Conservation patterns in angiosperm rDNA ITS2 sequences.

The two internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA have become commonly exploited sources of informative variation for interspecific-/intergeneric-level phylogenetic analyses among angiosperms and other eukaryotes. We present an alignment in which one-third to one-half of the ITS2 sequence is alignable above the family level in angiosperms and a phenetic analysis showing that ITS2 contains information sufficient to diagnose lineages at several hierarchical levels. Base compositional analysis shows that angiosperm ITS2 is inherently GC-rich, and that the proportion of T is much more variable than that for other bases. We propose a general model of angiosperm ITS2 secondary structure that shows common pairing relationships for most of the conserved sequence tracts. Variations in our secondary structure predictions for sequences from different taxa indicate that compensatory mutation is not limited to paired positions.     

The ITS2 Database

The internal transcribed spacer 2 (ITS2) has been used as a phylogenetic marker for more than two decades. As ITS2 research mainly focused on the very variable ITS2 sequence, it confined this marker to low-level phylogenetics only. However, the combination of the ITS2 sequence and its highly conserved secondary structure improves the phylogenetic resolution¹ and allows phylogenetic inference at multiple taxonomic ranks, including species delimitation^2-8.The ITS2 Database⁹ presents an exhaustive dataset of internal transcribed spacer 2 sequences from NCBI GenBank¹¹ accurately reannotated¹⁰. Following an annotation by profile Hidden Markov Models (HMMs), the secondary structure of each sequence is predicted. First, it is tested whether a minimum energy based fold¹² (direct fold) results in a correct, four helix conformation. If this is not the case, the structure is predicted by homology modeling¹³. In homology modeling, an already known secondary structure is transferred to another ITS2 sequence, whose secondary structure was not able to fold correctly in a direct fold.The ITS2 Database is not only a database for storage and retrieval of ITS2 sequence-structures. It also provides several tools to process your own ITS2 sequences, including annotation, structural prediction, motif detection and BLAST¹⁴ search on the combined sequence-structure information. Moreover, it integrates trimmed versions of 4SALE^15,16 and ProfDistS¹⁷ for multiple sequence-structure alignment calculation and Neighbor Joining¹⁸ tree reconstruction. Together they form a coherent analysis pipeline from an initial set of sequences to a phylogeny based on sequence and secondary structure.In a nutshell, this workbench simplifies first phylogenetic analyses to only a few mouse-clicks, while additionally providing tools and data for comprehensive large-scale analyses.     

Evolutionarily conserved structural features in the ITS2 of mammalian pre-rRNAs and potential interactions with the snoRNA U8 detected by comparative analysis of new mouse sequences.

Mechanisms of ITS2 excision from pre-rRNA remain largely elusive. In mammals, at least two endonucleolytic cleavages are involved, which result in the transient accumulation of precursors to 5.8S rRNA termed 8S and 12S RNAs. We have sequenced ITS2 in four new species of the Mus genus and investigated its secondary structure using thermodynamic prediction and comparative approach. Phylogenetic evidence supports an ITS2 folding organized in four domains of secondary structure extending from a preserved structural core. This folding is also largely conserved for the previously available mammalian ITS2 sequences, rat and human, despite their extensive sequence divergence relative to the Mus species. Conserved structural features include the structural core, containing the 3' end of 8S pre-rRNA within a single-stranded sequence, and a stem containing the 3' end of the 12S pre-rRNA species. A putative, phylogenetically preserved pseudoknot has been detected 1 nt downstream from the 12S 3' end. Two long complementarities have also been identified, in sequences conserved among vertebrates, between the pre-rRNA 32S and the snoRNA (small nucleolar RNA) U8 which is required for the excision of Xenopus ITS2. The first complementarity involves the 5.8S-ITS2 junction and 13 nt at the 5' end of U8, whereas the other one occurs between a mature 28S rRNA segment known to be required for ITS2 excision and positions 15-25 of snoRNA U8. These two potential interactions, in combination with ITS2 folding, could organize a functional pocket containing three cleavage sites and key elements for pre-rRNA processing, suggesting a chaperone role for the snoRNA U8.     

