我国赫坎按蚊复合体成员种的rDNA-ITS2区序列差异及系统发育分析

测定了我国赫坎按蚊复合体 9成员种的核糖体DNA第二内转录间隔区 (rDNA ITS2 )序列 ,根据序列差异分析各蚊种间的系统发育关系。结果显示 :( 1 )ITS2区序列最长的是中华按蚊 ( 4 6 8bp) ,最短的是克劳按蚊和赫坎按蚊 ( 4 36bp) ;GC含量为 4 4 9%～ 4 6 8% ;( 2 )发现该复合体 4成员种的ITS2区序列存在种内个体间差异 ,幅度为 0～ 3 8% ,明显小于种间差异 ;( 3)将各蚊种的ITS2区序列进行同源排序比较 ,发现其变异大多是简单重复单元的拷贝数不同 ;种间差异性最大的是克劳按蚊与嗜人按蚊( 32 3% ) ,最小的是贵阳按蚊与凉山按蚊 ( 9 0 % )平均差异率为 2 2 3% ;( 4 )根据ITS2区序列特征 ,用 3种方法构建的树状图拟合一致。以上结果表明赫坎按蚊复合体各成员种rDNA ITS2序列在种内非常保守 ,以种间序列差异分析为基础的分子鉴别技术是甄别蚊种分类地位混淆和错误的有效方法。     

Sequence Variation of Ribosomal Internal Transcribed Spacers (ITS) in Commercially Important Phytoseiidae Mites

Preliminary work is needed to assess the usefulness of different markers at different taxonomic scales when a new group is analyzed, such as the commercially important Phytoseiidae mites. We investigate here the level of sequence variation of the nuclear ribosomal spacers ITS 1 and 2 and the 5.8S gene in six species of Phytoseiidae: Neoseiulus californicus, N. fallacis, Euseius concordis, Metaseiulus occidentalis, Typhlodromus pyri and Phytoseiulus persimilis. As expected, the 5.8S gene (148 base pairs) is markedly conserved and displays little variation in between genera comparisons. ITS1 and ITS2 show contrasting patterns: while the ITS2 is short (80–89 bp) and shows little variation, the ITS1 is longer (303–404 bp) and is very variable in sequence. This fact compromises reliable nucleotide homologies when comparing the genera. The comparison of ITS1 sequence similarity at the species level might be useful for species identification, however, the value of ITS in taxonomic studies does not extend to the level of the family. The intraspecific variations of ITS were investigated in three species: N. californicus, N. fallacis and E. concordis. The first species has identical ITS1 sequences and the last two display low polymorphism (2 nucleotide substitutions). The ITS2 and 5.8S sequences were identical in all three subspecies comparisons.     

韩国赫坎按蚊复合体3成员种核糖体DNA内转录间隔2区的比较(英文)

本文对韩国中华按蚊、雷氏按蚊和八代按蚊核糖体DNA (rDNA)内转录间隔 2区 (ITS2 )序列进行了比较研究。用PCR扩增的rDNA ITS2片段直接测序 ,每种蚊测定 3个个体 ,结果显示 :韩国中华按蚊、雷氏按蚊和八代按蚊的rDNA ITS2序列长度分别为 4 6 8bp、 4 51bp和 4 53bp ,GC含量分别为 4 4 .87%、 4 6 .2 %和 4 5.7% ,3种按蚊序列差异范围为 12 .16 %— 30 .74 %。研究表明 ,rDNA ITS2序列差异可用于韩国中华按蚊、雷氏按蚊和八代按蚊的分子鉴别。     

DIFFERENCES IN SEQUENCES OF RIBOSOMAL DNA SECOND INTERNAL TRANSCRIBED SPACER AMONG THREE MEMBERS OF ANOPHELES HYRCANUS COMPLEX FROM THE REPUBLIC OF KOREA

Abstract  Differences in sequences of ribosomal DNA second internal transcribed spacer (ITS) among Anopheles sinensis, An. lesteri and An. yatsushiroensis from Korea were compared. The PCR amplified rDNA-ITS2 fragments were sequenced directly. Three samples of each species were individually determined.
Lengths of the ITS2 regions were 468bp in An sinensis , 451bp in An lesteri and 453bp in An yatsushiroenesis and GC contents were 44.87 % 46.2 % and 45.7 % respectively. Variations of the sequences ranged from 12.16 % to 30.7 % among the three species. The differences of the rDNA-ITS2 sequences would be useful for molecular identification of the three members of Anopheles hycanus complex from Korea.     

