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.     

Heterogeneity of ITS1 sequences in the biting midge Culicoides impunctatus (Goetghebuer) suggests a population in Argyll, Scotland, may be genetically distinct.

Ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) is a useful genomic region for understanding evolutionary and genetic relationships. In the current study, variation in ITS1 from eight Culicoides species was analysed by PCR, DNA restriction analysis, cloning, and sequencing. ITS1 variants were essentially homogenized within a species, as sequences were identical or closely related. However, Culicoides impunctatus ITS1 sequences derived from one (Argyll) of five populations contained considerable genomic diversity. The secondary structure of each ITS1 was computed. The structure aided the production of an accurate alignment and the identification of a large indel. A phylogenetic analysis was performed. Some of the sequences from the diverse Argyll C. impunctatus population were more related to Culicoides imicola, a vector of animal pathogens in the Old World, than they were to the other C. impunctatus sequences. Thus, the rDNA ITS1 regions of individuals in the Argyll C. impunctatus population were not conforming to the general theory of rDNA homogenization through molecular drive.     

Heterogeneity of the internal transcribed spacer 1 (ITS1) inTulipa (Liliaceae)

Heterogeneity of the internal transcribed spacer ITS1 of the rDNA within individuals ofTulipa gesneriana L.,T. kaufmanniana Regel, and their interspecific hybrids was analyzed by PCRRFLP, using the polymorphic restriction enzymesRsaI andHinfI, and by nucleotide sequence analysis. In most cases, the sum of the sizes of the restriction fragments was higher than the entire length of the undigested ITS fragment, indicating heterogeneity at the restriction sites within an individual. Differences in band intensities within the restriction patterns indicate the occurrence of variation in copy number of these different ITS1 variants within individuals. Automated sequencing without a visual inspection often failed to detect existing heterogeneity within sequences, resulting in a discrepancy between the sequencing and restriction analysis results. By visual interpretation of the sequences, the restriction patterns could mostly be predicted well. Fluorescence in situ hybridization (FISH) experiments in fourTulipa species revealed the occurrence of several rDNA spots. The number of rDNA loci varied from seven inT. gesneriana Christmas Marvel to ten inT. australis Link. This might explain the occurrence of heterogeneity in ITS sequences inTulipa, as homogenization of variants has to take place over different loci.     

Ribosomal DNA polymorphisms in the yeast Geotrichum candidum

The dimorphic yeast Geotrichum candidum (teleomorph: Galactomyces candidus) is commonly used to inoculate washed-rind and bloomy-rind cheeses. However, little is known about the phylogenetic lineage of this microorganism. We have sequenced the complete 18S, 5.8S, 26S ribosomal RNA genes and their internal transcribed spacers (ITS1) and ITS2 regions (5126 nucleotides) from 18 G. candidum strains from various environmental niches, with a focus on dairy strains. Multiple sequence alignments revealed the presence of 60 polymorphic sites, which is generally unusual for ribosomal DNA (rDNA) within a given species because of the concerted evolution mechanism. This mechanism drives genetic homogenization to prevent the divergent evolution of rDNA copies within individuals. While the polymorphisms observed were mainly substitutions, one insertion/deletion (indel) polymorphism was detected in ITS1. No polymorphic sites were detected downstream from this indel site, that is, in 5.8S and ITS2. More surprisingly, many sequence electrophoregrams generated during the sequencing of the rDNA had dual peaks, suggesting that many individuals exhibited intragenomic rDNA variability. The ITS1-5.8S-ITS2 regions of four strains were cloned. The sequence analysis of 68 clones revealed 32 different ITS1-5.8S-ITS2 variants within these four strains. Depending on the strain, from four to twelve variants were detected, indicating that multiple rDNA copies were present in the genomes of these G. candidum strains. These results contribute to the debate concerning the use of the ITS region for barcoding fungi and suggest that community profiling techniques based on rDNA should be used with caution.     

