rDNA Internal Transcribed Spacer Region as a Taxonomic Marker for Nematodes

The ITS region from a wide taxonomic range of nematodes, including secernentean and adenophorean taxa, and free-living, entomopathogenic, and plant-parasitic species, was evaluated as a taxonomic marker. Size of the amplified product aided in the initial determination of group membership, and also suggested groups that may require taxonomic reevaluation. Congeneric species often displayed identically sized ITS regions, but genera such as Pratylenchus and Tylenchorhynchus had species with large differences in size. ITS heterogeneity in individuals and populations was identified in several nematode taxa. PCR-RFLP of ITS1 is advocated as a method of taxonomic analysis in genera such as Helicotylenchus that contain numerous species with few diagnostic morphological characteristics.     

除虫菊和茼蒿核糖体ＤＮＡ ＩＴＳ区的序列

除虫菊(Pyrethrum cinerariifolium),又称白花除虫菊,是众所周知的著名杀虫植物,也是目前世界上唯一集约化栽培的天然杀虫剂原料,至今仍为肯尼亚、厄瓜多尔等一些国家的支柱产业。除虫菊的头状花序中含有6种0.4 %～2 %的杀虫成分,即除虫菊酯Ⅰ,Ⅱ(pyrethrin-I,II),瓜菊酯Ⅰ,Ⅱ(cinerin-I,II)和茉莉菊酯Ⅰ,Ⅱ(Jasmolin-I,II),6个成分组成的复合植物杀虫剂,对家蝇的杀虫活性LD50为15～20μg/g,对大白鼠的毒性LD50为2124～2416 mg/kg,对温血动物毒性更低。面对呼唤绿色产业的21世纪,除虫菊的产业开发又成为天然农药开发的一个热点。我们…     

Phylogeny of the GenusTrichodermaBased on Sequence Analysis of the Internal Transcribed Spacer Region 1 of the rDNA Cluster

Sequences of the internal transcribed spacer region 1 (ITS1) of the ribosomal DNA were used to determine the phylogenetic relationships of species ofTrichodermasect.Pachybasium.To this end, 85 strains—including all the availableex-type strains—were analyzed. Parsimony analysis demonstrated that the section is nonmonophyletic, distributing the 85 strains among three main groups that were supported by bootstrap values. Group A comprises two clades (A1 and A2), with A1 includingT. polysporum, T. piluliferum,andT. minutisporum,while A2 includedT. hamatum, T. pubescens,andT. strigosumin addition to species previously included in sect.Trichoderma(i.e.,T. viride, T. atroviride,andT. koningii). Theex-type strain ofT. fasciculatumformed a separate branch basal to clade A. Clade B contained the sect.PachybasiummembersT. harzianum, T. fertile, T. croceum, T. longipile, T. strictipile, T. tomentosum, T. oblongisporum, T. flavofuscum, T. spirale,and the anamorphs ofHypocrea semiorbisandH. cf. gelatinosa.Sequence differences among clades A1, A2, and B were in the same order of magnitude as between each of them andT. longibrachiatum,which was used as an outgroup in these analyses. Sequence differences within clades A1, A2, and B were considerably smaller: in some cases (i.e.,T. virensandT. flavofuscum; T. strictipileandH. cf. gelatinosa), the ITS1-sequences were identical, suggesting conspecifity. In other cases (e.g.,T. crassumandT. longipile; T. harzianum, T. inhamatum, T. croceum, T. fertile,andH. semiorbis; T. hamatumandT. pubescens;andT. viride, T. atroviride,andT. koningii) differences were in the range of 1–3 nt only, suggesting a very close phylogenetic relationship. The sequence of a previously described aggressive mushroom competitor group ofT. harzianumstrains (Th2) was strikingly different from that of theex-type strain ofT. harzianumand closely related species and is likely to be a separate species.     