ITS-2 secondary structures and phylogeny of Anopheles culicifacies species

Background: Second internal transcribed spacer (ITS2) has proven to contain useful biological information at higher taxonomic levels. Objectives: This study was carried out to unravel the biological information in the ITS2 region of An. culicifacies and the internal relationships between the five species of Anopheles culicifacies. Methodology: In achieving these objectives, twenty two ITS2 sequences (~370bp) of An. culicifacies species were retrieved from GenBank and secondary structures were generated. For the refinement of the primary structures, i.e. nucleotide sequence of ITS2 sequences, generated secondary structures were used. The improved ITS2 primary structures sequences were then aligned and used for the construction of phylogenetic trees. Results and discussions: ITS2 secondary structures of culicifacies closely resembled near universal eukaryotes secondary structure and had three helices, and the structures of helix II and distal region of helix III of ITS2 of An. culicifacies were strikingly similar to those regions of other organisms strengthening possible involvement of these regions in rRNA biogenesis. Phylogenetic analysis of improved ITS2 sequences revealed two main clades one representing sibling B, C and E and A and D in the other. Conclusions: Near sequence identity of ITS2 regions of the members in a particular clade indicate that this region is undergoing parallel evolution to perform clade specific RNA biogenesis. The divergence of certain isolates of An. culicifacies from main clades in phylogenetic analyses suggests the possible existence of camouflaged sub-species within the complex of culicifacies. Using the fixed nucleotide differences, we estimate that these two clades have diverged nearly 3.3 million years ago, while the sibling species in clade 2 are under less evolutionary pressure, which may have evolved much later than the members in clade 1.     

The utility of internally transcribed spacer 2 DNA sequences of the nuclear ribosomal gene for distinguishing sibling species of Trichogramma

The usefulness of the internally transcribed spacer 2 (ITS2) of the nuclear ribosomal gene complex is tested for providing taxonomic characters to identify Trichogramma species. The ITS2 sequences of a group of sibling species of the T. deion/T. pretiosum complexes were determined. A simple and precise identification key to the species of these assemblages was constructed using as taxonomic characters the size of the ITS2 and the difference in restriction length polymorphism of species with similarly sized ITS2. Individual wasps can be identified by amplification of their ITS2 with general primers, determining the size of the PCR product using standard agarose electrophoresis, followed in some species by a DNA-digestion with a restriction enzyme. Because this system works well for a number of closely related species we are hopeful that similar PCR-based identification can be extended to all species of the genus once their ITS2 sequences have been determined. The advantage of this identification system over the morphology-based system is that non-specialists are able to quickly and cheaply identify individual specimens. In addition, species specific primers were tested for the two most common species of these groups (i.e. T. pretiosum and T. deion). These primers can be used either as a direct identification tool or as a method to confirm the identification using the general key. The phylogeny of this group of wasps was also analyzed based on the ITS2 sequence.     

Clustering of fungal community internal transcribed spacer sequence data obscures taxonomic diversity

Next‐generation DNA sequencing has enabled a rapid expansion in the size of molecular fungal ecology studies employing the nuclear internal transcribed spacer (ITS) region. Many sequence‐processing pipelines and protocols require sequence clustering to generate operational taxonomic units (OTUs) based on sequence similarity as a step to reduce total data quantity and complexity prior to taxonomic assignment. However, the consequences of ITS sequence clustering in regard to sample taxonomic coverage have not been carefully examined. Here we demonstrate that typically used clustering thresholds for fungal ITS sequences result in statistically significant losses in taxonomic coverage. Analyses using environmentally derived fungal sequences indicated an average of 3.1% of species went undetected (P < 0.05) if the sequences were denoised and clustered at a 97% threshold prior to taxonomic assignment. Additionally, an in silico analysis using a reference fungal ITS database suggested that approximately 25% of species went undetected if the sequences were clustered prior to taxonomic assignment. Finally, analysis of sequences derived from pure‐cultured fungal isolates of known identity indicated sequence denoising and clustering were not critical in improving identification accuracy.     