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     

Extensive 5.8S nrDNA polymorphism in <Emphasis Type="Italic">Mammillaria</Emphasis> (Cactaceae) with special reference to the identification of pseudogenic internal transcribed spacer regions

The internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, ITS2) represents the most widely applied nuclear marker in eukaryotic phylogenetics. Although this region has been assumed to evolve in concert, the number of investigations revealing high degrees of intra-individual polymorphism connected with the presence of pseudogenes has risen. The 5.8S rDNA is the most important diagnostic marker for functionality of the ITS region. In Mammillaria, intra-individual 5.8S rDNA polymorphisms of up to 36% and up to nine different types have been found. Twenty-eight of 30 cloned genomic Mammillaria sequences were identified as putative pseudogenes. For the identification of pseudogenic ITS regions, in addition to formal tests based on substitution rates, we attempted to focus on functional features of the 5.8S rDNA (5.8S motif, secondary structure). The importance of functional data for the identification of pseudogenes is outlined and discussed. The identification of pseudogenes is essential, because they may cause erroneous phylogenies and taxonomic problems. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Within- and between-individual sequence variation among ITS1 copies in the meadow grasshopper Chorthippus parallelus indicates frequent intrachromosomal gene conversion

Sequencing multiple copies of the ITS1 region revealed the coexistence of two or more haplotypes within the genome of Chorthippus parallelus. Using a PCR-RFLP approach, the ITS1 numbers and frequencies of haplotypes present in each of 40 individuals were investigated, revealing a consistent lack of homogeneity. For each individual, the level of intra-individual variation was estimated from a sample of 20 ITS1 copies. The level of differentiation in haplotype frequency among individuals was then estimated by maximum likelihood using models based on the Dirichlet distribution. This confirmed the existence of significant levels of variation among individuals within each population studied. The most likely turnover mechanism that could generate this pattern of variation is gene conversion, operating at the intrachromosomal level. Furthermore, the discovery of linkage disequilibrium among the ITS1 haplotypes of C. parallelus suggests that intrachromosomal gene conversion occurs more frequently than interchromosomal recombination. Subspecies of C. parallelus showed significantly different haplotype distributions following about 0.5 Myr of divergence. With respect to the process of concerted evolution, we show that homogenization of repeats is slow relative to speciation, and the standing variation among individuals is sufficient for selection to operate.     

The sequence of the isoepoxydon dehydrogenase gene of the patulin biosynthetic pathway in <Emphasis Type="Italic">Penicillium</Emphasis> species

Interest in species of the genus Penicillium is related to their ability to produce the mycotoxin patulin and to cause spoilage of fruit products worldwide. The sequence of the isoepoxydon dehydrogenase (idh) gene, a gene in the patulin biosynthetic pathway, was determined for 28 strains representing 12 different Penicillium species known to produce the mycotoxin patulin. Isolates of Penicillium carneum, Penicillium clavigerum, Penicillium concentricum, Penicillium coprobium, Penicillium dipodomyicola, Penicillium expansum, Penicillium gladioli, Penicillium glandicola, Penicillium griseofulvum, Penicillium paneum, Penicillium sclerotigenum and Penicillium vulpinum were compared. Primer pairs for DNA amplification and sequencing were designed from the P. griseofulvum idh gene (GenBank AF006680). The two introns present were removed from the nucleotide sequences, which were translated to produce the IDH sequences of the 12 species for comparison. Phylogenetic relationships among the species were determined from rDNA (ITS1, 5.8 S, ITS2 and partial sequence of 28S rDNA) and from the idh nucleotide sequences minus the two introns. Maximum parsimony analysis showed trees based on rDNA and idh sequences to be congruent. It is anticipated that the genetic information obtained in the present study will aid in the design of probes, specific for patulin biosynthetic pathway genes, to identify the presence of these mycotoxigenic fungi. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Ribosomal DNA variation within and among individuals ofLisianthius (Gentianaceae) populations

Restriction endonuclease fragment analysis of nuclear ribosomal DNA (rDNA) was completed on 25 individuals each from seven populations of theLisianthius skinneri (Gentianaceae) species complex in Panama. Seven restriction enzymes were used to determine the amount and type of rDNA variation within and among individuals of the populations. No restriction site variation was seen within populations or individuals although site differences were seen among populations. Spacer length variation within and among individuals of populations was mapped to the internal transcribed spacer (ITS) region between the 18S and 5.8S rRNA genes, a region inLisianthius rDNA that previously was shown to exhibit length differences among populations. This is the first reported case of such variation within and among individuals of populations for the ITS region. Presence or absence of ITS spacer length variation is not correlated with levels of isozymic heterozygosity within populations. No detectable length variation within individuals or populations was seen in the larger intergenic spacer (IGS). Although populations varied with respect to IGS length, all individuals of a given population had a single and equivalent IGS length.     

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.     

