Additive polymorphisms and reticulation in an ITS phylogeny of thrifts (Armeria,Plumbaginaceae)

A parsimony analysis of 133 sequences of the nuclear ribosomal DNA ITS1+5.8S+ITS2 region from 71 taxa in Armeria was carried out. The presence of additive polymorphic sites (APS; occurring in 14 accessions) fits the reticulate scenario proposed in previous work for explaining the ITS pattern of variation on a much smaller scale and is based mainly on the geographical structure of the data, irrespective of taxonomic boundaries. Despite the relatively low bootstrap values and large polytomies, part of which are likely due to disruptive effects of reticulation and concerted evolution in these multicopy sequences, the ITS analysis has phylogenetic and biogeographic implications. APS detected in this study are consistent with hypothesized hybridization events, although biased concerted evolution, previously documented in the genus, needs to be invoked for specific cases and may be responsible for a possible "sink" effect in terminals from a large clade. The causes for sequences of the same species appearing in different clades (here termed transclade) are discussed.     

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。     

Nuclear ribosomal DNA sequence polymorphism and hybridization in checker mallows (Sidalcea, Malvaceae)

Checker mallows (Sidalcea, Malvaceae) constitute a western North American genus of annuals and perennials that have been regarded as taxonomically difficult because of complex patterns of morphological variation putatively stemming from hybridization and polyploidy. In recent molecular phylogenetic investigations extensive polymorphism was observed in the internal and external transcribed spacers (ITS and ETS) of 18S–26S nuclear ribosomal DNA for some Sidalcea samples. To resolve the evolutionary basis for this polymorphism and to readdress the evolutionary impact of hybridization in Sidalcea we cloned and sequenced the polymorphic DNAs and included the clones in phylogenetic analyses together with direct sequences of non-polymorphic samples. The positions of cloned spacer sequences in the phylogenetic trees suggest that S. reptans and two subspecies of S. malviflora may have been influenced by past hybridization with lineages of the “glaucescens” clade. Polymorphic sequence patterns in other taxa may be a result of extensive interbreeding within young clades, in keeping with the minimal sequence divergence, largely overlapping geographic distributions and morphology, and ploidy variation in these groups. Other possible explanations for polymorphic sequences in members of Sidalcea include slow concerted evolution relative to mutation rates, incomplete lineage sorting, and recent pseudogene formation.     

ITS sequence variation and concerted evolution in the natural hybrid species Malus toringoides

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) is one of the most used molecular characters in plant systematics. Our previous studies based on morphological analysis and ITS sequence variation suggested that Malus toringoides (Rehd.) Hughes is derived from hybridization between M. transitoria (Batal.) Schneid. and M. kansuensis (Batal.) Schneid. To further understand the variation pattern of ITS sequences in M. toringoides, and to elucidate the evolutionary processes that affect ITS sequence variation after hybridization, we sampled 99 accessions from multiple populations of the hybrid and parental species, and then obtained totally 254 ITS sequences by cloning and sequencing. Our ITS variation data demonstrates three outcomes of ITS repeats after hybrid speciation. ～ 27–41% of M. toringoides have only M. transitoria type ITS sequence, ～ 40–70% have M. transitoria type ITS sequence plus one or two chimeric ITS sequences generated by recombination between parental ITS sequences, and six accessions retain both parental type ITS sequences. The plausible evolutionary processes that created the observed ITS variations were inferred to be the joint actions of recombination, concerted evolution, pseudogenization and backcrossing. Our study provides further understandings of the variation model of ITS repeats after hybridization as well as the evolution of M. toringoides after its hybrid speciation.     

Can Extensive Reticulation and Concerted Evolution Result in a Cladistically Structured Molecular Data Set?