DNA barcodes identify Chinese medicinal plants and detect geographical patterns of Sinosenecio (Asteraceae)

DNA barcodes have proved to be efficient for plants species discrimination and identification using short and standardized genomic regions. The genus Sinosenecio(Asteraceae) is used for traditional medicinal purposes in China. Most species of the genus occur in restricted geographical regions and exhibit a wide range of morphological variations within species, making them difficult to differentiate in the field. Previously, taxonomic revisions have been made based on morphological and cytological evidence. In the present study, barcoding analysis was performed on 107 individuals representing 38 species in this genus to evaluate the performance of four candidate barcoding loci (matK, rbcL, trnH-psbA and internal transcribed spacer [ITS]) and detect geographical patterns. Three different methods based on genetic distance, sequence similarity, and the phylogenetic tree were used. Comparably high species discrimination power was detected in species-level taxonomic process by the ITS dataset alone or combined with other loci, which was suggested to be the most suitable barcode for Sinosenecio. Our results are congruent with previous taxonomic studies concerning the monophyly of the S. oldhamianus group. The present study provides an empirical paradigm for the identification of medicinal plant species and their geographical patterns, ascertaining the congruence between taxonomical studies and barcoding analysis inSinosenecio.     

荒川库蠓rDNA ITS2的序列测定与分析

库蠓种类繁多,给人畜健康带来巨大危害.荒川库蠓(Culicoides arakawae)是卡氏住白虫的重要传播媒介,引起鸡的卡氏住白虫病,导致蛋鸡产蛋量下降、肉鸡体重下降甚至死亡(Li et al.,2000),给养鸡业带来巨大经济损失.如何对各种媒介库蠓进行正确的区分或鉴定,是摆在各位寄生虫学家面前的具体问题.目前,寄生虫的分类鉴别主要依据形态学特征,一旦遇到形态学相似的近缘种就非常棘手.     

A Nucleotide Signature for Identification of Aglaia stellatopilosa Pannell

Members of the genus Aglaia have been reported to contain bioactive phytochemicals. The genus, belonging to the Meliaceae family, is represented by at least 120 known species of woody trees or shrubs in the tropical rain forest. As some of these species are very similar in their morphology, taxonomic identification can be difficult. A reliable and definitive molecular method which can identify Aglaia to the level of the species will hence be useful in comparing the content of specific bioactive compounds between the species of this genus. Here, we report the analysis of DNA sequences in the internal transcribed spacer (ITS) of the nuclear ribosomal DNA and the observation of a unique nucleotide signature in the ITS that can be used for the identification of Aglaia stellatopilosa. The nucleotide signature consists of nine bases over the length of the ITS sequence (654 bp). This uniqueness was validated in 37 samples identified as Aglaia stellatopilosa by an expert taxonomist, whereas the nucleotide signature was lacking in a selection of other Aglaia species and non-Aglaia genera. This finding suggests that molecular typing could be utilized in the identification of Aglaia stellatopilosa.     

Towards a unified paradigm for sequence‐based identification of fungi

The nuclear ribosomal internal transcribed spacer (ITS) region is the formal fungal barcode and in most cases the marker of choice for the exploration of fungal diversity in environmental samples. Two problems are particularly acute in the pursuit of satisfactory taxonomic assignment of newly generated ITS sequences: (i) the lack of an inclusive, reliable public reference data set and (ii) the lack of means to refer to fungal species, for which no Latin name is available in a standardized stable way. Here, we report on progress in these regards through further development of the UNITE database ( http://unite.ut.ee ) for molecular identification of fungi. All fungal species represented by at least two ITS sequences in the international nucleotide sequence databases are now given a unique, stable name of the accession number type (e.g. Hymenoscyphus pseudoalbidus|GU586904|SH133781.05FU), and their taxonomic and ecological annotations were corrected as far as possible through a distributed, third‐party annotation effort. We introduce the term ‘species hypothesis’ (SH) for the taxa discovered in clustering on different similarity thresholds (97–99%). An automatically or manually designated sequence is chosen to represent each such SH. These reference sequences are released ( http://unite.ut.ee/repository.php ) for use by the scientific community in, for example, local sequence similarity searches and in the QIIME pipeline. The system and the data will be updated automatically as the number of public fungal ITS sequences grows. We invite everybody in the position to improve the annotation or metadata associated with their particular fungal lineages of expertise to do so through the new Web‐based sequence management system in UNITE.     

