Nuclear ribosomal DNA internal transcribed spacer 1 (ITS1) in Picea (Pinaceae): sequence divergence and structure

The nrDNA ITS1 of Picea is 2747-3271 bp, the longest known of all plants. We obtained 24 cloned ITS1 sequences from six individuals of Picea glehnii, Picea mariana, Picea orientalis, and Picea rubens. Mean sequence divergence within these individuals (0.018+/-0.009) is more than half that between the species (0.031+/-0.011) and may be maintained against concerted evolution by separation of Picea 18S-26S rDNA repeats on multiple chromosomes. Picea ITS1 contains three subrepeats with a motif (5'-GGCCACCCTAGTC) that is conserved across Pinaceae. Two subrepeats are tandem, remote from the third, and more closely related and significantly more similar to one another than either is to the third subrepeat. This correlation between similarity and proximity may be the result of subrepeat duplication or concerted evolution within rDNA repeats. In inferred secondary structures, subrepeats generally form long hairpins, with a portion of the Pinaceae conserved motif in the terminal loop, and tandem subrepeats pair with one another over most of their length. Coalescence of ITS1 sequences occurs in P. orientalis but not in the other species.     

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.     

Differentiation of two ovine Babesia based on the ribosomal DNA internal transcribed spacer (ITS) sequences

The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene for six Babesia spp. isolated from different geographic origins were characterized. Varying degrees of ITS1 and ITS2 intra- and inter-species sequence polymorphism were found among these isolates. Phylogenetic analysis of the ITS1-5.8S gene-ITS2 region clearly separated the isolates into two clusters. One held an unidentified Babesia sp. transmitted by Hyalomma anatolicum anatolicum. The second held five other isolates, which were considered to be Babesia motasi. Each Babesia species cluster possessed ITS1 and ITS2 of unique size(s) and species specific nucleotide sequences. The results showed that ITS1, ITS2 and the complete ITS1-5.8S-ITS2 region could be used to discriminate these ovine Babesia spp. effectively.     

Origin and relationships of Saintpaulia (Gesneriaceae) based on ribosomal DNA internal transcribed spacer (ITS) sequences

Phylogenetic relationships of eight species of Saintpaulia H. Wendl., 19 species of Streptocarpus Lindl. (representing all major growth forms within the genus), and two outgroups (Haberlea rhodopensis Friv., Chirita spadiciformis W. T. Wang) were examined using comparative nucleotide sequences from the two internal transcribed spacers (ITS) of nuclear ribosomal DNA. The length of the ITS 1 region ranged from 228 to 249 base pairs (bp) and the ITS 2 region from 196 to 245 bp. Pairwise sequence divergence across both spacers for ingroup and outgroup species ranged from 0 to 29%. Streptocarpus is not monophyletic, and Saintpaulia is nested within Streptocarpus subgenus Streptocarpella. Streptocarpus subgenus Streptocarpus is monophyletic. The ITS sequence data demonstrate that the unifoliate Streptocarpus species form a clade, and are also characterized by a unique 47-bp deletion in ITS 2. The results strongly support the monophyly of (1) Saintpaulia, and (2) Saintpaulia plus the African members of the subgenus Streptocarpella of Streptocarpus. The data suggest the evolution of Saintpaulia from Streptocarpus subgenus Streptocarpella. The differences in flower and vegetative characters are probably due to ecological adaptation leading to a relatively rapid radiation of Saintpaulia.     

Phylogenetic relationships in Lupinus (Fabaceae: Papilionoideae) based on internal transcribed spacer sequences (ITS) of nuclear ribosomal DNA

Internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA from 44 taxa of the genus Lupinus and five outgroup taxa were used for phylogenetic analysis. Lupinus appears as a strongly supported monophyletic genus, which is unambiguously part of the Genisteae. The lupines are distributed into five main clades in general accordance with their geographical origin. In the Old World, almost all the recognized taxonomic units are well resolved. The ITS data reveal an unexpectedly close relationship between the diverse sections Angustifoli and Lutei. The ITS results suggest a geographical division between the western New World lupines and the eastern ones. They also indicate the presence of some moderately to strongly supported groups of taxa, such as the Microcarpi-Pusilli group, the L. spariflorus-L. arizonicus group, the L. mexicanus-L. elegans group in the western New World, and the notable L. multiflorus-L. paraguariensis group in the eastern New World. The latter group strongly suggests that the eastern South American compound- and simple-leaved perennial lupines derive from a common ancestor. However, apart from some exceptions, relationships within the genus still remain largely unresolved based on ITS data. The lack of resolution at the base of the genus is suggestive of a rapid initial radiation of the lupines subsequent to the dispersal of their common ancestor. Relative rate tests demonstrate the presence of rate heterogeneity of ITS sequences within Lupinus. In many pairwise comparisons between taxa, substitution rate inequalities are correlated with the habit (annual, perennial), suggesting some role for the generation time effects in the evolutionary history of lupines.     

Evolutionary relationships of the salmonid fish genus Salvelinus inferred from DNA sequences of the first internal transcribed spacer (ITS 1) of ribosomal DNA.

DNA sequences of the ribosomal DNA (rDNA) first internal transcribed spacer region (ITS 1) of six species of the salmonid fish genus Salvelinus (alpinus, malma, confluentus, leucomaenis, fontinalis, and namaycush) and the closely related species Hucho perryi were determined. Phylogenetic analysis of the aligned sequences by both phenetic and cladistic methods with H. perryi as an outgroup generated one best topology which pairs S. alpinus with S. malma as the most recently derived species, and pairs S. confluentus with S. leucomaenis. Three other possible topologies favor the pairing of S. namaycush and S. fontinalis, with one tree placing them on separate branches, and vary the branching order of the interior groups. These results agree with previous studies based on comparisons of morphologies, isozymes, karyotypes, and restriction sites showing a close genetic relationship and possible hybridization between the members of this genus.     

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.     

Lengths and nucleotide sequences of the internal spacers of nuclear ribosomal DNA in gymnosperms and pteridophytes

The nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA were analyzed in species belonging to gymnosperms and pteridophytes. The lengths of the ITSs of sixteen species of gymnosperms and seven species of pteridophytes were estimated. The gymnosperms have ITS1 regions larger than those observed in the pteridophytes and angiosperms (ca. 610–3100 bp versus 159–360 bp). On the other hand, the ITS2 regions appear to be of a conserved length (182–370 bp). We have determined the complete nucleotide sequences of ITS regions from four gymnosperm species and five pteridophyte species by cloning the PCR products. Sequence analysis showed the presence of three short tandem arranged subrepeats of about 70 bp in the 1112 bp ITS1 ofEphedra fragilis. Pyrimidine rich (about 90%) DNA segments of 40–50 bp were observed in the ITS1 ofGinkgo biloba. A highly conserved 16 bp long sequence known to be present in the ITS1 of the angiosperm species has been also found in the ITS1 ofCycas revoluta, Taxus baccata andEphedra fragilis. Dedicated to Prof.Emilio Battaglia.     

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.     

Delineation of the European Armillaria species based on the sequences of the internal transcribed spacer (ITS) of ribosomal DNA

Variation within the internal transcribed spacer (ITS) of the ribosomal RNA gene of 15 isolates representing seven European Armillaria species, was examined by sequencing of the PCR-amplified products. The analysis of an 744-bp region showed that the 5.8S gene appeared to be highly conserved in the 15 isolates and in other Basidiomycetes and Ascomycetes, whereas ITS1 and especially ITS2 spacers exhibited polymorphisms due to base substitutions, insertions or deletions of up to eight nucleotides. An initial dendrogram for the full sequence was drawn using cluster analysis (UPGMA), and a tree was constructed using the maximum parsimony method. Both methods indicated that the isolates could be divided into four major groups. One group, consisting of A. ectypa , was distinct from all the other species. Examination of the other groups indicated that A. tabescens and A. mellea were in a separated cluster, with a significant variation between the two isolates of the latter species. A. gallica and A. cepistipes constituted another closely related group distinguishable from A. ostoyae and A. borealis , these latter two species exhibiting the highest similarity. The results are consistent with, and discussed in regard to, the relationships estimated previously by pairing tests, morphological and physiological comparisons, as well as by restriction fragment length polymorphism of the rDNA.     

