BIOLUMINESCENCE IN THE MARINE DINOFLAGELLATE PYROCYSTIS LUNULA

Intragenomic nrDNA ITS variability was examined in representative species of the Stephanodiscus niagarae complex. PCR was performed on DNA extracted from monoclonal cultures, and sequence variability determined using standard cloning techniques. Preliminary data from all species reveal intragenomic polymorphism. ITS sequence data is being used to delineate closely related species of algae and polymorphisms are potentially problematic for phylogenetic reconstruction and identity. We are exploring the extent of polymorphism in other freshwater Thalassiorsiraceae, including Stephanodiscus hantzschii fo. tenuis and Cyclostephanos invisitatus.     

The rDNA ITS Region in the Lessepsian Marine Angiosperm <Emphasis Type="BoldItalic">Halophila stipulacea</Emphasis> (Forssk.) Aschers. (Hydrocharitaceae): Intragenomic Variability and Putative Pseudogenic Sequences

Halophila stipulacea is a dioecious marine angiosperm, widely distributed along the western coasts of the Indian Ocean and the Red Sea. This species is thought to be a Lessepsian immigrant that entered the Mediterranean Sea from the Red Sea after the opening of the Suez Canal (1869). Previous studies have revealed both high phenotypic and genetic variability in Halophila stipulacea populations from the western Mediterranean basin. In order to test the hypothesis of a Lessepsian introduction, we compare genetic polymorphism between putative native (Red Sea) and introduced (Mediterranean) populations through rDNA ITS region (ITS1-5.8S-ITS2) sequence analysis. A high degree of intraindividual variability of ITS sequences was found. Most of the intragenomic polymorphism was due to pseudogenic sequences, present in almost all individuals. Features of ITS functional sequences and pseudogenes are described. Possible causes for the lack of homogenization of ITS paralogues within individuals are discussed.     

Identification of species in the genus Pichia by restriction of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal DNA gene

In this study, the variability within the ribosomal DNA region spanning the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene (5.8S-ITS rDNA) was used to differentiate species in the genus Pichia. The 5.8S-ITS rDNA region was PCR-amplified and the PCR product digested with the enzymes CfoI, HinfI, and HaeIII. The variability in the size of the amplified product and in the restriction patterns enabled differentiation between species in the genus Pichia, and between Pichia species and yeast species from other genera in the Yeast-id database (). Moreover, the restriction fragment length polymorphism (RFLP) patterns of the 5.8S-ITS enabled misidentified strains to be detected and revealed genetic heterogeneity between strains within the Pichia membranifaciens and Pichia nakazawae species. Ultimately, the RFLP patterns of the 5.8S-ITS rDNA failed to differentiate between some Pichia and Candida species that could be distinguished on the basis of the sequence of the 5.8S-ITS rRNA region or the sequence of the D1/D2 domain of the 26S rDNA gene.     

Sequencing of the intergenic 16S-23S rRNA spacer (ITS) region of Mollicutes species and their identification using microarray-based assay and DNA sequencing

Abstract We have completed sequencing the 16S-23S rRNA intergenic transcribed spacer (ITS) region of most known Mycoplasma , Acholeplasma , Ureaplasma , Mesoplasma , and Spiroplasma species. Analysis of the sequence data revealed a significant interspecies variability and low intraspecies polymorphism of the ITS region among Mollicutes . This finding enabled the application of a combined polymerase chain reaction–microarray technology for identifying Mollicutes species. The microarray included individual species-specific oligonucleotide probes for characterizing human Mollicutes species and other species known to be common cell line contaminants. Evaluation of the microarray was conducted using multiple, previously characterized, Mollicutes species. The microarray analysis of the samples used demonstrated a highly specific assay, which is capable of rapid and accurate discrimination among Mollicutes species.     