Internal transcribed spacer 2 (nu ITS2 rRNA) sequence-structure phylogenetics: towards an automated reconstruction of the green algal tree of life

Background

Chloroplast-encoded genes (matK and rbcL) have been formally proposed for use in DNA barcoding efforts targeting embryophytes. Extending such a protocol to chlorophytan green algae, though, is fraught with problems including non homology (matK) and heterogeneity that prevents the creation of a universal PCR toolkit (rbcL). Some have advocated the use of the nuclear-encoded, internal transcribed spacer two (ITS2) as an alternative to the traditional chloroplast markers. However, the ITS2 is broadly perceived to be insufficiently conserved or to be confounded by introgression or biparental inheritance patterns, precluding its broad use in phylogenetic reconstruction or as a DNA barcode. A growing body of evidence has shown that simultaneous analysis of nucleotide data with secondary structure information can overcome at least some of the limitations of ITS2. The goal of this investigation was to assess the feasibility of an automated, sequence-structure approach for analysis of IT2 data from a large sampling of phylum Chlorophyta.

Methodology/Principal Findings

Sequences and secondary structures from 591 chlorophycean, 741 trebouxiophycean and 938 ulvophycean algae, all obtained from the ITS2 Database, were aligned using a sequence structure-specific scoring matrix. Phylogenetic relationships were reconstructed by Profile Neighbor-Joining coupled with a sequence structure-specific, general time reversible substitution model. Results from analyses of the ITS2 data were robust at multiple nodes and showed considerable congruence with results from published phylogenetic analyses.

Conclusions/Significance

Our observations on the power of automated, sequence-structure analyses of ITS2 to reconstruct phylum-level phylogenies of the green algae validate this approach to assessing diversity for large sets of chlorophytan taxa. Moreover, our results indicate that objections to the use of ITS2 for DNA barcoding should be weighed against the utility of an automated, data analysis approach with demonstrated power to reconstruct evolutionary patterns for highly divergent lineages.     

Evaluation of the internal transcribed spacer 2 (ITS2) as a molecular marker for phylogenetic inference using sequence and secondary structure information in blow flies (Diptera: Calliphoridae)

The internal transcribed spacer 2 (ITS2) is a small non-coding region located inside the nuclear ribosomal DNA cluster. ITS2 sequence variability is thought to be appropriate to differentiate species and for phylogenetic reconstructions analyses, which can be further improved if structural information is considered. We evaluated the potential of ITS2 as a molecular marker for phylogenetic inference in Calliphoridae (Diptera: Brachycera) using a broad range of inference methods and different substitution models, accounting or not for structural information. Sequence analyses revealed a hierarchically organized pattern of sequence variation and a small level of nucleotide substitution saturation. Intragenomic variation due to small sequence repeats was found mainly in the most variable domain (IV), but it has no significant impact on the phylogenetic signal at the species level. Inferred secondary structures revealed that GC pairs are more frequently found flanking bulges and loops regions in more conserved domains, thus ensuring structure stability. In the phylogenetic analyses, the use of substitution models accounting for structural information significantly improves phylogenetic inference in both neighbour-joining and Bayesian analyses, although the former provides limited resolution for dealing with highly divergent sequences. For Bayesian analyses, a significant improvement in likelihood was observed when considering structure information, although with small changes in topology and overall support, probably reflecting better evolutionary rates estimates. Based on these findings, ITS2 is a suitable molecular marker for phylogenetic analyses in Calliphoridae, at both species and generic level.     