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.     

A phylogeny of Thermopsideae (Leguminosae: Papilionoideae) inferred from nuclear ribosomal internal transcribed spacer (ITS) sequences

Based on nuclear ribosomal DNA internal transcribed spacer (ITS) sequences, Thermopsideae is phylogenetically studied within a genistoid background. Analysis reveals that the tribe is not supported as a monophyletic group. Some species of Sophora s.s are nested within it. The central Asian desert Ammopiptanthus forms an isolated clade but is relatively remote to other Thermopsideae members. Piptanthus , Anagyris , Baptisia , and Thermopsis are clustered together into a robust clade. We hence propose that the tribe could either be reduced to just the four 'core genera' with Ammopiptanthus excluded, or, as an alternative, that Thermopsideae could become part of a new Sophoreae s.s. if it is re-circumscribed in the future. Both Piptanthus and Baptisia appear as monophyletic. The genus Anagyris is closer to some east Asian Thermopsis species than to Piptanthus . The east Asian and North American disjunct Thermopsis is not monophyletic. The ITS results suggest a geographical division between the Old World and New World Thermopsis . The east Asian species are clustered with Piptanthus and Anagyris , whereas the North American species are allied to Baptisia . Nonetheless, the only two north-eastern east Asian native Thermopsis species appear to be more related to the North American group than to the east Asian one. The related biogeographical significance has therefore been additionally discussed.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 151 , 365–373.     

Yeast gene SRP1 (serine-rich protein). Intragenic repeat structure and identification of a family of SRP1-related DNA sequences

We have isolated and sequenced a yeast gene encoding a protein (Mr 24,875) very rich in serine (SRP) and alanine residues that accounted for 25% and 20% of the total amino acids, respectively. The SRP1 gene is highly expressed in culture conditions leading to glucose repression (Marguet & Lauquin, 1986), the amount of SRP1 mRNA representing about 1 to 2% of total poly(A)+ RNA. A repetitive structure of eight direct tandem repeats 36-base long, also reflected in the amino acid sequence, was found in the second half of the open reading frame. The consensus amino acid sequence of the repeat was Ser-Ser-Ser-Ala-Ala-Pro-Ser-Ser-Ser-Glu-Ala-Lys. Replacing the genomic copy of the cloned gene with a disrupted SRP1 gene indicated that the SRP1 gene was not essential for viability in yeast, but several SRP1-homologous sequences were found within the yeast genome, raising the possibility that the disrupted SRP1 gene is rescued by one of the other SRP-homologous sequences. Complete separation of yeast chromosomes by contour-clamped homogeneous field electrophoresis indicated that, apart from chromosome V, which carries the SRP1 gene, 12 chromosomes have SRP-related sequences with various degrees of homology. These sequences were located on chromosomes XV, VII and XI under stringent conditions of hybridization (tm -20 degrees C), and observed on chromosomes I, II, III, IV, VI, VIII, X, XI and XII, only under low-stringency conditions (tm -40 degrees C). Northern blot analysis of both the wild type and SRP1-disrupted strains indicated that along with SRP1 at least one more member of the SRP family was transcribed to a 0.7 kb (1 kb = 10(3) bases) polyadenylated RNA species clearly distinct from the SRP1-specific mRNA (1 kb long). Analyses of the SRP1 repeat domain suggested a model for the divergent evolution of the repeats in the SRP1 sequence.     