Selection of Enzymes for Terminal Restriction Fragment Length Polymorphism Analysis of Fungal Internally Transcribed Spacer Sequences

Terminal restriction fragment length polymorphism (TRFLP) profiling of the internally transcribed spacer (ITS) ribosomal DNA of unknown fungal communities is currently unsupported by a broad-range enzyme-choosing rationale. An in silico study of terminal fragment size distribution was therefore performed following virtual digestion (by use of a set of commercially available 135 type IIP restriction endonucleases) of all published fungal ITS sequences putatively annealing to primers ITS1 and ITS4. Different diversity measurements were used to rank primer-enzyme pairs according to the richness and evenness that they showed. Top-performing pairs were hierarchically clustered to test for data dependency. The enzyme set composed of MaeII, BfaI, and BstNI returned much better results than randomly chosen enzyme sets in computer simulations and is therefore recommended for in vitro TRFLP profiling of fungal ITSs.Terminal restriction fragment length polymorphism (TRFLP) profiling was originally developed as a means of genotyping mixed DNA samples (30) and is currently being employed in fungal community ecology studies (3, 5, 6, 7, 10, 13, 19, 22, 26, 27, 29, 33, 38), despite a number of technical and conceptual difficulties (11). Briefly, TRFLP profiling involves amplifying the DNA in pools of mixed genetic material with fluorescently labeled primers, digesting the products with restriction endonucleases, and sizing the labeled terminal fragments in a sequencer. The difference in the positions at which the different restriction enzymes cleave DNA is thought to provide enough variability for such DNA mixtures to be characterized and the contributing organisms to be identified.However, the technique is not without its problems. DNA extraction and PCR amplification biases burden most modern molecular techniques, including TRFLPs (18, 25). Additionally, concerns exist regarding the ability of the differences between primer-enzyme pairs (PEPs) to generate sufficiently different fragment sizes (2), the success of enzymatic cleavage (2), the dependency on the detection threshold of the sequencer (4), and the accuracy of DNA sizing (1). The choice of the primer pairs and restriction enzymes to be used has also been a matter of concern since the appearance of TRFLP profiling. Liu et al. (30) performed virtual digestion of all the bacterial RNA sequences in the Ribosomal Database Project database (release V) with 10 different enzymes and four primer pairs. This pioneering work showed the importance of avoiding enzymes with highly conserved target motifs, something that later became recognized as a major source of TRFLP bias (2, 14, 16, 32). Similar studies have been performed by Osborn et al. (36), Dunbar et al. (12), Engebretson and Moyer (15), and Cardinale et al. (8).The first virtual TRFLP analysis involving a database of fungal DNA sequences was performed by Edwards and Turco (14). This consisted of virtual digestion, by use of six restriction endonucleases, of 316 internally transcribed spacer (ITS) sequences belonging to a number of ectomycorrhizal genera. Avis et al. (2) found only small differences in the diversity of the TRFLPs produced in silico by three PEPs when using their own fungal ITS database, although these differences increased with sample number in iterative analysis. Recent advances using automated resources, such as REPK software (9), have allowed optimal enzyme selection for TRFLP profiling of previously defined communities of organisms. This software selects up to four restriction endonucleases capable of discriminating a desired number of sequence groups. However, this system relies on a priori information, which in real biological communities may not available.The aim of the present work was to improve selection of restriction enzymes for use in the TRFLP profiling of the ITS sequences of unknown fungal communities.     

Sequence Variation in the Ribosomal DNA Internal Transcribed Spacer of Tridacna crocea

DNA-based genetic markers are needed to augment existing allozyme markers in the assessment of genetic diversity of wild giant clam populations. The dearth of polymorphic mitochondrial DNA regions amplified from known universal polymerase chain reaction (PCR) primers has led us to search other regions of the genome for viable sources of DNA polymorphism. We have designed tridacnid-specific PCR primers for the amplification of internal transcribed spacer regions. Sequences of the first internal transcribed spacer segment (ITS-1) revealed very high polymorphism, showing 29% variation arising from base substitutions alone. Preliminary restriction analysis of the ITS regions using 8 restriction enzymes revealed cryptic changes in the DNA sequence. These mutations are promising as marker tools for differentiating geographically separated populations. Such variation in the ITS region can possibly be used for population genetic analysis. Received February 1, 2000; accepted May 8, 2000.     