DNA barcodes and microsatellites: How they complement for species identification in the complex genus Tamarix (Tamaricaceae)

DNA barcoding allows the identification of an organism by comparing the sequence of selected DNA regions (barcodes) with a previously compiled database, and it can be useful for taxonomic identification of species in complex genera, such as Tamarix. Many species of this genus show convergent morphology, which leads to frequent errors in their identification. Highly variable genetic markers, such as microsatellites or short sequence repeats (SSR), could be used to differentiate species where DNA barcodes fail. Here, we tested the ability of both, 5 different marker regions (rbcL, matK, ITS, trnH-psbA, and ycf1), and 14 microsatellites, to properly identify Tamarix species, especially those from the Mediterranean Basin, and compared the pros and cons of the different analytical methods for species identification. DNA barcoding allows the genetic identification of certain species in Tamarix. The two-locus barcodes matK + ITS and ITS + ycf1 were the best-performing combinations, allowing up to 69% and 70%, respectively, correct identification. However, DNA barcoding failed in phylogenetically close groups, such as many Mediterranean species. The use of SSR can aid the identification of species, and the combination of both types of data (DNA barcoding and SSR) improved the success. The combination of data was especially relevant in detecting the presence of hybridization processes, which are common in the genus. However, caution must be exercised when choosing the clustering methods for the SSR datasince different methods can lead to very different results.     

Intragenomic rDNA ITS2 variation in the neotropical Anopheles (Nyssorhynchus) albitarsis complex (Diptera: Culicidae)

We cloned and sequenced the rDNA internal transcribed spacer 2 (ITS2) of 4 species belonging to the neotropical Anopheles (Nyssorhynchus) albitarsis complex, that is, A. albitarsis; A. albitarsis B; Anopheles marajoara, a proven malaria vector; and Anopheles deaneorum, a suspected vector. Even though the ITS2 sequences of these species were very similar (< or =1.17% divergence), we found differences suitable for species identification and intragenomic variation of possible consequence in phylogenetic reconstruction. Variation came from 2 microsatellite regions and a number of indels and base substitutions. The existence of partially correlated subsets of clones in A. albitarsis is hypothesized either to be separate rDNA loci or to be semi-independently evolving portions of a single rDNA locus. No differences were found between males and females, suggesting that similar rDNA arrays exist on both the X and Y chromosomes. In addition, highly variant clones, possibly pseudogenes, were found in A. marajoara from Venezuela.     

Amplicon –Based Metagenomic Analysis of Mixed Fungal Samples Using Proton Release Amplicon Sequencing

Next generation sequencing technology has revolutionised microbiology by allowing concurrent analysis of whole microbial communities. Here we developed and verified similar methods for the analysis of fungal communities using a proton release sequencing platform with the ability to sequence reads of up to 400 bp in length at significant depth. This read length permits the sequencing of amplicons from commonly used fungal identification regions and thereby taxonomic classification. Using the 400 bp sequencing capability, we have sequenced amplicons from the ITS1, ITS2 and LSU fungal regions to a depth of approximately 700,000 raw reads per sample. Representative operational taxonomic units (OTUs) were chosen by the USEARCH algorithm, and identified taxonomically through nucleotide blast (BLASTn). Combination of this sequencing technology with the bioinformatics pipeline allowed species recognition in two controlled fungal spore populations containing members of known identity and concentration. Each species included within the two controlled populations was found to correspond to a representative OTU, and these OTUs were found to be highly accurate representations of true biological sequences. However, the absolute number of reads attributed to each OTU differed among species. The majority of species were represented by an OTU derived from all three genomic regions although in some cases, species were only represented in two of the regions due to the absence of conserved primer binding sites or due to sequence composition. It is apparent from our data that proton release sequencing technologies can deliver a qualitative assessment of the fungal members comprising a sample. The fact that some fungi cannot be amplified by specific “conserved” primer pairs confirms our recommendation that a multi-region approach be taken for other amplicon-based metagenomic studies.     

