Multiple polymorphic sites at the ITS and ATAN loci in cultured isolates of Perkinsus marinus

Sequence analysis of genomic DNA from the protozoan parasite Perkinsus marinus at two loci revealed genetic polymorphisms within and among different cultured isolates. Genomic DNA from 12 Perkinsus marinus isolates was amplified at the internal transcribed spacer region and at an anonymous locus previously identified to contain polymorphisms by restriction fragment length polymorphism analysis. Fourteen polymorphic nucleotide positions were identified at the internal transcribed spacer region; eight in internal transcribed spacer 1 and six in internal transcribed spacer 2. Thirteen polymorphic nucleotide sites were identified within the anonymous locus. In some instances, more than three different sequences were observed at both the internal transcribed spacer region and at the anonymous locus from a single clonal isolate, suggesting the possibility of recombination in cultured cells and/or strand jumping during the polymerase chain reaction. Intra-isolate sequence variation (3.46% for the anonymous locus and 3.08% for internal transcribed spacer 1) was in several cases as high as inter-isolate sequence variation, even in one isolate where recombination was not evident. High intra- and inter-isolate variation detected at both loci demonstrates the importance of determining the genetic variation of each locus prior to development of sequence-based molecular diagnostics.     

Identification of herbal medicinal materials using DNA barcodes

Herbal medicinal materials have been used worldwide for centuries to maintain health and to treat disease. However, adulteration of herbal medicines remains a major concern of users and industry for reasons of safety and efficacy. Identification of herbal medicinal materials by DNA technology has been widely applied,started from the mid-1990s. In recent years, DNA barcoding of global plant species using four standard barcodes (rbcL, matK, trnH-psbA and ITS) has been a major focus in the fields of biodiversity and conservation. These DNA barcodes can also be used as reliable tools to facilitate the identification of herbal medicinal materials for the safe use of herbs, quality control, and forensic investigation. Many studies have applied these DNA barcodes for the identification of herbal medicinal species and their adulterants. The present article reviews efforts in the identification of herbal medicinal materials using the standard DNA barcodes and other DNA sequence-based markers.     

Molecular prospecting for cryptic species of nematodes: mitochondrial DNA versus internal transcribed spacer

DNA sequence divergence at internal transcribed spacer regions (ITS-1 and ITS-2) was compared with divergence at mitochondrial cox1 or nad4 loci in pairs of congeneric nematode species. Mitochondrial sequences accumulate substitutions much more quickly than internal transcribed spacer, the difference being most striking in the most closely related species pairs. Thus, mitochondrial DNA may be the best choice for applications in which one is using sequence data on small numbers of individuals to search for potential cryptic species. On the other hand, internal transcribed spacer remains an excellent tool for DNA diagnostics (quickly distinguishing between known species) owing to its lower level of intraspecific polymorphism.     

GC balance in the internal transcribed spacers ITS 1 and ITS 2 of nuclear ribosomal RNA genes

Summary The internal transcribed spacer (ITS) 1 and 2, the 5.8S rRNA gene, and adjacent 18S rRNA and 25S rRNA coding regions of two Cucurbitaceae (Cucurbita pepo, zucchini, ITS 1: 187 bp, and ITS 2: 252 bp in length, andCucumis sativus, cucumber, ITS 1: 229 bp, and ITS 2: 245 bp in length) have been sequenced. The evolutionary pattern shown by the ITSs of these plants is different from that found in vertebrates. Deletions, insertions, and base substitutions have occurred in both spacers; however, it is obvious that some selection pressure is responsible for the preservation of stem-loop structures. The dissimilarity of the 5 region of ITS 2 found in higher plants has consequences for proposed models on U3 snRNA-ITS 2 interaction in higher eukaryotes.The two investigated Cucurbitaceae species show a G+C content of ITS 1 that nearly equals that of ITS 2. An analysis of the ITS sequences reveals that in 19 out of 20 organisms published, the G+C content of ITS 1 nearly equals that of ITS 2, although it ranges from 20% to 90% in different organisms (GC balance). Moreover, the balanced G+C content of the ITSs in a given species seems to be similar to that of so-called expansion segments (ESs) in the 25/28S rRNA coding region. Thus, ITSs show a phenomenon called molecular coevolution with respect to each other and to the ESs. In the ITSs of Cucurbitaceae the balanced G+C composition is at least partly achieved by C to T transitions, via deamination of 5-methylcytosine. Other mutational events must be taken into account. The appearance of this phenomenon is discussed in terms of functional constraints linked to the structures of these spacers.     