Two new species of <Emphasis Type="Italic">Trichoderma</Emphasis> from Yunnan,China

Two new species of the fungal genus Trichoderma, Trichoderma compactum and Trichoderma yunnanense, isolated from rhizosphere of tobacco in Yunnan Province, China are described based on morphological characters and phylogenetic analyses of nucleotide sequences. Our DNA sequences included the internal transcribed spacer (ITS) regions of the rDNA cluster (ITS1 and ITS2), and partial sequences of the translation elongation factor 1-alpha (tef1) and a fragment of the gene coding for endochitinase 42 (ech42). The analyses show that T. compactum belongs to the Harzianum clade, and T. yunnanense belongs to the Hamatum clade.     

Genetic relationship of Tricholoma matsutake and T. nauseosum from the Northern Hemisphere based on analyses of ribosomal DNA spacer regions

The genetic relationship among Tricholoma matsutake and T. nauseosum strains collected from various parts of the Northern Hemisphere was investigated using sequence analysis of the rDNA ITS region and PCR-RFLP analysis of the rDNA IGS-1 region. ITS sequence similarity between T. matsutake and T. nauseosum ranged between 98.1% and 100%. The strains of T. matsutake from coniferous forests and those from broad-leaved forests showed more than 99.8% similarity in their ITS sequences. Three distinct RFLP types were detected when IGS-1 regions were digested with Cfr13I. RFLP patterns showed no variability among the strains of T. nauseosum and those of T. matsutake from broad-leaved forests. This pattern corresponded to the dominant RFLP type in the Japanese population of T. matsutake. Thus, strains belonging to this RFLP type are widely distributed throughout East Asia and Europe and associated with many tree species of Pinaceae and Fagaceae. The result suggests that T. matsutake in coniferous and broad-leaved forests and T. nauseosum should be treated as the same species genetically.     

Intragenomic diversity and phylogenetic systematics of wild rosemaries (Rosmarinus officinalis L. s.l., Lamiaceae) assessed by nuclear ribosomal DNA sequences (ITS)

Nuclear ribosomal sequences (ITS) were used to study species boundaries and to infer phylogenetic patterns in wild rosemaries (Rosmarinus officinalis, R. eriocalyx, R. tomentosus). Intragenomic polymorphisms (overlapping peaks and in some cases unreadable sequences) were found throughout the sequencing electrophoretograms of most Rosmarinus accessions. Sequencing the cloned ITS products from representative individuals resulted in 25 ribotypes differing at 59 variable sites. Average sequence divergence among clones was 1.75%, and the most divergent sequences differed by 3.48%. No single ribotype was shared between any two-paired species. The highest values of intragenomic divergence were similar in R. officinalis (1.63%) and R. eriocalyx (1.14%–2.12%), and contrast with those shown by R. tomentosus (0.97%). Sequence data suggest that most divergent rDNA sequences within individuals belong to paralogous loci that apparently are not pseudogenes. A detailed inspection of direct and cloned sequences does not show evidence that the intragenomic polymorphism found is due to interspecific hybridization. Phylogenetic analyses of cloned sequences suggested that both R. officinalis and R. tomentosus were monophyletic, whereas R. tomentosus clones were nested within a paraphyletic R. eriocalyx.     

四照花亚属的nrDNA ITS致同进化不完全

四照花亚属(Cornus subg.Syncarpea)隶属于山茱萸科山茱萸属(Cornus),我国该亚属共有5种8亚种。为探讨四照花亚属nrDNA ITS序列的致同进化不完全现象及假基因产生的可能原因,分析了该亚属4种(每种1~2个居群)共21个个体的nrDNA ITS序列。结果表明,这些类群的nrDNA ITS存在多态性,通过分析这些nrDNA ITS克隆序列的G+C含量、5.8S保守基序和二级结构最小自由能,推测其可能存在假基因。系统发育研究结果显示所有nrDNA ITS序列分成5个分支,同一个体的不同拷贝被分别置于两个甚至多个分支中,且不同分支显示了不同种间关系。四照花亚属物种个体内部存在nrDNA ITS不完全致同进化,可能归咎于不完全的世系分选(incomplete lineage sorting)、种间杂交或多倍化等进化事件,从而导致基因组内nrITS区序列出现多态性,同时也导致难以通过外部形态来划分亚属内种间界限。     

The evolution and utility of ribosomal ITS sequences in Bambusinae and related species: divergence, pseudogenes, and implications for phylogeny

