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.     

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.     

Analysis of the sequences of internal transcribed spacers ITS1, ITS2 and the 5.8S ribosomal gene of species of the Amaranthus genus

Analysis of the sequence ITS1-5.8S-ITS2 in 11 samples of the amaranth species (Amaranthus caudatus, A. cruentus, A. hybridus, A. tricolor, A. paniculatus, A. hypohondriacus) was performed. It has been shown that the variability of the sequences of the intergenic spacers ITS1, ITS2 and 5.8S rRNA gene of the amaranth species analyzed is extremely low. A possible secondary structure of the 5.8S rRNA molecule was determined for the first time; three conservative motifs were identified. A single nucleotide substitution found in A. hybridus did not change the loop topology. In the sample of Celosia cristata taken as an external group, a four-nucleotide insertion in the 5′-end of the gene and a one-nucleotide deletion in the fourth hairpin not affecting the general topology of the 5.8S rRNA molecule were found.     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA

The phylogenetic relationships of the genus Sorghum and related genera were studied by sequencing the nuclear ribosomal DNA (rDNA) internal transcribed spacer region (ITS). DNA was extracted from 15 Sorghum accessions, including one accession from each of the sections Chaetosorghum and Heterosorghum, four accessions from Parasorghum, two accessions from Stiposorghum, and seven representatives from three species of the section Sorghum (one accession from each of S. propinquum and S. halepense, and five races of S. bicolor). The maize (Zea mays) line, H95, and an accession from Cleistachne sorghoides were also included in the study. Variable nucleotides were used to construct a strict consensus phylogenetic tree. The analyses indicate that S. propinquum, S. halepense and S. bicolor subsp. arundinaceum race aethiopicum may be the closest wild relatives of cultivated sorghum; Sorghum nitidum may be the closest 2n=10 relative to S. bicolor, the sections Chaetosorghum and Heterosorghum appear closely related to each other and more closely related to the section Sorghum than Parasorghum; and the section Parasorghum is not monophyletic. The results also indicate that the genus Sorghum is a very ancient and diverse group.This research was partially supported by a Third Country Scholarship from Pioneer Hi-Bred International Incorporated Contribution 94-182-J from Kansas Agricultural Experiment Station     

Evolutionary divergence of the ribosomal internal transcribed spacer 2 (ITS2) in lizards

During the pre-rRNA cleavage pathway, the excision of ITS2, a eukaryotic specific insertion, remains the most elusive processing step, even in yeast. Comparison of the ITS2 sequences in different organisms permits to reveal conservative, presumably functionally important elements as well as obtain new information about ITS2 divergence in evolution. We have cloned and sequenced the ITS2 of three lizard species, Agama caucasia (Agamidae), Darevskia armeniaca and Lacerta strigata (Lacertidae) and detected in them a set of specific and conservative structural elements employing secondary structure consensus for vertebrate ITS2. Furthermore, we have performed an alignment and comparative analysis of the ITS2 sequences for the two lizards families. It enables us to propose that modern lizard species formation in evolution was accompanied by ITS2 duplication in the rDNA of their common progenitors.     

Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution

The ITS sequences of Acropora spp. are the shortest so far identified in any metazoan and are among the shortest seen in eukaryotes; ITS1 was 70-80 bases, and ITS2 was 100-112 bases. The ITS sequences were also highly variable, but base composition and secondary structure prediction indicate that divergent sequence variants are unlikely to be pseudogenes. The pattern of variation was unusual in several other respects: (1) two distinct ITS2 types were detected in both A. hyacinthus and A. cytherea, species known to hybridize in vitro with high success rates, and a putative intermediate ITS2 form was also detected in A. cytherea; (2) A. valida was found to contain highly (29%) diverged ITS1 variants; and (3) A. longicyathus contained two distinct 5.8S rDNA types. These data are consistent with a reticulate evolutionary history for the genus Acropora.      