Reticulate evolution and phylogeography in Asarum sect. Asiasarum (Aristolochiaceae) documented in internal transcribed spacer sequences (ITS) of nuclear ribosomal DNA

Phylogenetic analyses were performed for the taxonomically complicated group, Asarum sect. Asiasarum, using internal transcribed spacers (ITS) of nuclear ribosomal DNA. Direct sequences for 99 samples of a total of 14 taxa and geographic races and cloning analyses for 32 of these samples provided new insights that extensive reticulate evolution has occurred in this group. Eight taxa had slight or no polymorphism of the ITS sequences. On the other hand, the other five taxa had polymorphic ITS sequences composed of two ribotypes that were completely the same or almost the same as the sequences recognized in the taxa with only slight or no polymorphism, and were probably of diploid hybrid origin and to have retained their parental ribotypes. In terms of biogeographic implications, at least four interactions including migration, hybridization, and introgression, were presumed between the Japanese Archipelago and the continents, two times via a southern route, from the Korean Peninsula, and two times via a northern route, from Sakhalin or directly from the Eurasian continent.     

Molecular phylogeny ofCheirolophus (Asteraceae:Cardueae-Centaureinae) based on ITS sequences of nuclear ribosomal DNA

The genusCheirolophus has an interesting western Mediterranean and Macaronesian distribution. Here we investigate the delimitation of the genus and its exclusion from the large genusCentaurea, the systematic position of the related genusPaleocyanus, the delimitation of some species and the phylogeny of the group. We have carried out a phylogenetic analysis of the PCR-generated sequences of the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. The results suggest that the genus, includingPaleocyanus crassifolius is monophyletic; thus, a new combination of this species underCheirolophus is proposed. The Macaronesian group of species is also monophyletic, indicating a single colonization of the archipelago. The poor resolution of microspecies in the Macaronesian group reinforces the hypothesis of a very recent differentiation of the group.     

Sterols of the heterotrophic dinoflagellate,pfiesteria piscicida (dinophyceae): is there a lipid biomarker?1

Within U.S. waters, blooms of the dinoflagellate, Pfiesteria piscicida, have been recorded on an almost regular basis in the Chesapeake Bay and surrounding mid‐Atlantic regions for the last two decades. Despite the apparent significance of such blooms to the environment and human health and the attendant economic consequences, little work has addressed the physiology and biochemistry, particularly that of sterol composition, of P. piscicida. GC‐MS characterization of trimethylsilyl ether derivatives of sterols from free sterol and sterol ester fractions was performed in an effort to determine whether P. piscicida produces unique sterols that may serve as potential biomarkers. This characterization revealed that like most dinoflagellates, the majority of sterols was present as free sterols. Furthermore, the profile of free sterols was found to resemble those of photosynthetic dinoflagellates, with the dominant compound being the previously reported dinoflagellate sterol, dinosterol. A number of other 4α‐methyl‐substituted sterols and steroidal ketones common to other dinoflagellates were also identified. No strong candidate(s) for a unique sterol biomarker was present.     

Studies on the origin and evolution of tetraploid wheats based on the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA

In this study, the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA in the tetraploid wheats, Triticum turgidum (AABB) and Triticum timopheevii (AAGG), their possible diploid donors, i.e., Triticum monococcum (AA), Triticum urartu (AA), and five species in Aegilops sect. Sitopsis (SS genome), and a related species Aegilops tauschii were cloned and sequenced. ITS1 and ITS2 regions of 24 clones from the above species were compared. Phylogenetic analysis demonstrated that Aegilops speltoides was distinct from other species in Aegilops sect. Sitopsis and was the most-likely donor of the B and G genomes to tetraploid wheats. Two types of ITS repeats were cloned from Triticum turgidum ssp. dicoccoides, one markedly similar to that from T. monococcum ssp. boeoticum (AA), and the other to that from Ae. speltoides (SS). The former might have resulted from a recent integression event. The results also indicated that T. turgidum and T. timopheevii might have simultaneously originated from a common ancestral tetraploid species or be derived from two hybridization events but within a very short interval time. ITS paralogues in tetraploid wheats have not been uniformly homogenized by concerted evolution, and high heterogeneity has been found among repeats within individuals of tetraploid wheats. In some tetraploid wheats, the observed heterogeneity originated from the same genome (B or G). Three kinds of ITS repeats from the G genome of an individual of T. timopheevii ssp. araraticum were more divergent than that from inter-specific taxa. This study also demonstrated that hybridization and polyploidization might accelerate the evolution rate of ITS repeats in tetraploid wheats.