蓼属头状蓼组rDNA-ITS的序列扩增及分析

以贵州境内蓼属头状蓼组6种(含1变种)植物为材料,对其rDNA的内转录间隔区(ITS)序列进行PCR扩增,得到6种植物的ITS序列,分别为:赤胫散2个居群(Polygonum runcinatum var.sinense,GenBank登录号FJ606887、FJ648802),平卧蓼(P.strindbergii,GenBank登录号FJ648803 ),尼泊尔蓼(P.nepalense,GenBank登录号FJ648804),羽叶蓼(P.runcinatum,GenBank登录号FJ648805),火炭母(P.chinense,GenBank登录号FJ648806)和头花蓼(P.capitatum,GenBank登录号FJ648807).其中赤胫散与平卧蓼的ITS序列为首次报道.序列分析结果表明,蓼属头状蓼组6种植物ITS序列总长度为661～666 bp,ITS1区序列长度为243～246 bp,5.8 S rDNA区序列长度165 bp,ITS2区序列长度253～258 bp,6种植物的差异主要集中在ITS1和ITS2区.聚类分析显示,6种头状蓼组植物具有共同起源,结果支持赤胫散从羽叶蓼变种上升为独立物种.     

Resolution of Prochlorococcus and Synechococcus Ecotypes by Using 16S-23S Ribosomal DNA Internal Transcribed Spacer Sequences

Cultured isolates of the marine cyanobacteria Prochlorococcus and Synechococcus vary widely in their pigment compositions and growth responses to light and nutrients, yet show greater than 96% identity in their 16S ribosomal DNA (rDNA) sequences. In order to better define the genetic variation that accompanies their physiological diversity, sequences for the 16S-23S rDNA internal transcribed spacer (ITS) region were determined in 32 Prochlorococcus isolates and 25 Synechococcus isolates from around the globe. Each strain examined yielded one ITS sequence that contained two tRNA genes. Dramatic variations in the length and G+C content of the spacer were observed among the strains, particularly among Prochlorococcus strains. Secondary-structure models of the ITS were predicted in order to facilitate alignment of the sequences for phylogenetic analyses. The previously observed division of Prochlorococcus into two ecotypes (called high and low-B/A after their differences in chlorophyll content) were supported, as was the subdivision of the high-B/A ecotype into four genetically distinct clades. ITS-based phylogenies partitioned marine cluster A Synechococcus into six clades, three of which can be associated with a particular phenotype (motility, chromatic adaptation, and lack of phycourobilin). The pattern of sequence divergence within and between clades is suggestive of a mode of evolution driven by adaptive sweeps and implies that each clade represents an ecologically distinct population. Furthermore, many of the clades consist of strains isolated from disparate regions of the world's oceans, implying that they are geographically widely distributed. These results provide further evidence that natural populations of Prochlorococcus and Synechococcus consist of multiple coexisting ecotypes, genetically closely related but physiologically distinct, which may vary in relative abundance with changing environmental conditions.     

Identification of Closely Related Hydrobaenus Species (Diptera: Chironomidae) Using the Second Internal Transcribed Spacer (ITS2) Region of Ribosomal DNA

Three species of Japanese Hydrobaenus Fries (H. Kondoi, H. biwaquartus and H. kisosecundus) can be separated by variation in the sequence of the second internal transcribed spacer (ITS2) regions of ribosomal DNA. Suggested synonymy of H. kondoi with H. biwaquartus is refuted, and previously suggested diagnostic morphology (virga length, antennal, leg and venarum ratios, shape of tergite IX and gonostylus length) distinguishes the taxa. No difference in larval morphology was found. H. kisosecundus is readily distinguished on adult and larval morphology from H. kondoi and H. biwaquartus, and is more distant by molecular similarity measures. ITS2 regions apparently provide useful information for distinguishing closely related species in Chironomidae.     

Improved Molecular Detection of Angiostrongylus cantonensis in Mollusks and Other Environmental Samples with a Species-Specific Internal Transcribed Spacer 1-Based TaqMan Assay