Ribosomal internal transcribed spacer (ITS) sequences are commonly used for phylogenetic reconstruction because they are highly reiterated as components of rDNA repeats, and hence are often subject to rapid homogenization through concerted evolution. Concerted evolution leads to intragenomic uniformity of repeats even between loci on nonhomologous chromosomes. However, a number of studies have shown that the ITS polymorphism within individuals is quite common. The molecular systematics of Bambusinae and related species were recently assessed by different teams using independently generated ITS sequences, and the results disagreed in some remarkable features. Here we compared the ITS sequences of the members of Bambusa s. l., the genera Dendrocalamus, Dinochloa, Gigantochloa, Guadua, Melocalamus, Monocladus, Oxytenanthera, Thyrsostachys, Pleioblastus, Pseudosasa and Schizostachyum.We have reanalysed the ITS sequences used by different research teams to reveal the underlying patterns of their different results. After excluding the sequences suspected to represent paralogous loci, a phylogenetic analysis of the subtribe Bambusinae species were performed using maximum parsimony and maximum-likelihood methods. The implications of the findings are discussed. The risk of incorporating ITS paralogues in plant evolutionary studies that can distort the phylogenetic signal should caution molecular systematists.     

Phylogenetic analysis of Allium subg. Melanocrommyum infers cryptic species and demands a new sectional classification

Allium subgenus Melanocrommyum (Alliaceae) from Eurasia comprises about 150 mostly diploid species adapted to arid conditions. The group is taxonomically complicated with different and contradictory taxonomic treatments, and was thought to include a considerable number of hybrid species, as the taxa show an admixture of assumed morphological key characters. We studied the phylogeny of the subgenus, covering all existing taxonomic groups and their entire geographic distribution. We analyzed sequences of the nuclear rDNA internal transcribed spacer region (ITS) for multiple individuals of more than 100 species. Phylogenetic analyses of cloned and directly sequenced PCR products confirmed the monophyly of the subgenus, while most sections were either para- or polyphyletic. The splits of the large sections are supported by differences in the anatomy of flower nectaries. ITS data (i) demand a new treatment at sectional level, (ii) do not support the hypotheses of frequent gene flow among species, (iii) indicate that multiple rapid radiations occurred within different monophyletic groups of the subgenus, and (iv) detected separately evolving lineages within three morphologically clearly defined species (cryptic species). In two cases these lineages were close relatives, while in Allium darwasicum they fall in quite different clades in the phylogenetic tree. Fingerprint markers show that this result is not due to ongoing introgression of rDNA (ITS capture) but that genome-wide differences between both lineages exist. Thus, we report one of the rare cases in plants where morphologically indistinguishable diploid species occurring in mixed populations are non-sister cryptic species.     

Conserved 5S and variable 45S rDNA chromosomal localisation revealed by FISH in <Emphasis Type="Italic">Astyanax scabripinnis</Emphasis> (Pisces,Characidae)

Fluorescence in situhybridisation (FISH) and double FISH experiments were carried out to ascertain the chromosomal distribution pattern of the 45S and 5S ribosomal (r) DNAs in four populations of the characid fish Astyanax scabripinnis – a group considered to be a species complex for its wide karyotypical and morphological diversity. The results regarding the 45S rDNA agreed with this hypothesis, since these sites showed intra- and inter-populational, numerical and positional variations. However, the data obtained with the 5S rDNA probe revealed a highly conserved chromosomal distribution pattern of these sequences among individuals of each population, as well as among the populations analysed. We consider this contrasting situation as a functional divergence between 45S and 5S ribosomal DNAs, which may reflect the localisation of these sequences in distinct nuclear compartments, leading them to undergo differentiated evolutionary processes.     

Genetic structure of the endangered species <Emphasis Type="Italic">Pinna nobilis</Emphasis> (Mollusca: Bivalvia) inferred from mtDNA sequences

This study examines the population genetic structure of the endangered bivalve Pinna nobilis (Mollusca: Bivalvia), based on novel mtDNA sequences (partial COI and 16S rDNA mtDNA genes). The analyzed nucleotide sequences of COI were 729 bp in size, coding for a 243 amino acid peptide, while the analyzed nucleotide sequences of 16S rDNA were 489 bp in size. These sequences of P. nobilis were the first DNA sequences of the species submitted to any Genetic Data Base. Population samples from four geographic regions from Greece, as well as a population sample of Atrina fragilis (as an outgroup) were used. High values of haplotypic diversity were found in the population samples of P. nobilis, based on the COI sequences. A single base in the analyzed 16S rDNA sequences was different in all analyzed individuals from a single population sample (Chios island) differentiating it from the other ones. These mtDNA sequences could be informative for further genetic analyses of the endangered species, contributing in conservation plans for its protection and/or aquaculture investigations.     

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.