Phylogeny of calcareous dinoflagellates as inferred from ITS and ribosomal sequence data

The phylogenetic relationships of calcareous dinoflagellates (i.e., Calciodinellaceae and Thoracosphaera) are investigated. Molecular data from the ribosomal 5.8S rRNA and highly conserved motifs of the ITS1 show Calciodinellaceae s.l. to be monophyletic when few non-calcareous taxa are included. They segregate into three monophyletic assemblages in a molecular analysis that considers the 5.8S rRNA and both the Internal Transcribed Spacer regions ITS1 and ITS2: a clade comprising species of Ensiculifera and Pentapharsodinium (E/P-clade), Scrippsiella s.l. (including fossil-based taxa such as Calciodinellum and Calcigonellum), and a heterogeneous group (T/P-clade) of calcareous (e.g., Thoracosphaera) and non-calcareous taxa (e.g., the highly toxic Pfiesteria). The potential to produce calcareous structures is considered as apomorphic within alveolates, and non-calcareous taxa nesting with calcareous dinoflagellates may have reduced calcification secondarily. Molecular results do not contradict general evolutionary scenarios provided by previous morphological (mainly paleontological) investigations.     

Comprehensive DNA barcodes for species identification and discovery of cryptic diversity in mayfly larvae from South Korea: Implications for freshwater ecosystem biomonitoring

DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing baseline reference for phylogenetic analysis and aquatic ecosystem biomonitoring. We obtained 112 novel sequences of the barcode region of the mitochondrial DNA cytochrome c oxidase subunit I gene representing 11 families, 25 genera, and 43 species of mayfly (Insecta: Ephemeroptera) from South Korea. No species shared barcode sequences and all can be identified with barcodes with a possible exception of some species. Minimum levels of interspecific genetic distances ranged from 6.7 to 32.9% (mean: 23.7%), whereas average levels of intraspecific divergence was 3.7%. The latter value was inflated by the presence of very high divergences within some taxa. In fact, approximately 33.3% (15/45) of the species included two or more haplotype clusters showing greater than 5.0% sequence divergence and some values were as high as 32.9%. Many of the species with high intraspecific divergences are para‐ or polyphyletic and represent the possibility of species complexes. Our study suggests that type or topotype specimens should be sequenced to identify accurate barcoding clusters with morphological species concepts and also to determine the status of currently synonymized species.     

Identification of species in the angiosperm family Apiaceae using DNA barcodes

Apiaceae (Umbelliferae) is a large angiosperm family that includes many medicinally important species. The ability to identify these species and their adulterants is important, yet difficult to do so because of their subtle fruit morphological differences and often lack of diagnostic features in preserved specimens. Moreover, dried roots are often the official medical organs, making visual identification to species almost impossible. DNA barcoding has been proposed as a powerful taxonomic tool for species identification. The Consortium for the Barcode of Life (CBOL) Plant Working Group has recommended the combination of rbcL+matK as the core plant barcode. Recently, the China Plant BOL Group proposed that the nuclear ribosomal DNA internal transcribed spacer (ITS), as well as a subset of this marker (ITS2), be incorporated alongside rbcL+matK into the core barcode for seed plants, particularly angiosperms. In this study, we assess the effectiveness of these four markers plus psbA‐trnH as Apiaceae barcodes. A total of 6032 sequences representing 1957 species in 385 diverse genera were sampled, of which 211 sequences from 50 individuals (representing seven species) were newly obtained. Of these five markers, ITS and ITS2 showed superior results in intra‐ and interspecific divergence and DNA barcoding gap assessments. For the matched data set (173 samples representing 45 species in five genera), the ITS locus had the highest identification efficiency (73.3%), yet ITS2 also performed relatively well with 66.7% identification efficiency. The identification efficiency increased to 82.2% when using an ITS+psbA‐trnH marker combination (ITS2+psbA‐trnH was 80%), which was significantly higher than that of rbcL+matK (40%). For the full sample data set (3052 ITS sequences, 3732 ITS2 sequences, 1011 psbA‐trnH sequences, 567 matK sequences and 566 rbcL sequences), ITS, ITS2, psbA‐trnH, matK and rbcL had 70.0%, 64.3%, 49.5%, 38.6% and 32.1% discrimination abilities, respectively. These results confirm that ITS or its subset ITS2 be incorporated into the core barcode for Apiaceae and that the combination of ITS/ITS2+psbA‐trnH has much potential value as a powerful, standard DNA barcode for Apiaceae identification.     