The rpb2 gene represents a viable alternative molecular marker for the analysis of environmental fungal communities

Although the commonly used internal transcribed spacer region of rDNA (ITS) is well suited for taxonomic identification of fungi, the information on the relative abundance of taxa and diversity is negatively affected by the multicopy nature of rDNA and the existence of ITS paralogues. Moreover, due to high variability, ITS sequences cannot be used for phylogenetic analyses of unrelated taxa. The part of single‐copy gene encoding the second largest subunit of RNA polymerase II (rpb2) was thus compared with first spacer of ITS as an alternative marker for the analysis of fungal communities in spruce forest topsoil, and their applicability was tested on a comprehensive mock community. In soil, rpb2 exhibited broad taxonomic coverage of the entire fungal tree of life including basal fungal lineages. The gene exhibited sufficient variation for the use in phylogenetic analyses and taxonomic assignments, although it amplifies also paralogues. The fungal taxon spectra obtained with rbp2 region and ITS1 corresponded, but sequence abundance differed widely, especially in the basal lineages. The proportions of OTU counts and read counts of major fungal groups were close to the reality when rpb2 was used as a molecular marker while they were strongly biased towards the Basidiomycota when using the ITS primers ITS1/ITS4. Although the taxonomic placement of rbp2 sequences is currently more difficult than that of the ITS sequences, its discriminative power, quantitative representation of community composition and suitability for phylogenetic analyses represent significant advantages.     

Cryptic diversity of the symbiotic cyanobacterium Synechococcus spongiarum among sponge hosts

Cyanobacteria are common members of sponge-associated bacterial communities and are particularly abundant symbionts of coral reef sponges. The unicellular cyanobacterium Synechococcus spongiarum is the most prevalent photosynthetic symbiont in marine sponges and inhabits taxonomically diverse hosts from tropical and temperate reefs worldwide. Despite the global distribution of S. spongiarum , molecular analyses report low levels of genetic divergence among 16S ribosomal RNA (rRNA) gene sequences from diverse sponge hosts, resulting either from the widespread dispersal ability of these symbionts or the low phylogenetic resolution of a conserved molecular marker. Partial 16S rRNA and entire 16S–23S rRNA internal transcribed spacer (ITS) genes were sequenced from cyanobacteria inhabiting 32 sponges (representing 18 species, six families and four orders) from six geographical regions. ITS phylogenies revealed 12 distinct clades of S. spongiarum that displayed 9% mean sequence divergence among clades and less than 1% sequence divergence within clades. Symbiont clades ranged in specificity from generalists to specialists, with most (10 of 12) clades detected in one or several closely related hosts. Although multiple symbiont clades inhabited some host sponges, symbiont communities appear to be structured by both geography and host phylogeny. In contrast, 16S rRNA sequences were highly conserved, exhibiting less than 1% sequence divergence among symbiont clades. ITS gene sequences displayed much higher variability than 16S rRNA sequences, highlighting the utility of ITS sequences in determining the genetic diversity and host specificity of S. spongiarum populations among reef sponges. The genetic diversity of S. spongiarum revealed by ITS sequences may be correlated with different physiological capabilities and environmental preferences that may generate variable host–symbiont interactions.     

Sequence Analysis of the Internal Transcribed Spacer 2 (ITS2) from Yeast Species Within the Genus Candida

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) regions were determined for 13 species within the genus Candida, representing a collection of those species pathogenic for humans. No two species had identical sequences and the sizes of ITS2 varied fourfold, representing an apparent continuous gradient of nucleotides. When present, sequence homologies were observed in the 5′ end of ITS2, and many species exhibited more limited homologies within three known conserved domains found in other yeasts. Cluster analysis of primary sequence revealed a concordance with a known taxonomic subfamily and suggests that certain species within the genus form a similar grouping. A majority of species exhibited similar presumptive RNA secondary structures, consistent with the hypothesis that these spacer regions are essential for correct processing of the 5.8S and 28S subunits. Received: 14 April 1997 / Accepted: 22 July 1997     

Molecular Evolution and Phylogenetic Utility of the Internal Transcribed Spacer 2 (ITS2) in Calyptratae (Diptera: Brachycera)

The resolution potential of internal transcribed spacer 2 (ITS2) at deeper levels remains controversial. In this study, 105 ITS2 sequences of 55 species in Calyptratae were analyzed to examine the phylogenetic utility of the spacer above the subfamily level and to further understand its evolutionary characteristics. We predicted the secondary structure of each sequence using the minimum-energy algorithm and constructed two data matrixes for phylogenetic analysis. The ITS2 regions of Calyptratae display strong A-T bias and slight variation in length. The tandem and dispersed repeats embedded in the spacers possibly resulted from replication slippage or transposition. Most foldings conformed to the four-domain model. Sequence comparison in combination with the secondary structures revealed six conserved motifs. Covariation analysis from the conserved motifs indicated that the secondary structure restrains the sequence evolution of the spacer. The deep-level phylogeny derived from the ITS2 data largely agreed with the phylogenetic hypotheses from morphologic and other molecular evidence. Our analyses suggest that the accordant resolutions generated from different analyses can be used to infer deep-level phylogenetic relations.     