Polyploid speciation inHedera (Araliaceae): Phylogenetic and biogeographic insights based on chromosome counts and ITS sequences

Variation in chromosome number and internal transcribed sequences (ITS) of nrDNA is used to infer phylogenetic relationships of a wide range ofHedera species. Polyploidy was found to be frequent inHedera, with diploid, tetraploid, hexaploid and octoploid populations being detected. Nucleotide additivity occurs in the ITS sequences of one tetraploid (H. hibernica) and two hexaploid species (H. maderensis, H. pastuchovii), suggesting that all three species originated by allopolyploidisation. ITS sequence polymorphism and nucleotide characters may indicate the presence of an ancient genome persistent only in some allopolyploid species. Phylogenetic analyses of ITS sequence data reveal two lineages ofHedera: one containing all sequences belonging to extant diploids plus the tetraploidH. algeriensis, and a second that includes this ancient ITS type and others exclusive to several polyploid species. The origin of the polyploids is evaluated on the basis of morphology, chromosome counts, ITS sequence polymorphism, and phylogenetic analyses. Reconstruction of reticulate evolution inHedera agrees with two allopolyploid areas on both sides of the Mediterranean basin. Morphological, molecular and cytological evidence also suggests an active dispersal ofHedera populations that may account for three independent introductions in Macaronesia.     

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccard's coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.     

Molecular characterization of some Indian Aloe vera populations through RAPD and ITS markers

The most important and well-known medicinal plant among ~400 species of the genus Aloe is Aloe vera. It is widely used in pharmaceutical, cosmetic, and food industries. Identification and assessment of genetic relationship among the populations and cultivars is needed for conservation and sustainable utilization of this commercially important plant. DNA fingerprinting with random amplified polymorphic DNA (RAPD) marker and Internal Transcribed Spacer (ITS1 and ITS2) of ribosomal DNA sequence analysis were carried out to assess the genetic diversity among populations of Aloe vera collected from geographically different four districts of West Bengal and Jodhpur, Rajasthan. RAPD profiles yielded 158 amplicons showing ~87.34% polymorphism. Analyses of ITS sequences showed that in contrast to ITS2, the length and %GC content (53.6–77.3%) of ITS1 varied within populations. Multiple sequence alignment data reveal that substitutions, insertions, and deletions have arisen at various positions in the ITS regions suggesting polymorphism. A 5′-GGCGCGATGGGCGCCAAGGAA-3′ sequence in ITS1 is conserved in all populations, except AvS4. RAPD dendrogram and topologies of the NJ, Parsimony and ML tree generated from ITS1 sequence revealed that there is a close genetic similarity among AvS1, AvS4, and AvS7 populations. These genetic studies may contribute to plant improvement programs of A. vera.     

Sequence analysis of the ribosomal internal transcribed spacers region in spider mites (Prostigmata: Tetranychidae) occurring in citrus orchards in Eastern Spain: use for species discrimination

Tetranychus urticae is a polyphagous mite which is an important pest of citrus worldwide. This mite can be found feeding on many plant species occurring in the citrus agrosystem moving from weeds to trees. Because field samples consist of a mixture of different Tetranychidae species, as a first step necessary to further implement population characterisation of T. urticae, species‐discriminating criteria based on molecular techniques are needed. In this study, the nucleotide variation of the internal transcribed spacers (ITS) 1 and 2 and the intergenic 5.8S fragment of nuclear rDNA of T. urticae, Tetranychus turkestani, Tetranychus evansi, Tetranychus ludeni and Panonychus citri have been determined. Results demonstrate that for these species, the rDNA ITS2 regions are much more conserved than the corresponding rDNA ITS1. The high homogeneity of the ITS2 sequence observed among the specimens of T. urticae obtained from the same ecoregion makes this DNA sequence an excellent tool for species discrimination. ITS sequences differentiate not only species but also specimens from different geographical origin. Furthermore, polymerase chain reaction–restriction fragment length polymorphism analysis of the ITS2 proved adequate for a quick screening of high numbers of field samples.     

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

Restriction analysis of the amplified ribosomal DNA spacers ITS1 and ITS2 of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> isolates

The purpose of this study was to examine the genotypic variability of Bipolaris sorokiniana by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using rDNA. Fifty B. sorokiniana isolates from Brazil and other countries, one Bipolaris oryzae and six Drechslera teres isolates were used. The intragenic spacer regions (ITS1 and ITS2) were the regions used for characterization of isolates. The amplification products for both ITS regions, showed two DNA fragments for all isolates. Two B. sorokiniana isolates presented an intraspecific variability showing a third fragment for the ITS1 region. The dendrograms generated with PCR-RFLP data showed intra- and inter-specific groups. The dendrograms showed that most of Brazilian isolates clustered together forming groups between them, and this behavior was repeated with most isolates from other countries. The dendrograms did not enable the separation of B. sorokiniana isolates by their geographic origin or host type. These results suggest the occurrence of gene flow between different populations of the fungus isolated in geographically distant regions and lends cogency to the occurrence of gene flow between species.     