DNA barcodes for Mexican Cactaceae, plants under pressure from wild collecting

DNA barcodes could be a useful tool for plant conservation. Of particular importance is the ability to identify unknown plant material, such as from customs seizures of illegally collected specimens. Mexican cacti are an example of a threatened group, under pressure because of wild collection for the xeriscaping trade and private collectors. Mexican cacti also provide a taxonomically and geographically coherent group with which to test DNA barcodes. Here, we sample the matK barcode for 528 species of Cactaceae including approximately 75% of Mexican species and test the utility of the matK region for species-level identification. We find that the matK DNA barcode can be used to identify uniquely 77% of species sampled, and 79-87% of species of particular conservation importance. However, this is far below the desired rate of 95% and there are significant issues for PCR amplification because of the variability of primer sites. Additionally, we test the nuclear ITS regions for the cactus subfamily Opuntioideae and for the genus Ariocarpus (subfamily Cactoideae). We observed higher rates of variation for ITS (86% unique for Opuntioideae sampled) but a much lower PCR success, encountering significant intra-individual polymorphism in Ariocarpus precluding the use of this marker in this taxon. We conclude that the matK region should provide useful information as a DNA barcode for Cactaceae if the problems with primers can be addressed, but matK alone is not sufficiently variable to achieve species-level identification. Additional complementary regions should be investigated as ITS is shown to be unsuitable.     

帘蛤科贝类rDNA内转录间隔区序列的研究

根据18SrDNA、5．8SrDNA和28SrDNA保守序列设计引物,应用聚合酶链式反应（PCR）扩增了文蛤（Meretrix meretrix L．）、青蛤（Cyclina sinensis G）、硬壳蛤（Mercenaria mercenaria L．）和江户布目蛤（Protothaca jedoensis L．）4种帘蛤科贝类的第一内转录间隔区（ITS1）和第二内转录间隔区（ITS2）序列,并进行了测序。结果表明,文蛤、青蛤、硬壳蛤和江户布目蛤的ITS1扩增产物大小分别为978bp、663bp、757bp和942bp,GC含量分别为61．55％、60．78％、62．48％和64．86％～64．97％,其中ITS1序列长度分别为900bp、585bp、679bp和864bp,是迄今已报道双壳贝类中变化范围最大的,GC含量分别为61．67％、61．03％、63．03％和65．51％～65．62％,江户布目蛤种内ITS1序列有个体差异;ITS2扩增产物大小分别为644bp、618～620bp、593bp和513～514bp,GC含量分别为61．18％、61．29％～61．81％、62．73％和61．48％61．60％,其中ITS2序列长度分别为412bp、386～388bp、361bp和281～282bp,GC含量分别为65．29％、65．21％～66．06％、67．87％和67．38％～67．62％,青蛤和江户布目蛤种内ITS2序列有个体差异。4种蛤ITS1和ITS2序列种间差异很大,有明显的长度多态性,ITS2种间序列相似度73．0％～89．1％,与ITS1的种间序列相似度48．7％～81．5％相比略高。此外,在4种蛤ITS1和ITS2序列中各发现2个与rRNA加工有关的保守区。通过对ITS1和ITS2序列的组装获得了4种蛤5．8SrRNA基因完整序列,序列长度都是157bp,GC含量57．96％～58．60％,4种蛤5．8SrRNA基因相对保守,种间序列差异度0-6．0％,共有10个变异位点,其中转换4处,颠换6处,硬壳蛤和江户布目蛤5．8SrRNA基因序列完全相同。以ITS2序列（包含5．8SrRNA和28SrRNA基因部分序列）为标记,调用北极蛤科的Arctica islandica相应序列数据作外群,构建了帘蛤科贝类的系统发育树,其拓扑结构显示江户布目蛤与硬壳蛤亲缘关系最近,青蛤与其他3物种的亲缘关系最远。     

Molecular barcodes for soil nematode identification

Using a molecular barcode, derived from single-specimen polymerase chain reaction (PCR) and sequencing of the 5' segment of the small subunit ribosomal RNA (SSU) gene, we have developed a molecular operational taxonomic unit (MOTU) scheme for soil nematodes. Individual specimens were considered to belong to the same MOTU when the sequenced segment of 450 bases was > 99.5% identical. A Scottish upland Agrostis-Festuca grassland soil was sampled, using both culture-based and random selection methods. One hundred and sixty-six cultured isolates were sequenced, and clustered into five MOTU. From 74 randomly sampled individuals across the study site, 19 MOTU were defined. A subsequent sample of 18 individuals from a single subplot contained eight MOTU, four of which were unique to the single subplot sample. Interestingly, seven of these MOTU were not present in the culture-independent sampling. Overall, a total of 23 MOTU were defined from only 240 sequences. Many MOTU could readily be assigned to classical, morphologically defined taxonomic units using a database of SSU sequences from named nematode species. The MOTU technique allows a rapid assessment of nematode taxon diversity in soils. Correlation with a database of sequences from known species offers a route to application of the technique in ecological surveys addressing biological as well as genetic diversity.     