Length variation in the internal transcribed spacers of ribosomal DNA in Picea abies and related species

The structure and variation of nuclear ribosomal DNA (rDNA) units of Picea abies, (L.) Karst. was studied by restriction mapping and Southern hybridization. Conspicuous length variation was found in the internal transcribed spacer (ITS) region of P. abies, although the length of this region is highly conserved both within and among most of the plant species. Two types of ITS variants (A and B), displaying a size difference of 0.5 kb in the ITS2 region, were present within individuals of P. abies from Sweden, Central Europe and Siberia. A preliminary survey of 14 additional Eurasian and North American species of Picea suggested that length variation in the ITS region is widespread in this genus. Alltogether three length variants (A, B and C) were identified. Within individuals of eight Picea species, two length variants were present within the genome (combinations of A and B variants in P. glehnii, P. maximowiczii, P. omorika, P. polita and P. sitchensis and variants B and C in P. jezoensis, P. likiangensis and P. spinulosa). Within individuals from five species, however only one rDNA variant was present in their genome (variant A in P. aurantiaca, P. engelmannii, P. glauca, P. koraiensis and P. koyamai; variant B in P. bicolor). The ITS length variation will be useful as a molecular marker in evolutionary studies of the Picea species complex, whose phylogeny is controversial. The presence of intraindividual variation in, and shared polymorphism of the, ITS length variants raises questions about the occurrence of interspecific hybridization during the evolutionary history of Picea.     

A phylogenetic analysis of the Illiciaceae based on sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

Sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA were determined for 15 species ofIllicium (Illiciaceae) to examine phylogenetic relationships. The ITS trees show a major dichotomy between the two North American species (I. floridanum andI. parviflorum) and the remaining east Asian species. This suggests that the existing division between two sections (sect.Illicium and sect.Cymbostemon) ofIllicium based on tepal characters in unnatural. The ITS phylogeny shows congruence with palynology: of the species examined, the three species (I. angustisepalum, I. anisatum andI. fargesii) from sect.Illicium that possess trizonocolpate pollen consistently form a clade, although nesting within a clade consisting of the species of sect.Cymbostemon, which generally have trisyncolpate pollen. The low ITS sequence divergence and the close relationship among east Asian species suggest a recent diversification of this group of species or an unusual slowdown of sequence mutations.     

Molecular phylogenetics of the subtribeGentianinae (Gentianaceae) inferred from the sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA from representatives of 23 species of the subtribeGentianinae and one outgroup species (Centaurium capitatum) were analyzed by polymerase chain reaction amplification and direct DNA sequencing. Within the taxa analyzed, the length of the ITS1 region varied from 221 to 233 bp, ITS2 from 226 to 234 bp. Of the aligned sequences of 497 positions, 151 sites involved gaps or nucleotide ambiguity, 133 were invariable and 213 showed divergence. In pairwise comparisons among the taxa of the subtribeGentianinae and the outgroup, sequence divergence ranged from 1.3% to 34.1% in ITS1, from 0 to 28.1% in ITS2 and from 0.6% to 27.5% in combined ITS1 and ITS2. Phylogenetic trees generated from ITS sequences were highly resolutive and principally concordant with morphological classifications for the major phylogenetic divisions in the subtribe. An ancient divergence leading to two evolutionary lines was suggested in the subtribe by both DNA sequence and morphological data. One line encompasses the generaGentiana, Crawfurdia andTripterospermum, morphologically characterized by their glands on the base of ovary and their plicate corolla, while the other line involves all other members of the subcribe surveyed, characterized by their epipetalous glands and simple corolla without plicae.Megacodon, with glands on the base of ovary but without plicae on its corolla, was revealed to be more related to the latter group than to the former.Comastoma, Gentianella andGentianopsis were shown to be well-defined monophyletic genera.Pterygocalyx showed much closer affinity toGentianopsis than to any other genus. Some conflictions were detected in the genusSwertia.     