Sequencing of the internal transcribed spacer region ITS1 as a molecular tool detecting variation in the Stylosanthes guianensis species complex

 The internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal DNA from Stylosanthes guianensis CIAT 1283 and cv ‘Schofield’ were amplified by polymerase chain reaction using conserved ITS primers from the 18S, 5.8S and 26S ribosomal genes flanking those regions. The entire region of 683 bp long was cloned, and seven clones were sequenced. Comparison of the ITS spacer regions with published DNA sequences of other plant species revealed limited homology only; this was in contrast to their comparison with the 5.8S rDNA sequences. The ITS1 region of 45 S. guianensis accessions was amplified by PCR and sequenced on both strands using the conserved primers ITS2-ITS5. These sequences, ranging from 201 to 204 bp, were aligned to each other to assess intra-specific polymorphism. Within the S. guianensis (Aubl.) Sw. species complex, 11 DNA sequence types could be distinguished based on an insertion/deletion (indel) event and 15 single base-pair substitutions. In 1 of the S. guianensis types, two kinds of ITS1 sequence were observed in each individual, reminiscent of an incomplete homogenization of the repeat structure in this type. Polymorphisms in the sequence of the ITS1 region were used to define molecular markers for S. guianensis on the basis of PCR-restriction fragment length polymorphism and selective PCR. Received: 24 June 1997 / Accepted: 31 October 1997     

Addition of centric diatoms to the flora in waterbodies of the northern slope of the Polar Ural

In rivers, lakes, and streams located in the region of the northern slope of the Polar Ural, 15 species of centric diatoms belonging to genera Aulacoseira, Cyclotella, Discostella, Melosira, and Stephanodiscus have been recorded. The species Cyclotella tripartita, Stephanodiscus hantzschii, S. minutulus, Aulacoseira tenella, Cyclotella arctica, C. ocellata, Stephanodiscus alpinus, and S. cf. niagarae, which are new for the flora of the region, have been found. The high morphological variability of Cyclotella ocellata is shown.     

Analysis of nucleotide sequence polymorphism of internal transcribed spacers of ribosomal genes in diploid <Emphasis Type="Italic">Aegilops</Emphasis> (L.) species

The nucleotide sequence of the fragment of the internal transcribed spacer (ITS) of rDNA comprising the full-length ITS1, the gene encoding 5.8S rRNA, and part of the ITS2 sequence was determined in 22 samples of five diploid Aegilops species. The full alignment length of compared sequences was 524 bp. Species-specific substitutions were found in the ITS nucleotide sequence of rDNA of different Aegilops species. Intraspecific differences in ITS structure in diploid Aegilops species were detected for the first time. Polymorphism of the ITS nucleotide sequence within the same sample was revealed, which might be due either to differences between the genomes of individual plants comprising the sample or to the presence of several types of ribosomal genes in the genome of one plant. In general, both interspecific and intraspecific variability of the ITS nucleotide sequences of rDNA is extremely low. In total, 26 variable sites, twelve of which were informative, were identified.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 193–197.Original Russian Text Copyright © 2005 by Goryunova, Chikida, Gori, Kochieva.     

Molecular authentication of ginseng cultivars by comparison of internal transcribed spacer and 5.8S rDNA sequences

Molecular authentication among three Panax species and within cultivars and accessions of P. ginseng was investigated using the DNA sequence in the ribosomal ITS1–5.8S–ITS2 region. Four single-nucleotide polymorphisms were identified between P. ginseng and other Panax species. In the electrophoresis profile, obtained after digestion with the enzyme TaqI, three fingerprinting patterns were obtained from cultivars and accessions of Panax species. Consequently, this authentication procedure based upon the restriction fragment length polymorphism in the ribosomal ITS1–5.8S–ITS2 region can now be utilized to differentiate these Panax species as well as major Korean cultivars such as Gopoong and Kumpoong from other cultivars and accessions in Panax species at the DNA level. O. T. Kim and K. H. Bang contributed equally to this paper.     