Angiostrongylus cantonensis is the most common cause of human eosinophilic meningitis. Humans become infected by ingesting food items contaminated with third-stage larvae that develop in mollusks. We report the development of a real-time PCR assay for the species-specific identification of A. cantonensis in mollusk tissue.Angiostrongylus cantonensis is the most common agent associated with eosinophilic meningitis in humans. Young adult worms develop in the brains of rodents and are carried to pulmonary arteries to reach sexual maturity. Eggs are laid in lung tissues, and first-stage (L1) larvae break into air spaces, migrate to the trachea, are swallowed, and are passed with rodent feces. The L1 larvae must infect mollusks to develop into third-stage (L3) larvae; L3 is the infective stage for rodents and other mammals. Humans become infected by ingesting raw produce contaminated with L3 larvae or infected raw or undercooked mollusks or paratenic hosts. The immature worms remain in the human brain, creating tissue damage and inflammation (2, 19, 21).A. cantonensis is endemic in Southeast Asia, parts of the Caribbean, and the Pacific Islands, including Hawaii (7, 12, 15-17). The worm has been detected in host animals in Louisiana (5, 14) and in one human patient from New Orleans (18), but it is currently unclear to what extent the nematode has spread into other U.S. states (8, 9). Ascertaining the geographic presence of the parasite is important to manage and prevent new cases of eosinophilic meningitis associated with ingestion of infective larvae (12, 18).Detection of A. cantonensis in mollusks can be performed by releasing the larvae from the tissue with pepsin digestion (11). However, that procedure requires access to living mollusks, which complicates analysis of large numbers of samples. After a recent outbreak of angiostrongyliasis in Hawaii (12), we developed a conventional PCR assay and applied it to survey the Hawaiian mollusk population using frozen tissue (20). That PCR assay, as well as morphological identification using pepsin digestion, can only identify the larvae on the superfamily level, so additional molecular work is required for species-specific classification. Here we describe a new real-time PCR assay that allows for a direct detection of A. cantonensis at the species level.The 18S rRNA gene is too conserved among nematode species to allow species-specific detection. The first and second internal transcribed spacers (ITS1 and ITS2) are comparatively more variable than the rRNA coding regions and have thus been used for differentiation of closely related species (1, 4, 6, 10, 22, 23). We PCR amplified and sequenced ITS1 from A. costaricensis (two laboratory strains from Costa Rica and Brazil), A. vasorum (from naturally infected hosts in United Kingdom), and A. cantonensis from three geographical regions (one laboratory strain from Japan plus nine environmental isolates from Hawaii and New Orleans, LA) to assess the variability of this potential PCR target. The oligonucleotide primers used were AngioF1674 (5′-GTCGTAACAAGGTATCTGTAGGTG-3′) and 58SR4 (5′-TAGCTGCGTTTTTCATCGATA-3′). The reaction mixtures contained 0.4 μM each primer and AmpliTaq Gold PCR master mix (Applied Biosystems, Foster City, CA) and were cycled 45 times at 94°C for 30 s, 65°C for 30 s, and 72°C for 1 min. PCR products were cloned into pCR2.1 vectors using the TOPO cloning technique (Invitrogen, Carlsbad, CA) and sequenced on both strands as described elsewhere (20).The sequence analysis revealed high interspecific and low intraspecific variability. A TaqMan assay targeting ITS1 was then designed using Primer Express version 2.3 (Applied Biosystems, Foster City, CA). The real-time PCR assay was performed in a 20-μl total volume containing Platinum qPCR Supermix (Invitrogen, Carlsbad, CA), 0.2 μM (each) primers AcanITS1F1 (5′-TTCATGGATGGCGAACTGATAG-3′) and AcanITS1R1 (5′-GCGCCCATTGAAACATTATACTT-3′), and 0.05 μM the TaqMan probe AcanITS1P1 (5′-6-carboxyfluorescein-ATCGCATATCTACTATACGCATGTGACACCTG-BHQ-3′). The standard cycling conditions for TaqMan assays were used (i.e., 40 cycles of 95°C for 15 s and 60°C for 1 min).We evaluated the real-time PCR assay with a set of 26 Parmarion martensi slugs from Hawaii. Seventeen slugs were positive for L3 larvae as determined by pepsin digestion, and nine slugs were negative. DNA was extracted from approximately 25 mg of tissue of each slug using the DNeasy tissue and blood DNA extraction kit (Qiagen, Inc., Valencia, CA). The real-time PCR performed on this set of samples returned an identical result to the morphological analysis. The real-time PCR amplified only DNA from A. cantonensis and did not react with DNA from other nematode species (Table ​(Table1).1). The detection limit of the assay was determined by serially diluting a recombinant plasmid containing the ITS1 sequence to less than 1 copy per μl of sample. The real-time PCR reliably detected down to 10 plasmid copies in the reaction.

TABLE 1.

Comparison of conventional and real-time PCR for detection of Angiostrongylus cantonensis in mollusks and nematode samples

Length and Sequence Heterogeneity in the Mitochondrial Internal Transcribed Spacer of Acanthamoeba spp.