Hierarchy is the main criterion for informativeness in a data set, even if no explicit reference to evolution as a causal process is provided. Sequence data (nuclear ribosomal DNA ITS) from Armeria (Plumbaginaceae) contains a certain amount of hierarchical structure as suggested by data decisiveness and distribution of tree lengths. However, ancillary evidence suggests that extensive gene flow and biased concerted evolution in these multicopy regions have significantly shaped the ITS data set. This argument is discussed using parsimony analysis of four data sets, constructed by combining wild sequences with those from different generations of artificial hybrids (wild + F₁, F₂, and backcrosses; wild + backcrosses; wild + F₁; wild + F₂). Compared to the F₁ hybrids, F₂ show a certain degree of homogenization in polymorphic sites. This effect reduces topological disruption caused by F₁ and is considered to be illustrative of how extensive gene flow and biased concerted evolution may have modeled the wild ITS data. The possibility that hierarchy has arisen as a result of—or despite a significant contribution from—those two such potentially perturbing forces raises the question of what kind of signal are we recovering from this molecular data set.     

Fine-scale geographical structure, intra-individual polymorphism and recombination in nuclear ribosomal internal transcribed spacers in Armeria (Plumbaginaceae)

BACKGROUND AND AIMS: Isolation and drift are the main causes for geographic structure of molecular variation. In contrast, the one found in a previous survey in Armeria (Plumbaginaceae) for nuclear ribosomal ITS multicopy regions was species-independent and has been hypothesized to be due to extensive gene-flow and biased concerted evolution. Since this was inferred from a genus-level phylogenetic analysis, the aim of this study was to check for the occurrence of such structure and the validity of the proposed model at a local scale, in a southern Spanish massif (Sierra Nevada), as well as to examine the evolutionary implications at the organism level. METHODS: In addition to 117 sequences of direct PCR products from genomic DNA, 50 sequences of PCR products from cloned DNA were obtained to analyse cases of intragenomic polymorphisms for the ITS regions. KEY RESULTS: Sequence data confirm the occurrence of a species-independent structure at a local scale and reveal insights through the analysis of contact areas between different ITS copies (ribotypes). A comparison between cloned and direct sequences (a) confirms that, within these contact areas, ITS copies co-occur both in different individuals and within single genomes; and (b) reveals recombination between different copies. CONCLUSIONS: This study supports the utility of direct sequences for detecting intra-individual polymorphism and for partially inferring the ITS copies involved, given previous knowledge of the variability. The main evolutionary implication at the organism level is that gene-flow and concerted evolution shape the geographic structure of ITS variation.     

Taxonomy, phylogeny, and distribution of Puccinia graminis, the black stem rust: new insights based on rDNA sequence data

Puccinia graminis (Uredinales) is an economically important and common host-alternating rust species on Berberidaceae/Poaceae (subfamilies Pooideae and Panicoideae) that has been spread globally by human activities from an unknown center of origin. To evaluate the taxonomic implications, phylogenetic relationships, and distribution/spread of this complex species, we sequenced and cladistically analyzed the ITS1, 5.8S, and ITS2 regions from herbarium specimens on various host plants from Iran (17), Europe (1), and North America (4). The ITS region plus the 5.8S gene ranged from 686 to 701 bp, including the flanking partial sequences of the 18S and 28S rDNA. Our phylogenetic analysis included 54 bp of the 18S sequence, the entire ITS1 + 5.8S + ITS2, and 58 bp of the 28S sequence. A second analysis used only the last 42 bp of ITS1, and all the 5.8S and ITS2, to incorporate data from additional sequences downloaded from GenBank. In addition to variation in sequence length, there was variation in sequence content. The analysis does not support classical morphology-based taxonomic concepts of the P. graminis complex. Also, host range, host taxonomy, and geographic origin provide minor information on taxonomic relationships. Puccinia graminis is most probably monophyletic. Coevolutionary aspects can hardly be discussed because of lack of sequence data from alternate host specimens. The occurrence of unrelated fungal taxa on the same host species suggests that, besides coevolution with the host, host jumps and hybridization may have played an important role in the evolution of P. graminis. From rDNA data we conclude that the pathogen was introduced to North America at least twice independently. For a new taxonomic concept, we think the complex has to be split into at least two species. New morphological features and further features other than sequence data, however, must be checked for taxonomic value first and, if necessary, be considered.     