Intraspecific invariability of the internal transcribed spacer region of rDNA of the truffleTerfezia terfezioides in Europe

ITS regions (internal transcribed spacers—ITS1 andITS2—with the 5.8S gene of the nuclear rDNA) of 25 fruit body samples ofTerfezia terfezioides, originating from Hungary and Italy, were compared. The amplification and sequencing of the ITS region was successful with both theITS1-ITS4 andITSIF-ITS4 primer pairs. No differences of the restriction fragment length polymorphism profiles were detected among 19 samples collected in one place at the same time. The sequences of the ITS region of 9 samples collected in different localities were highly invariable, differing in only two bases. Thus the intraspecific homogeneity of the ITS region seems to be an important species-specific characteristic ofT. terfezioides in contrast to otherTerfezia species. As the samples of the species were collected from different and distant localities of Europe, the ITS sequence ofT. terfezioides can be considered a very conservative, reliable molecular marker of the fungus. *** DIRECT SUPPORT *** A00EN076 00008     

77 Cryptic speciation of Peridinium Limbatum

Cosmopolitanism vs. endemism of microbial diversity in water has been debated for a long time. Finlay (2002) argued that reported similarities in microbial biodiversity in different lakes and oceans support cosmopolitanism of microorganisms. Lee and Patterson (1998) suggested that many endemism studies are flawed due to underrepresentation of taxa. On the other hand, Theriot and Stoermer (1984) showed that Stephanodiscus niagrarae, the centric diatom once thought to be a cosmopolitan species, actually consists of four slightly different species. Mating tests of Paramecium aurelia revealed 14 sibling species, which are morphologically almost identical (Sonneborn, 1957). Here, I present evidence for the cryptic speciation of Peridinium limbatum from two water bodies (Crystal Lake and Crystal Bog) that are near each other but chemically distinct. We observed that the two Peridinium limbatum had different responses to the same growth medium. Previous works at the Microbial Observatory program showed that their seasonal population dynamics were also different; while a summer bloom of Crystal Bog Peridinium limbatum was correlated with a dramatic decrease in phytoplankton diversity, and an extreme drop in bacterial diversity, a bloom of Crystal Lake Peridinium limbatum was not. To see the genetic variations in this study, I sequenced internal transcribed spacer region 1 and 2 from the two Peridinium limbatum. The sequence variations were 8.53% in ITS1 and 8.06% in ITS2, which were much larger than within variations. In another ongoing study that I expect to complete before the meeting, I've been testing their possible allelopathic differences on Cryptomonas.     

Cryptic species in the <Emphasis Type="Italic">Terfezia boudieri</Emphasis> complex

Phylogenetic analyses have corroborated the discovery of three internal transcribed spacer (ITS) Types in Terfezia boudieri isolates in the course of earlier studies and have emphasized the divergence of Type 2 from Types 1 and 3. The application of molecular and physiological tools described below, revealed the existence of cryptic species within T. boudieri. The markers used include sequences taken from the 5′ end of the ribosomal large subunit gene, a chitin synthase partial sequence, β-tubulin partial sequence and amplified fragment length polymorphism (AFLP)-based markers. Following initial sequencing of a single PCR amplified sample for each Type, mass analysis of specimens relied on RFLP differences between the Types. Over 100 fruit bodies, 30 or more specimens for each ITS Type, were tested with each of the markers. The markers analysis divided the isolates into three groups, each correlated to a specific ITS Type. Two of the physiological traits examined: mycelial proliferation and mycorrhiza formation, consistently showed responses paralleling the ITS Types; the data presented suggest that T. boudieri is comprised of three cryptic species.     