ABSTRACT. The internal transcribed spacer (ITS) between the mitochondrial large (23S rRNA; rnl ) and small (16S rRNA; rns ) subunit ribosomal RNA genes of Acanthamoeba castellanii strain Neff was sequenced previously and was uniquely interesting because it contained tRNA genes with acceptor stem mismatches that underwent RNA editing repair. Our interest in this ITS region was to determine its phylogenetic potential in differentiating between closely related isolates. We analyzed the mitochondrial ITS region for 17 Acanthamoeba isolates and observed extensive sequence and length variability, making this region difficult to align. Acanthamoeba griffini strain S-7 had the shortest ITS (i.e. 559 base pairs [bp]) compared with Acanthamoeba palestinensis strain Reich, which had the longest (i.e. 1,360 bp). The length disparity occurred predominantly between the spacer region of the aspartic acid ( trnD ) and methionine ( trnM ) tRNA genes. Unexpectedly, this region in A. palestinensis Reich was found to contain a duplication of the trnM gene. Additionally, like A. castellanii strain Neff, all isolates examined had tRNAs with mismatches in their acceptor stem. Also, the potential for an additional type of editing not described previously for Acanthamoeba , involving purine to pyrimidine transversions was observed.     

A Real-Time PCR Method for Quantifying Viable Ascaris Eggs Using the First Internally Transcribed Spacer Region of Ribosomal DNA

Worldwide, 1.4 billion people are infected with the intestinal worm Ascaris lumbricoides. As a result, Ascaris eggs are commonly found in wastewater and sludges. The current microscopy method for detecting viable Ascaris eggs is time- and labor-intensive. The goal of this study was to develop a real-time quantitative PCR (qPCR) method to determine the levels of total and viable Ascaris eggs in laboratory solutions using the first internally transcribed spacer (ITS-1) region of ribosomal DNA (rDNA) and rRNA. ITS-1 rDNA levels were proportional to Ascaris egg cell numbers, increasing as eggs developed from single cells to mature larvae and ultimately reaching a constant level per egg. Treatments causing >99% inactivation (high heat, moderate heat, ammonia, and UV) eliminated this increase in ITS-1 rDNA levels and caused decreases that were dependent on the treatment type. By taking advantage of this difference in ITS-1 rDNA level between viable, larvated eggs and inactivated, single-celled eggs, qPCR results were used to develop inactivation profiles for the different treatments. No statistical difference from the standard microscopy method was found in 75% of the samples (12 of 16). ITS-1 rRNA was detected only in samples containing viable eggs, but the levels were more variable than rDNA levels and ITS-1 rRNA could not be used for quantification. The detection limit of the rDNA-based method was approximately one larvated egg or 90 single-celled eggs; the detection limit for the rRNA-based method was several orders of magnitude higher. The rDNA qPCR method is promising for both research and regulatory applications.     

Nucleotide polymorphisms associated with Internal Transcribed Spacer (ITS) regions of ocular isolates of Aspergillus flavus

PURPOSE: To analyse the genetic similarity among ocular isolates of Aspergillus flavus by Polymerase chain reaction based Restriction Fragment Length Polymorphism (PCR-RFLP) and DNA sequencing. MATERIALS AND METHODS: Seven ocular isolates of A. flavus from 5 patients (3 from paient 1, and four isolates from patients no. 2, 3, 4, and 5 respectively) consisting of 2 Aqueous Humor (AH), 2 Vitreous fluid (VF), 1 eviscerated material, 1 corneal button were included in the study. The three specimens from 1 were one each of AH, VF and corneal button. The fungal isolates were amplified using primers targeting ITS region and the amplicons were subjected to PCR-RFLP using Hae-III enzyme and DNA sequencing to analyse the genetic similarity. RESULTS: A. flavus isolates yielded a specific product of 595 bp after amplification. All the seven A. flavus isolates showed similar pattern of digestion with Hae-III . However, DNA sequencing of ITS amplicons revealed 97.7% genetic similarity and 2.3% dissimilarity with nucleotide polymorphisms -- single, double and multiple pertaining to inversion, substitution, insertion and deletion in comparison with that of standard strain of A. flavus ATCC 16883 [Accession Number ]. A. flavus isolated from AH, VF and corneal button from the same patient showed similar nucleotide polymorphisms as against other isolates which exhibited distinct polymorphisms. This pattern of nucleotide polymorphisms in A. flavus isolates is novel and first time reported in literature to the best of our knowledge. CONCLUSION: DNA sequencing proves to be a useful molecular tool in screening for nucleotide polymorphisms among fungal isolates.     