Molecular differentiation of three closely related members of the mosquito species complex, Anopheles moucheti, by mitochondrial and ribosomal DNA polymorphism

Distinction between members of the equatorial Africa malaria vector Anopheles moucheti (Evans) s.l. (Diptera: Culicidae) has been based mainly on doubtful morphological features. To determine the level of genetic differentiation between the three morphological forms of this complex, we investigated molecular polymorphism in the gene encoding for mitochondrial cytochrome oxidase b (CytB) and in the ribosomal internal transcribed spacers (ITS1 and ITS2). The three genomic regions revealed sequence differences between the three morphological forms similar in degree to the differences shown previously for members of other anopheline species groups or complexes (genetic distance d = 0.047-0.05 for CytB, 0.084-0.166 for ITS1 and 0.03-0.05 for ITS2). Using sequence variation in the ITS1 region, we set up a diagnostic polymerase chain reaction (PCR) for rapid and reliable identification of each subspecies within the An. moucheti complex. Specimens of An. moucheti s.l. collected in Cameroon, the Democratic Republic of Congo (DRC), Uganda and Nigeria were successfully identified, demonstrating the general applicability of this technique.     

Species identification of Alnus (Betulaceae) using nrDNA and cpDNA genetic markers

One nuclear and three chloroplast DNA regions (ITS, rbcL, matK and trnH-psbA) were used to identify the species of Alnus (Betulaceae). The results showed that 23 out of all 26 Alnus species in the world, represented by 131 samples, had their own specific molecular character states, especially for three morphologically confused species (Alnus formosana, Alnus japonica and Alnus maritima). The discriminating power of the four markers at the species level was 10% (rbcL), 31.25% (matK), 63.6% (trnH-psbA) and 76.9% (ITS). For ITS, the mean value of genetic distance between species was more than 10 times the intraspecific distance (0.009%), and 13 species had unique character states that differentiated them from other species of Alnus. The trnH-psbA region had higher mean values of genetic distance between and within species (2.1% and 0.68% respectively) than any other region tested. Using the trnH-psbA region, 13 species are distinguished from 22 species, and seven species have a single diagnostic site. The combination of two regions, ITS and trnH-psbA, is the best choice for DNA identification of Alnus species, as an improvement and supplement for morphologically based taxonomy. This study illustrates the potential for certain DNA regions to be used as novel internet biological information carrier through combining DNA sequences with existing morphological character and suggests a relatively reliable and open taxonomic system based on the linked DNA and morphological data.     

Diversity and phylogenetic affinities of foliar fungal endophytes in loblolly pine inferred by culturing and environmental PCR

We examined endophytic fungi in asymptomatic foliage of loblolly pine (Pinus taeda) in North Carolina, U.S.A., with four goals: (i) to evaluate morphotaxa, BLAST matches and groups based on sequence similarity as functional taxonomic units; (ii) to explore methods to maximize phylogenetic signal for environmental datasets, which typically contain many taxa but few characters; (iii) to compare culturing vs. culture-free methods (environmental PCR of surface sterilized foliage) for estimating endophyte diversity and species composition; and (iv) to investigate the relationships between traditional ecological indices (e.g. Shannon index) and phylogenetic diversity (PD) in estimating endophyte diversity and spatial heterogeneity. Endophytes were recovered in culture from 87 of 90 P. taeda leaves sampled, yielding 439 isolates that represented 24 morphotaxa. Sequence data from the nuclear ribosomal internal transcribed spacer (ITS) for 150 isolates revealed 59 distinct ITS genotypes that represented 24 and 37 unique groups based on 90% and 95% sequence similarity, respectively. By recoding ambiguously aligned regions to extract phylogenetic signal and implementing a conservative phylogenetic backbone constraint, we recovered well supported phylogenies based on ca. 600 bp of the nuclear ribosomal large subunit (LSUrDNA) for 72 Ascomycota and Basidiomycota, 145 cultured endophytes and 33 environmental PCR samples. Comparisons with LSUrDNA-delimited species showed that morphotaxa adequately estimated total species richness but rarely corresponded to biologically meaningful groups. ITS BLAST results were variable in their utility, but ITS genotype groups based on 90% sequence similarity were concordant with LSUrDNA-delimited species. Environmental PCR yielded more genotypes per sampling effort and recovered several distinct clades relative to culturing, but some commonly cultured clades were never found (Sordariomycetes) or were rare relative to their high frequency among cultures (Leotiomycetes). In contrast to traditional indices, PD demonstrated spatial heterogeneity in endophyte assemblages among P. taeda trees and study plots. Our results highlight the need for caution in designating taxonomic units based on gross cultural morphology or ITS BLAST matches, the utility of phylogenetic tools for extracting robust phylogenies from environmental samples, the complementarity of culturing and environmental PCR, the utility of PD relative to traditional ecological indices, and the remarkably high diversity of foliar fungal endophytes in this simplified temperate ecosystem.     