Nuclear ribosomal internal transcribed spacer 1 (ITS1) variation in the Anastrepha fraterculus cryptic species complex (Diptera,Tephritidae) of the Andean region

The nuclear ribosomal internal transcribed spacer 1 (ITS1) was sequenced for Anastrepha fraterculus (Wiedemann, 1830) originating from 85 collections from the northern and central Andean countries of South America including Argentina (Tucumán), Bolivia, Perú, Ecuador, Colombia, and Venezuela. The ITS1 regions of additional specimens (17 collections) from Central America (México, Guatemala, Costa Rica, and Panamá), Brazil, Caribbean Colombia, and coastal Venezuela were sequenced and together with published sequences (Paraguay) provided context for interpretation. A total of six ITS1 sequence variants were recognized in the Andean region comprising four groups. Type I predominates in the southernmost range of Anastrepha fraterculus. Type II predominates in its northernmost range. In the central and northern Andes, the geographic distributions overlap and interdigitate with a strong elevational effect. A discussion of relationships between observed ITS1 types and morphometric types is included.     

Natural variation for drought-response traits in the Mimulus guttatus species complex

Soil moisture is a key factor affecting plant abundance and distribution, both across and within species. In response to water limitation, plants have evolved numerous morphological, physiological, and phenological adaptations. In both well-watered and water-limited conditions, we identified considerable natural variation in drought-related whole-plant and leaf-level traits among closely related members of the Mimulus guttatus species complex that occupy a diversity of habitats in the field. The self-fertilizing Mimulus nasutus and serpentine-endemic Mimulus nudatus demonstrated the overall greatest tolerance to soil water limitation, exhibiting the smallest reduction in seed set relative to well-watered conditions. This may be due in part to early flowering, faster fruit development, and low stomatal density. In contrast, flowering of coastal M. guttatus was so delayed that it precluded any seed production in water-limited conditions. This range of phenotypic responses to soil water deficit in Mimulus, coupled with developing genomic resources, holds considerable promise for identifying genomic variation responsible for adaptive responses to soil water availability.     

Sequence differences in the internal transcribed spacers of DNA among four species of hookworm (Ancylostomatoidea: Ancylostoma)

The two ribosomal DNA internal transcribed spacers (1 and 2) of the hookworms Ancylostoma caninum, A. tubaeforme, A. ceylanicum and A. duodenale were sequenced. The sequence lengths were similar among the four species, except that A. ceylanicum had slightly longer (by 5–7 bp) internal transcribed spacer 1 and 2 sequences. The predicted secondary structure of the internal transcribed spacer 2 precursor rRNA was similar for all species, despite interspecific differences in primary sequence ranging from 0.9% to 13.2%. Interspecific differences in internal transcribed spacer 1 sequence ranged from 0.9% to 7.5%. A cladistic analysis of the sequence data, using the human hookworm Necator americanus as the outgroup, provided little resolution of the phylogenetic relationships, except that A. ceylanicum occurred on a branch external to the other three species. Nonetheless, internal transcribed spacers 1 and 2 may provide useful phylogenetic information at higher taxonomic levels within the superfamily Ancylostomatoidea.     

Sequence analysis of the ribosomal internal transcribed spacers region in psocids (Psocoptera: Liposcelididae) for phylogenetic inference and species discrimination