Intragenomic Profiling Using Multicopy Genes: The rDNA Internal Transcribed Spacer Sequences of the Freshwater Sponge Ephydatia fluviatilis

Multicopy genes, like ribosomal RNA genes (rDNA), are widely used to describe and distinguish individuals. Despite concerted evolution that homogenizes a large number of rDNA gene copies, the presence of different gene variants within a genome has been reported. Characterization of an organism by defining every single variant of tens to thousands of rDNA repeat units present in a eukaryotic genome would be quite unreasonable. Here we provide an alternative approach for the characterization of a set of internal transcribed spacer sequences found within every rDNA repeat unit by implementing direct sequencing methodology. The prominent allelic variants and their relative amounts characterizing an individual can be described by a single sequencing electropherogram of the mixed amplicon containing the variants present within the genome. We propose a method for rational analysis of heterogeneity of multicopy genes by compiling a profile based on quantification of different sequence variants of the internal transcribed spacers of the freshwater sponge Ephydatia fluviatilis as an example. In addition to using conventional substitution analysis, we have developed a mathematical method, the proportion model method, to quantify the relative amounts of allelic variants of different length using data from direct sequencing of the heterogeneous amplicon. This method is based on determining the expected signal intensity values (corresponding to peak heights from the sequencing electropherogram) by sequencing clones from the same or highly similar amplicon and comparing hypothesized combinations against the values obtained by direct sequencing of the heterogeneous amplicon. This method allowed to differentiate between all specimens analysed.     

Phylogenetic Analysis ofBambusa (Poaceae: Bambusoideae) Based on Internal Transcribed Spacer Sequences of Nuclear Ribosomal DNA

Phylogenetic analyses of Bambusa species were performed using internal transcribed spacer sequences of nuclear ribosomal DNA. The 21 species sampled included members of Bambusa (sensu stricto), Dendrocalamopsis, Dendrocalamus, Guadua, Leleba, and Lingnania. Arundinaria gigantea was used as an outgroup. Using the maximum parsimony method with PAUP*, gaps were treated as missing states or new states. Parsimonious analysis revealed that Dendrocalamus latiflorus was closely related to the members of Dendrocalamopsis. Dendrocalamus membranaceus was a sister species to Dendrocalamus strictus. Three Dendrocalamus species were closely related to and nested in a polyphyletic Bambusa. Bambusa subaequalis was a sister species to B. multiplex, B. emeiensis to B. chungii, B. contracta to B. hainanensis, and B. flexuosa was a sister species to B. sinospinosa, B. tuldoides, B. surrecta, B. intermedia, and B. valida group, which raised doubts about the monophyly of the subgenera Bambusa (sensu stricto), Dendrocalamopsis, Leleba, and Lingnania under the genus Bambusa.     

Molecular Evolution and Phylogenetic Utility of the Internal Transcribed Spacer 2 (ITS2) in Calyptratae (Diptera: Brachycera)

The resolution potential of internal transcribed spacer 2 (ITS2) at deeper levels remains controversial. In this study, 105 ITS2 sequences of 55 species in Calyptratae were analyzed to examine the phylogenetic utility of the spacer above the subfamily level and to further understand its evolutionary characteristics. We predicted the secondary structure of each sequence using the minimum-energy algorithm and constructed two data matrixes for phylogenetic analysis. The ITS2 regions of Calyptratae display strong A-T bias and slight variation in length. The tandem and dispersed repeats embedded in the spacers possibly resulted from replication slippage or transposition. Most foldings conformed to the four-domain model. Sequence comparison in combination with the secondary structures revealed six conserved motifs. Covariation analysis from the conserved motifs indicated that the secondary structure restrains the sequence evolution of the spacer. The deep-level phylogeny derived from the ITS2 data largely agreed with the phylogenetic hypotheses from morphologic and other molecular evidence. Our analyses suggest that the accordant resolutions generated from different analyses can be used to infer deep-level phylogenetic relations.