基于ITS序列的东亚当归属植物的分类学研究

采用PCR直接测序法,测定了东亚地区狭义当归属Angelica s.s.及其近缘共7属40种代表植物的核糖体DNA ITS序列,并结合GenBank中相关植物的ITS序列(含外类群3种),应用遗传距离与系统树分析法对东亚地区狭义当归属植物内部以及当归属与其近缘属植物之间的亲缘关系进行了分析。结果表明:(1)广义当归属中的狭义当归属、柳叶芹属Czernaevia和高山芹属Coelopleurum之间的亲缘关系较近,山芹属Ostericum与它们的亲缘关系较远,这与果实形态、化学分析的结果一致,建议将山芹属作为一个相对独立的分类群处理。(2)ITS序列分析结果支持狭义当归属不是单起源的自然分类群,而应该被分成若干组的观点。(3)ITS序列以及化学成分分析结果表明,前胡属Peucedanum与狭义当归属之间的亲缘关系很近。(4)形态、化学成分以及ITS等多方面分析结果显示,当归A.sinensis与狭义当归属的多数植物之间均有一定的差距,其归属问题值得商榷。(5)ITS序列与化学成分的分析结果均显示藁本属Ligusticum不是一个自然类群。     

Evolution of rDNA ITS1 and ITS2 sequences and RNA secondary structures within members of the fungal genera Grosmannia and Leptographium

The two internal transcribed spacers (ITS) of the nuclear ribosomal (r) DNA tandem repeat were examined in ophiostomatoid fungi belonging to the genera Grosmannia and Leptographium and closely-related taxa. Although the DNA sequence of the ITS region evolves rapidly, core features of the RNA secondary structure of the ITS1 and ITS2 segments are conserved. The results demonstrate that structural conservation of GC-rich helical regions is facilitated primarily through compensatory base changes (CBCs), hemi-CBCs, and compensating insertions/deletions (indels), although slippage of the RNA strand is potentially an additional mechanism for maintaining basepairing interactions. The major conclusion of the structural analysis of both ITS segments is that two factors appear to be involved in limiting the type of changes observed: a high GC bias for both ITS1 and ITS2 and structural constraints at the RNA level.     

Secondary structure models of the nuclear internal transcribed spacer regions and 5.8S rRNA in Calciodinelloideae (Peridiniaceae) and other dinoflagellates

Secondary structure models of the 5.8S rRNA and both internal transcribed spacers (ITS1 and ITS2) are proposed for Calciodinelloideae (Peridiniaceae) and are also plausible for other dinoflagellates. The secondary structure of the 5.8S rRNA corresponds to previously developed models, with two internal paired regions and at least one 5.8S rRNA–28S rRNA interaction. A general secondary structure model of ITS1 for Calciodinelloideae (and other dinoflagellates), consisting of an open multibranch loop with three major helices, is proposed. The homology of these paired regions with those found in other taxa, published in previous studies (e.g. yeast, green algae and Platyhelmithes) remains to be determined. Finally, a general secondary structure model of ITS2 for Calciodinelloideae (and other dinoflagellates) is reconstructed. Based on the 5.8S rRNA–28S rRNA interaction, it consists of a closed multibranch loop, with four major helices. At least helix III and IV have homology with paired regions found in other eukaryotic taxa (e.g. yeast, green algae and vertebrates). Since the secondary structures of both ITS regions are more conserved than the nucleotide sequences, their analysis helps in understanding molecular evolution and increases the number of structural characters. Thus, the structure models developed in this study may be generally useful for future phylogenetic analyses.     