Study on Sequences of Ribosomal DNA Internal Transcribed Spacers of Clams Belonging to the Veneridae Family (Mollusca: Bivalvia)

The first and second internal transcribed spacer (ITS1 and ITS2) regions of the ribosomal DNA from four species, Meretrix meretrix L., Cyclina sinensis G., Mercenaria mercenaria L., and Protothaca jedoensis L., belonging to the family Veneridae were amplified by PCR and sequenced. The size of the ITS1 PCR amplification product ranged from 663 bp to 978 bp, with GC contents ranging from 60.78% to 64.97%. The size of the ITS1 sequence ranged from 585 bp to 900 bp, which is the largest range reported thus far in bivalve species, with GC contents ranging from 61.03% to 65.62%. The size of the ITS2 PCR amplification product ranged from 513 bp to 644 bp, with GC contents ranging from 61.29% to 62.73%. The size of the ITS2 sequence ranged from 281 bp to 412 bp, with GC contents ranging from 65.21% to 67.87%. Extensive sequence variation and obvious length polymorphisms were noted for both regions in these species, and sequence similarity of ITS2 was higher than that of ITS1 across species. The complete sequences of 5.8S ribosomal RNA gene were obtained by assembling ITS1 and ITS2 sequences, and the sequence length in all species was 157 bp. The phylogenetic tree of Veneridae clams was reconstructed using ITS2-containing partial sequences of both 5.8S and 28S ribosomal DNA as markers and the corresponding sequence information in Arctica islandica as the outgroup. Tree topologies indicated that P. jedoensis shared a close relationship with M. mercenaria and C. sinensis, a distant relationship with other species.     

Phylogenetic relationships between European and Chinese truffles based on parsimony and distance analysis of ITS sequences

Phylogenetic relationships among truffle species from Europe and China were investigated through parsimony analysis of the ITS sequences. Three major clades were obtained among the species analysed. The so-called white truffles appeared polyphyletic since Tuber magnatum was grouped with brown truffles and not with the other white species (T. maculatum, T. borchii, T. dryophilum, T. puberulum). The black truffles investigated in this study, T. brumale, T. melanosporum, T. indicum and T. himalayense, were grouped in an independent clade. The Périgord black truffle T. melanosporum and the Chinese black truffles T. indicum and T. himalayense, were very closely related. The delimitation of these species was estimated by a distance analysis on several isolates collected from different geographic areas. In spite of intraspecific variations of the internal transcribed spacers (ITS) sequences, T. melanosporum and the Chinese black truffles can be unambiguously attributed to distinct taxa.     

血卵涡鞭虫核糖体18SrDNA和ITS1-5.8SrDNA-ITS2序列测定与系统发育分析

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Efficiency of DNA barcodes for species delimitation: A case in Pterygiella Oliv. (Orobanchaceae)

DNA barcoding is becoming an increasingly popular means to identify species. The obscure discrimination in the genus Pterygiella calls into question the re-assessment of the criterion for species delimitation. We collected 20 individuals, representing all five described species of this genus in its distributional range. The aim was to use three proposed barcode DNA regions (rbcL, matK, and ITS) to diagnose Pterygiella species, and examine which barcode is more suitable for discerning the congeneric and related species. The results showed that the core barcodes matK and rbcL were comparatively less effective. However, the ITS region, especially ITS-1 and ITS-2, successfully identified all species in the genus. Furthermore, the secondary structure of ITS-2 RNA, especially compensatory base changes, appears complementary to classical primary sequence analysis for DNA barcoding.     

Identification of High γ-Aminobutyric Acid Producing Marine Yeast Strains by Physiological and Biochemical Characteristics and Gene Sequence Analyses

Four marine yeasts isolated from the Pacific Ocean off Japan (Siki No. 4, Siki No. 15, Hach No. 6, and Inub No. 11), which showed high γ-aminobutyric acid (GABA) producing abilities, were identified and classified by physiological and biochemical characteristics and gene sequence analyses. Analysis of biochemical data suggested that while Siki No. 15 was identical to Candida, the remaining three isolates belonged to the genus Pichia. However, these data were insufficient to resolve their identity at the species level. Subsequently, analysis of the 5.8S rRNA genes and the two internal transcribed spacer regions (ITS) sequences revealed that Siki No. 15 belongs to Pichia guilliermondii, while the remaining three isolates corresponded to Pichia anomala. Since Siki No. 4 showed slightly different biochemical properties than the other two isolates, which were otherwise identical, we sought to investigate the sequences of the intergenic spacer region 1 (IGS1). We observed few nucleotide changes, suggesting that the Hach No. 6 and Inub No. 11 isolates belong to different but new strains for which we propose the names P. anomola MR-1 and MR-2 respectively.     