The evolutionary history of Fagus in western Eurasia: Evidence from genes, morphology and the fossil record

 Fagus (beech) is among the most abundant and economically important genera of broad-leaved trees in northern hemisphere temperate forests. The number of modern taxa present in Europe and Asia Minor has long been a matter of debate and up to five species have been recognised. To resolve taxonomic and phylogenetic relationships we conducted morphological and molecular genetic analyses in western Eurasiatic taxa and evaluated palaeontological evidence. To place our findings from western Eurasiatic populations in a broader context additional East Asiatic and North American species of the same subgenus Fagus as well as two species of the subgenus Engleriana were included in our study. The morphological features exhibited in western Eurasiatic populations of Fagus show a west-east gradient that is characterised by strongly overlapping variability between geographical races. Fagus populations from Asia Minor exhibit an even higher variability that is also reflected in their genetic variability of nuclear rDNA internal transcribed spacer (ITS) sequences. The intraspecific genetic variability recorded here is in conflict with previous ITS studies in Fagus. The high amount of ITS polymorphism within Fagus from western Eurasia along with the clinal variation observed for morphological characters suggest the presence of only a single species, Fagus sylvatica L., in Europe and Asia Minor. Previously recognised taxa such as F. orientalis Lipsky and Fagus moesiaca (Maly) Czeczott should therefore be treated as synonyms of Fagus sylvatica. Although species belonging to the subgenus Engleriana were genetically distinct from species of the subgenus Fagus, relationships within the subgenus Fagus could not be clearly resolved. A reason for this could be the low rate of diversification in Fagus during the early phase of range expansion of the genus in the Oligocene period as indicated by the uniformity of leaf and cupule/nut fossils. This may account for the low overall ITS divergence and the high degree of polymorphism encountered in the subgenus Fagus and points to a late differentiation of western Eurasiatic and eastern Asiatic species. Area disruptions during the Pleistocene and the post-glacial recolonisation of western Europe appear to have caused the west-east gradient that is apparent in modern Fagus of western Eurasia but absent in Late Tertiary ancestors of Fagus sylvatica. Received June 22, 2001 Accepted February 25, 2002     

PHYLOGENY AND GENETIC VARIANCE IN TERRESTRIAL MICROCOLEUS (CYANOPHYCEAE) SPECIES BASED ON SEQUENCE ANALYSIS OF THE 16S rRNA GENE AND ASSOCIATED 16S–23S ITS REGION1

Thirty‐one strains of Microcoleus were isolated from desert soils in the United States. Although all these taxa fit the broad definition of Microcoleus vaginatus (Vaucher) Gomont in common usage by soil algal researchers, sequence data for the 16S rRNA gene and 16S–23S internal transcribed spacer (ITS) region indicated that more than one species was represented. Combined sequence and morphological data revealed the presence of two morphologically similar taxa, M. vaginatus and Microcoleus steenstrupii Boye‐Petersen. The rRNA operons of these taxa were sufficiently dissimilar that we suspect the two taxa belong in separate genera. The M. vaginatus clade was most similar to published sequences from Trichodesmium and Arthrospira. When 16S sequences from the isolates we identified as M. steenstrupii were compared with published sequences, our strains grouped with M. chthonoplastes (Mertens) Zanardini ex Gomont and may have closest relatives among several genera in the Phormidiaceae. Organization within the 16S–23S ITS regions was variable between the two taxa. Microcoleus vaginatus had either two tRNA genes (tRNA^Ile and tRNA^Ala) or a fragment of the tRNA^Ile gene in its ITS regions, whereas M. steenstrupii had rRNA operons with either the tRNA^Ile gene or no tRNA genes in its ITS regions. Microcoleus vaginatus showed no subspecific variation within the combined morphological and molecular characterizations, with 16S similarities ranging from 97.1% to 99.9%. Microcoleus steenstrupii showed considerable genetic variability, with 16S similarities ranging from 91.5% to 99.4%. In phylogenetic analyses, we found that this variability was not congruent with geography, and we suspect that our M. steenstrupii strains represent several cryptic species.     

Coding of intraspecific nucleotide polymorphisms: A tool to resolve reticulate evolutionary relationships in the ITS of beech trees (Fagus L., Fagaceae)

The internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA (rDNA) have recently been found to display remarkable intraspecific polymorphism, a feature suggested as limiting their value for phylogenetic reconstructions. A comparative study of oligonucleotide motives and intraindividual nucleotide variability across all species of the tree genus Fagus (beech) shows, however, that this intraspecific ITS polymorphism follows a particular pattern, which can be used to detect reticulation and ancient polymorphism within the genus. Coding ITS polymorphisms as phylogenetically informative characters, moreover, resulted in better‐resolved phylogenies than traditional ‘base‐per‐base’ maximum parsimony and maximum likelihood analyses.