Evolutionary divergence of ribosomal internal transcribed spacer 2 in lizards

Internal transcribed spacers 1 and 2 (ITS1 and ITS2) are known to play an important role in rRNA maturation, yet the mechanism of their action is still not completely understood. Comparison of the ITS1 and ITS2 nucleotide sequences for various organisms reveals conserved regions, which are potentially involved in rRNA biogenesis, and yields new information about the evolutionary divergence of the corresponding region of the genome. The rDNA fragments containing ITS2 were amplified, cloned, and sequenced for three lizard species: Darevskia armeniaca, Lacerta strigata (Lacertidae), and Agama caucasia (Agamidae). The lizard ITS2 sequences were compared with their counterparts from other organisms and proved to contain not only universally conserved elements characteristic of the consensus secondary structure of vertebrate ITS2, but also lizard-specific regions. Comparison of the ITS2 size and the distribution of homologous regions for the two lizard families made it possible to assume that evolution of the modern species involved duplication of ITS2 in the genome of their common ancestor.     

Phylogenetic relationships within the cyst-forming nematodes (Nematoda, Heteroderidae) based on analysis of sequences from the ITS regions of ribosomal DNA

The ITS1, ITS2, and 5.8S gene sequences of nuclear ribosomal DNA from 40 taxa of the family Heteroderidae (including the genera Afenestrata, Cactodera, Heterodera, Globodera, Punctodera, Meloidodera, Cryphodera, and Thecavermiculatus) were sequenced and analyzed. The ITS regions displayed high levels of sequence divergence within Heteroderinae and compared to outgroup taxa. Unlike recent findings in root knot nematodes, ITS sequence polymorphism does not appear to complicate phylogenetic analysis of cyst nematodes. Phylogenetic analyses with maximum-parsimony, minimum-evolution, and maximum-likelihood methods were performed with a range of computer alignments, including elision and culled alignments. All multiple alignments and phylogenetic methods yielded similar basic structure for phylogenetic relationships of Heteroderidae. The cyst-forming nematodes are represented by six main clades corresponding to morphological characters and host specialization, with certain clades assuming different positions depending on alignment procedure and/or method of phylogenetic inference. Hypotheses of monophyly of Punctoderinae and Heteroderinae are, respectively, strongly and moderately supported by the ITS data across most alignments. Close relationships were revealed between the Avenae and the Sacchari groups and between the Humuli group and the species H. salixophila within Heteroderinae. The Goettingiana group occupies a basal position within this subfamily. The validity of the genera Afenestrata and Bidera was tested and is discussed based on molecular data. We conclude that ITS sequence data are appropriate for studies of relationships within the different species groups and less so for recovery of more ancient speciations within Heteroderidae.     

The ITS1-5.8S-ITS2 sequence region in the Musaceae: structure, diversity and use in molecular phylogeny

Genes coding for 45S ribosomal RNA are organized in tandem arrays of up to several thousand copies and contain 18S, 5.8S and 26S rRNA units separated by internal transcribed spacers ITS1 and ITS2. While the rRNA units are evolutionary conserved, ITS show high level of interspecific divergence and have been used frequently in genetic diversity and phylogenetic studies. In this work we report on the structure and diversity of the ITS region in 87 representatives of the family Musaceae. We provide the first detailed information on ITS sequence diversity in the genus Musa and describe the presence of more than one type of ITS sequence within individual species. Both Sanger sequencing of amplified ITS regions and whole genome 454 sequencing lead to similar phylogenetic inferences. We show that it is necessary to identify putative pseudogenic ITS sequences, which may have negative effect on phylogenetic reconstruction at lower taxonomic levels. Phylogenetic reconstruction based on ITS sequence showed that the genus Musa is divided into two distinct clades--Callimusa and Australimusa and Eumusa and Rhodochlamys. Most of the intraspecific banana hybrids analyzed contain conserved parental ITS sequences, indicating incomplete concerted evolution of rDNA loci. Independent evolution of parental rDNA in hybrids enables determination of genomic constitution of hybrids using ITS. The observation of only one type of ITS sequence in some of the presumed interspecific hybrid clones warrants further study to confirm their hybrid origin and to unravel processes leading to evolution of their genomes.     