Ribosomal DNA difference between species A and D of the Anopheles dims complex of mosquitoes from China

Abstract. Species A and D of the Anopheles dints complex were found in China. Ribosomal DNA second internal transcribed spacers (ITS2) of both species A and D were sequenced and found to be 716 and 710 base-pairs in length, respectively, with 699c GC content. No evidence of intraspecific variation was detected in the ITS2 sequence of species A, whereas the sequence of species D showed variation at one position in the ITS2. A large number of simple repeat motifs were dispersed throughout the ITS2 sequences. The level of interspecific difference was 5.4% of the nucleotide sequences. Some of the interspecific differences were located in regions with subrepeat structure.     

Cryptic species within Anopheles longipalpis from southern Africa and phylogenetic comparison with members of the An. funestus group

House-resting Anopheles mosquitoes are targeted for vector control interventions; however, without proper species identification, the importance of these Anopheles to malaria transmission is unknown. Anopheles longipalpis, a non-vector species, has been found in significant numbers resting indoors in houses in southern Zambia, potentially impacting on the utilization of scarce resources for vector control. The identification of An. longipalpis is currently based on classical morphology using minor characteristics in the adult stage and major ones in the larval stage. The close similarity to the major malaria vector An. funestus led to investigations into the development of a molecular assay for identification of An. longipalpis. Molecular analysis of An. longipalpis from South Africa and Zambia revealed marked differences in size and nucleotide sequence in the second internal transcribed spacer (ITS2) region of ribosomal DNA between these two populations, leading to the conclusion that more than one species was being analysed. Phylogenetic analysis showed the Zambian samples aligned with An. funestus, An. vaneedeni and An. parensis, whereas the South African sample aligned with An. leesoni, a species that is considered to be more closely related to the Asian An. minimus subgroup than to the African An. funestus subgroup. Species-specific primers were designed to be used in a multiplex PCR assay to distinguish between these two cryptic species and members of the An. funestus subgroup for which there is already a multiplex PCR assay.     

Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences.

Laccate polypores of the Ganoderma lucidum species complex are widespread white rot fungi of economic importance, but isolates cannot be identified by traditional taxonomic methods. Parsimony analysis of nucleotide sequences from the internal transcribed spacers (ITS) of the ribosomal gene (rDNA) distinguished six lineages in this species complex. Each ITS lineage may represent one or more putative species. While some isolates have identical ITS sequences, all of them could be clearly differentiated by genetic fingerprinting using random amplified polymorphic DNA (RAPD). To investigate the suitability of RAPD markers for taxonomic identification and grouping of isolates of the G. lucidum complex, RAPD fragments (RAPDs) were used as phenotypic characters in numerical and parsimony analyses. Results show that data from RAPDS do not distinguish the same clades as ITS data do. Groupings based on analysis of RAPD data were very sensitive to the choice of the grouping method used, and no consistent grouping of isolates could be proposed. However, analysis with RAPDs did resolve several robust terminal clades containing putatively conspecific isolates, suggesting that RAPDs might be helpful for systematics at the lower taxonomic levels that are unresolved by ITS sequence data. The limitations of RAPDs for systematics are briefly discussed. The conclusion of this study is that ITS sequences can be used to identify isolates of the G. lucidum complex, whereas RAPDs can be used to differentiate between isolates having identical ITS sequences. The practical implications of these results are briefly illustrated.