Psocids (Psocoptera: Liposcelididae: Liposcelis spp.) are major pests of stored grain and commonly occur on a wide range of stored products. Increasingly, the genus of Liposcelis has gained recognition of their importance due to their feeding on stored grains, contaminating food, and agricultural commodities as well as transmitting harmful microorganisms, including fungi and bacteria. Psocids have close morphological similarities and often commix occur at the same ecosystems. Therefore, a first step necessary to further implement population studies is the accurate identification of species, based on molecular methods. In this study, we determined nucleotide sequences of the nuclear rDNA internal transcribed spacer (ITS)1-5.8S-ITS2 region in 100 individuals of six Liposcelis species (including Liposcelis bostrychophila Badonnel, Liposcelis entomophila (Enderlein), Liposcelis decolor (Pearman), Liposcelis tricolor Badonnel, Liposcelis paeta Pearman, and Liposcelis yunnaniensis Li & Li) from 16 locations of China. We evaluated the suitability of this marker for phylogenetic inference study in the Liposcelis species. We also developed a molecular identification method for six Liposcelis species based on ITS2 sequence. Results demonstrate that ITS1-5.8S-ITS2 sequences are a useful tool for the population genetic study and phylogeny estimation of Liposcelis species. The results of this study indicate that the ITS2 sequences can be a reliable tool for species discrimination of the six species of psocids tested here. In addition, the multiplex method described proved reliable when tested across different geographical populations.     

Variation in the nuclear ribosomal DNA internal transcribed spacer (ITS) region of Pinus rzedowskii revealed by PCR-RFLP

 In the genus Pinus the internal transcribed spacers (ITS1 and ITS2) and the 5.8s region of the nuclear ribosomal DNA are approximately 3000 bp in length. ITS1 is considerably longer than ITS2 and partial sequences of ITS1 indicate that this region is evolving rapidly and exhibits intraspecific variation. The ITS2 and 5.8s regions are relatively conserved. We surveyed restriction fragment length variability of PCR-amplified fragments (PCR-RFLP) of the ITS region in four populations (86 individuals) of Pinus rzedowskii, a pine endemic to western Michoacán, Mexico. Five of the restriction endonucleases assayed revealed variation, with a total of 13 variants, most of which were length mutations of 300–900 bp. A moderate degree of population differentiation was detected. The average diversity (Shannon’s index) of ITS fragment size patterns was 1.19, with 34% of the variation due to differences among populations and 66% due to differences among individuals within populations. The same individuals were assayed for nine polymorphic isozymes, which gave diversity measures similar to those of each restriction endonuclease. Received: 25 August 1997 / Accepted: 19 September 1997     

Infrageneric phylogeny of the genus Gentiana (Gentianaceae) inferred from nucleotide sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacers (ITSs) of nuclear ribosomal DNA have been sequenced for 20 species of Gentiana. By incorporating previously released sequence data of eight species, phylogenelic analyses using Fitch parsimony and character-state weighted parsimony were carried out. The length of ITS 1 in the taxa surveyed ranged from 223 to 238 bp and ITS2 from 216 to 234 bp. Sequence divergence between pairs of species ranged from 5.0% to 48.9% in ITS1, from 1.1% to 45.3% in ITS2, and from 3.2% to 46.1% in combined data of ITS1 and ITS2. The ITS phylogeny was generally congruent with morphological classifications except that G. asclepiadea was revealed to be closely related to section Gentiana instead of section Pneumonanthe and section Stenogyne was shown to be a paraphyletic group of the genus Gentiana that would be better excluded from the genus. A divergence among the three European endemic sections and the remaining sections of the genus other than section Stenogyne was revealed. Thus the European species of the genus together do not form a monophyletic group. A close relationship between the sections Chondrophyllae s. l. (including section Dolichocarpa), Cruciata and Pneumonanthe was suggested. The section Frigidae s. l. (including sections Monopodiae, Isomeria, Microsperma, and Phyllocalyx) contained two well-supported clades: section Frigidae s. str. and all others together. The monophyly of the typically dysploid group section Chondrophyllae s. l. was confirmed. Optimization of chromosome numbers on the ITS phylogeny suggested that 2/1 = 26 is a plesiomorphic state for the clade comprising sections Frigidae s. l., Cruciata, Pneumonanthe, and Chondrophyllae s. l., and probably 2n = 20 is a plesiomorphic state for the dysploid group, section Chondrophyllae s. l.