Evolution of Internal Transcribed Spacer Region of Nuclear Ribosomal DNA in Allopolyploids of Aegilops

Hybridization with subsequent polyploidy is a prominent process in evolution of higher plants, but few data address the evolution of homeologous sequences after polyploidy. The internal transcribed spacer (ITS) of nuclear ribosomal DNA (nrDNA) from eleven allopolyploid species in Aegilops was investigated by PCR amplification and direct sequencing. The sequences obtained were used to study the evolution of ITS region in allopolyploid species. The length of ITS region varied from 599 to 606 bp and the number of variable sites was 93, i.e. 51 and 42 for ITS1 and ITS2 re spectively. Some polymorphic sites were observed in polyploid species, and this indicated that the ancestral sequences had not been homogenized completely by concerted evolution. Distance matrix analysis of diploid and polyploid species by neighbor-joining method, using Triticum monococcum as outgroup, resulted in well-resolved neighbor-joining tree indicating that the ITS regions of UUMM and UUSS genome ( sect. Vertebrata) were homogenizing toward those of UU ancestal genome. This result is in agreement with the results of ctyogenetics of Aegilops. On the other hand, the neighbor joining tree including the D-genome group species (sect. Cylindropyrum and sect. Polyeides ) com prised three clades (CC-DDCC, UU-DDMM-DDMMSS-DDMMUU and MM-DDMvMv), which sug gested that concerted evolution was homogenizing the ITS region of the polyploid derivatives to either of their ancestors.     

A phylogenetic study ofSaxifraga sect.Saxifraga (Saxifragaceae) based on nrDNA ITS sequences

Parsimony analyses of 54 nrDNA ITS (Internal Transcribed Spacer) sequences ofSaxifraga sect.Saxifraga were performed. In addition to some unresolved clades, there is strong disagreement between the ITS phylogeny and previous classifications based primarily on morphology. The extensive cytological instability of sect.Saxifraga prevents previous cytotaxonomical results from resolving the incongruence between molecular and morphological data. Dissimilar topologies between chloroplast (matK) and nuclear (ITS) trees for eight species of sect.Saxifraga suggest that gene trees and the true species tree are not coincident. Recent and mid-term reticulation is proposed as an explanation for the incongruence between morphological, cytological, organellar, and nuclear data. Homogenization in multigene families, such as the ITS region, via concerted evolution may be the key to the interpretation of results based on ITS sequences within sect.Saxifraga. The use of organellar genes in a larger sample should help to determine whether extensive reticulation occurs in sect.Saxifraga, as has been documented in various genera of Saxifragaceae.     

Patterns of Concerted Evolution of the rDNA Family in a Natural Population of Zhikong Scallop, <Emphasis Type="Italic">Chlamys farreri</Emphasis>

Using denaturing high-performance liquid chromatography (DHPLC), we screened the insertion/deletion (indel) polymorphism in the internal transcribed spacer (ITS) sequences of 40 individuals from a natural population of Zhikong scallop (Chlamys farreri). Surprisingly, only 7.5% of individuals were homogeneous in ITS constitution, while the others (92.5%) were heterozygous. Based on different peak types in DHPLC analysis, seven individuals were randomly chosen to investigate indel polymorphism in the ITS sequences within individuals. Furthermore, indel polymorphism in the ITS sequences of single sperms was also investigated in more individuals belonging to different peak types. Based on these results, we concluded that rapid intrachromosomal recombination drove homogenization of rDNA arrays and interchromosomal recombination might contribute to form new variants, and that it may be less rare than previously thought although it was much less frequent than intrachromosomal recombination in the homogenization process. Further, we proposed an expanded model for concerted evolution of the rDNA family in a natural population of C. farreri. A pathway in the new model which homogenizes a variant unit, beginning with two-peak type individuals and ends with two-peak type individuals, is a larruping pathway in the natural population of C. farreri. As the highest proportion in natural populations, two-peak individuals with equal peak areas can be viewed as being in a steady and balanced state which is maintained by rapid intrachromosomal recombination.     