Evolution of nuclear rDNA its sequences in the Cladophora albida/sericea clade (Chlorophyta)

Ribosomal DNA ITS sequences were compared among 13 different species and biogeographic isolates from the monophyletic albida/sericea clade in the green algal genus Cladophora. Six distinct ITS sequence types were found, characterized by multiple insertions and deletions and high levels of nucleotide substitution. Conserved domains within the ITS regions indicate the presence of ITS secondary structure. Low transition/transversion ratios among the six types and nearly symmetrical tree-length frequency distributions indicate some saturation, and low phylogenetic signal. Although branching order among five of the six ITS sequence types could not be resolved, estimates of ITS sequence divergence as compared with 18S divergence in a subset of the taxa suggests that the origin of the different ITS types is probably in the mid-Miocene (12 Ma ago) but that biogeographic isolates within a single ITS type (including both Pacific and Atlantic representatives) have probably dispersed on a time scale of thousands rather than millions of years.Correspondence to: J.J. Olsen     

Toward a better understanding of the infrageneric relationships in Cortinarius (Agaricales, Basidiomycota)

Research on the molecular systematics of Cortinarius, a species-rich mushroom genus with nearly global distribution, is just beginning. The present study explores infrageneric relationships using rDNA ITS and LSU sequence data. One large dataset of 132 rDNA ITS sequences and one combined da-taset with 54 rDNA ITS and LSU sequences were generated. Hebeloma was used as outgroup. Bayesian analyses and maximum-likelihood (ML) analyses were carried out. Bayesian phylogenetic inference performed equally well or better than ML, especially in large datasets. The phylogenetic analysis of the combined dataset with species representing all currently recognized subgenera recovered seven well-supported clades (Bayesian posterior probabilities BPP > 90%). These major clades are: /Myxacium s.l., /subg. Cortinarius, the /phlegmacioid clade (including the subclades /Phlegmacium and /Delibuti), the /calochroid clade (/Calochroi, /Ochroleuci and /Allutus), the /telamonioid clade (/Telamonia, /Orellani, /Anomali), /Dermocybe s.l. and /Myxotelamonia. Our results show that Cortinarius consists of many lineages, but the relationships among these clades could not be elucidated. On one hand, the low divergence in rDNA sequences can be held responsible for this; on the other hand, taxon sampling is problematic in Cortinarius phylogeny. Because of the incredibly high diversity (~2000 Cortinarius species), our sampling included <5% of the known species. By choosing type species of subgenera and sections, our sampling is strongly biased toward Northern Hemisphere taxa. More extensive taxon sampling, especially of species from the Southern Hemisphere, is essential to resolve the phylogeny of this important genus of ectomycorrhizal fungi.     

Molecular phylogenetics in 2D: ITS2 rRNA evolution and sequence-structure barcode from Veneridae to Bivalvia

In this study, we analyzed the nuclear ITS2 rRNA primary sequence and secondary structure in Veneridae and comparatively with 20 Bivalvia taxa to test the phylogenetic resolution of this marker and its suitability for molecular diagnosis at different taxonomic levels. Maximum likelihood and Bayesian trees based on primary sequences were congruent with (profile-) neighbor-joining trees based on a combined model of sequence-structure evolution. ITS2 showed higher resolution below the subfamily level, providing a phylogenetic signal comparable to (mitochondrial/nuclear) gene fragments 2-5 times longer. Structural elements of the ITS2 folding, such as specific mismatch pairing and compensatory base changes, provided further support for the monophyly of some groups and for their phylogenetic relationships. Veneridae ITS2 folding is structured in six domains (DI-VI) and shows five striking sequence-structure features. Two of them, the Basal and Apical STEMs, are common to Bivalvia, while the presence of both the Branched STEM and the Y/R stretches occurs in five superfamilies of the two Heterodonta orders Myoida and Veneroida, thus questioning their reciprocal monophyly. Our results validated the ITS2 as a suitable marker for venerids phylogenetics and taxonomy, and underlined the significance of including secondary structure information for both applications at several systematic levels within bivalves.