Molecular definition and the ubiquity of species in the genus Naegleria

To investigate the variability within species of the genus Naegleria, the ITS1,5.8S and ITS2 rDNA were sequenced of several strains of N. lovaniensis and its Western Australian variants, N. australiensis, N. fowleri, N. andersoni, N. jamiesoni, N. tihangensis, N. pringsheimi, N. pagei, N. gruberi sensu lato and a Naegleria lineage that lost a group I intron from the SSUrDNA twintron. As a result, it is possible to define a molecular species within the Naegleria genus. In addition, one strain of each different allozyme cluster was sequenced to investigate whether they belong to described species or should be treated as distinct new species. This leads to the proposal of eleven new species. The sequencing results from those Naegleria spp. of which several strains are available indicate that these species are ubiquitous. The only exception might be the species represented by the WA variants. However, there are still many Naegleria spp. for which only one strain has been isolated, hence, it is important that the search for more isolates should be continued worldwide.     

New species Micractinium kostikovii (Chlorellaceae,Trebouxiophyceae) from Russia

Correctly identifying species of Chlorella-like microalgae is difficult because of their morphological simplicity and high phenotypic plasticity. The use of molecular tools has revolutionized research on algal diversity, enabling such advances as the discovery of numerous new taxa. This article presents the results of a study of strains that are newly isolated from a freshwater Lake Prudovikov (Samara region, Russian Federation). These strains had the typical Chlorella morphology, exhibiting spherical cells and a cup-shaped parietal chloroplast. The chloroplast contained a single pyrenoid enveloped by starch grains. However, 18S–ITS1–5.8S–ITS2 sequence analyses indicated that the studied strains are strongly allied with the well-supported genus Micractinium. Based on the results of a comparative analysis of the morphological, molecular and environmental characteristics of the studied strains (employing a polyphasic approach), we propose that they are new species of Micractinium: Micractinium kostikovii sp. nov.     

MOLECULAR DELINEATION OF SPECIES AND SPECIES RELATIONSHIPS IN THE RED ALGAL AGAROPHYTES GRACILARIOPSIS AND GRACILARIA (GRACILARIALES)1

Delineation of species in the economically important agarophyte genera Gracilaria and Gracilariopsis has proven extremely difficult using available morphological characteristics. In this study, we examine the usefulness of two transcribed spacers for molecular systematic studies of these genera. The polymerase chain reaction was used to amplify the internal transcribed spacers (ITSs) and the intervening 5.8S ribosomal DNA of the nuclear ribosomal repeat region. In addition, a plastid spacer region and flanking regions of coding genes were amplified from the RUBISCO operon. Both regions were sequenced for individuals and populations of Gracilariopsis lemaneiformis (Bory) Dawson, Acleto, et Foldvik to determine the usefulness of these spacers in delimiting populations. These studies reveal that there is as much variation among individuals of a population as there is between individuals of geographically separate populations. In addition, the ITS spacer regions were compared between different species of Gracilariopsis and Gracilaria. The nuclear ITS spacer region is conserved at a species level in both genera and provides phylogenetically informative characters that can be used to examine species interrelationships among relatively closely related taxa. However, because of the difficulties of aligning this entire region among species from the two genera, the ITS region is not useful for examining intergenera relationships. ITS interspecies sequence comparisons indicate that Gracilariopsis lemaneiformis from California is significantly different from G. lemaneiformis from China and that a species of Gracilariopsis from Peru is more closely related to G. lemaneiformis from North Carolina than it is to the other Gracilariopsis species examined. In addition, these studies indicate that Gracilaria chilensis Bird, McLachlan, et Oliveira from New Zealand and Gracilaria tenuistipitata Chang et Xia from southeast Asia are as closely related as are Gracilaria verrucosa (Hudson) Papenfuss, G. pacifica Abbott, and Gracilaria robusta Kylin. Phylogenetic analysis of aligned plastid spacer sequences from Gracilaria and Gracilariopsis taxa provide similar conclusions about species relationships.