苏铁nrDNA ITS区的序列多态性:不完全致同进化的证据

本研究对苏铁(Cycas revoluta) nrDNA ITS进行克隆测序, 并以cDNA ITS为参照, 比较分析获得的序列的碱基变异、GC含量、5.8S二级结构的稳定性和5.8S保守基序的有无以及系统发育关系。结果发现苏铁nrDNA ITS存在较高的基因组内多样性, 同时, 这些分化的nrDNA ITS拷贝中包含有假基因的存在, 而且假基因与功能拷贝之间已经形成了较大的遗传分化, 这暗示假基因起源有较长历史。苏铁核仁组织区不仅多达16个, 而且分布在13条染色体上, 这可能是其nrDNA ITS致同进化不完全的主要原因。     

The evolution pattern of rDNA ITS in Avena and phylogenetic relationship of the Avena species (Poaceae: Aveneae)

Ribosomal ITS sequences are commonly used for phylogenetic reconstruction because they are included in rDNA repeats, and these repeats often undergo rapid concerted evolution within and between arrays. Therefore, the rDNA ITS copies appear to be virtually identical and can sometimes be treated as a single gene. In this paper we examined ITS polymorphism within and among 13 diploid (A and C genomes), seven tetraploid (AB, AC and CC genomes) and four hexaploid (ACD genome) to infer the extent and direction of concerted evolution, and to reveal the phylogenetic and genome relationship among species of Avena. A total of 170 clones of the ITS1-5.8S-ITS2 fragment were sequenced to carry out haplotype and phylogenetic analysis. In addition, 111 Avena ITS sequences retrieved from GenBank were combined with 170 clones to construct a phylogeny and a network. We demonstrate the major divergence between the A and C genomes whereas the distinction among the A and B/D genomes was generally not possible. High affinity among the A(d) genome species A. damascena and the ACD genome species A. fatua was found, whereas the rest of the ACD genome hexaploids and the AACC tetraploids were highly affiliated with the A(l) genome diploid A. longiglumis. One of the AACC species A. murphyi showed the closest relationship with most of the hexaploid species. Both C(v) and C(p) genome species have been proposed as paternal donors of the C-genome carrying polyploids. Incomplete concerted evolution is responsible for the observed differences among different clones of a single Avena individual. The elimination of C-genome rRNA sequences and the resulting evolutionary inference of hexaploid species are discussed.     

Comparisons of phylogenetic hypotheses among different data sets in dwarf dandelions (Krigia,Asteraceae): Additional information from internal transcribed spacer sequences of nuclear ribosomal DNA

The internal transcribed spacer (ITS) region of the 18 S–25 S nuclear ribosomal DNA repeat was sequenced from 19 populations of the tribeLactuceae, including all species of dwarf dandelion (Krigia) and five outgroup genera. The incidence of length changes and base substitutions was at least two times higher for ITS 1 than ITS 2. Interspecific sequence divergence withinKrigia averaged 9.62% (1.61%–15.19%) and 4.26% (0%–6.64%) in ITS 1 and ITS 2, respectively. Intergeneric sequence divergence ranged from 15.6% to 44.5% in ITS 1 and from 8.0% to 28.6% in ITS 2. High sequence divergence and homoplasy among genera of tribeLactuceae suggest that the phylogenetic utility of ITS sequence data is limited to interspecific studies or comparisons among closely related genera. Trees generated from ITS sequences are essentially identical to those from restriction site comparisons of the entire nuclear ribosomal (nr) DNA region. The degree of tree resolution differed depending on how gaps were treated in phylogenetic analyses. The ITS trees were congruent with the chloroplast DNA and morphological phylogenies in three major ways: 1) the sister group relationship betweenKrigia andPyrrhopappus; 2) the recognition of two monophyletic sections,Krigia andCymbia, in genusKrigia; and 3) the monophyly of theK. occidentalis-K. cespitosa clade in sect.Cymbia. However, the two nrDNA-based trees are not congruent with morphology/chloroplast DNA-based trees for the interspecific relationships in sect.Krigia. An average of 22.5% incongruence was observed among fourKrigia data sets. The relatively high degree of incongruence among data sets is due primarily to conflict between trees based on nrDNA and morphological/cpDNA data. The incongruence is probably due to the concerted evolution of nrDNA repeating units. The results fromKrigia and theLactuceae suggest that nrDNA data may have limited utility in phylogenetic studies of plants, especially in groups which exhibit high levels of sequence divergence. Our combined phylogenetic analysis as a total evidence shows the least conflict to each of the individual data sets.     

Intragenomic Variation and Evolution of the Internal Transcribed Spacer of the rRNA Operon in Bacteria

Variation in the internal transcribed spacer (ITS) of the rRNA (rrn) operon is increasingly used to infer population-level diversity in bacterial communities. However, intragenomic ITS variation may skew diversity estimates that do not correct for multiple rrn operons within a genome. This study characterizes variation in ITS length, tRNA composition, and intragenomic nucleotide divergence across 155 Bacteria genomes. On average, these genomes encode 4.8 rrn operons (range: 2–15) and contain 2.4 unique ITS length variants (range: 1–12) and 2.8 unique sequence variants (range: 1–12). ITS variation stems primarily from differences in tRNA gene composition, with ITS regions containing tRNA-Ala + tRNA-Ile (48% of sequences), tRNA-Ala or tRNA-Ile (10%), tRNA-Glu (11%), other tRNAs (3%), or no tRNA genes (27%). Intragenomic divergence among paralogous ITS sequences grouped by tRNA composition ranges from 0% to 12.11% (mean: 0.94%). Low divergence values indicate extensive homogenization among ITS copies. In 78% of alignments, divergence is <1%, with 54% showing zero variation and 81% containing at least two identical sequences. ITS homogenization occurs over relatively long sequence tracts, frequently spanning the entire ITS, and is largely independent of the distance (basepairs) between operons. This study underscores the potential contribution of interoperon ITS variation to bacterial microdiversity studies, as well as unequivocally demonstrates the pervasiveness of concerted evolution in the rrn gene family. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Margaret Riley     

Molecular data suggest a hybrid origin for the invasive Caulerpa racemosa (Caulerpales,Chlorophyta) in the Mediterranean Sea

Morphological data has provided a basis for the hypothesis that three taxa belonging to the Caulerpa racemosa complex occur in the Mediterranean Sea: var. turbinata–uvifera, var. lamourouxi, and the `invasive variety'. In order to test this hypothesis and to determine the origin of the `invasive variety', the transcribed spacer ITS1–ITS2 and an 18S ribosomal DNA (rDNA) intron were analysed from 16 isolates of Caulerpa racemosa. The `invasive variety' shows intraindividual polymorphism for both types of sequences. The ITS1–ITS2 data confirm that the three morphological varieties of C. racemosa from the Mediterranean Sea are distinct taxonomic units. The 18S intron data suggest that the new `invasive variety' could be a recent hybrid between var. turbinata–uvifera and an unknown tropical strain. Incongruence between the phylogenetic tree computed from ITS1–ITS2 regions and the 18S intron indicates that homogenization processes of concerted evolution have run at different rates.     

Genetic Diversity in Pseudomonads Associated with Cereal Cultures Infected with Basal Bacteriosis

The genetic properties of 45 pseudomonad strains isolated from cereal cultures exhibiting symptoms of basal bacteriosis have been investigated. Considerable genetic diversity has been demonstrated using DNA fingerprints obtained by amplification with REP, ERIC, and BOX primers. Restriction analysis of the 16S–23S internal transcribed spacer (ITS1) allowed the strains to be subdivided into two major groups. In a phylogenetic tree, the ITS1s of these groups fell into two clusters, which also included the ITS1 of Pseudomonas syringae (“Syringae” cluster) and the ITS1 of P. fluorescens, P. tolaasii, P. reactans, P. gingeri, and P. agarici (“Fluorescens” cluster) from the GenBank database. Comparison of the ITS1 divergence levels within the “Fluorescens” cluster suggests expediency of treating P. tolaasii, P. reactans, various P. fluorescens groups, and, possibly, P. gingeri and P. agarici as subspecies of one genospecies. The intragenomic heterogeneity of ITS1s was observed in some of the pseudomonad strains studied. The results of amplification with specific primers and subsequent sequencing of the amplificate suggest the possibility of the presence of a functionally active syrB gene involved in syringomycin biosynthesis in the strains studied.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 537–544.Original Russian Text Copyright © 2005 by Bobrova, Milyutina, Troitskii.     

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.     

Evolutionary Diversification Indicated by Compensatory Base Changes in ITS2 Secondary Structures in a Complex Fungal Species, <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The rRNA cistron (18S–ITS1–5.8S–ITS2–28S) is used widely for phylogenetic analyses. Recent studies show that compensatory base changes (CBC) in the secondary structure of ITS2 correlate with genetic incompatibility between organisms. Rhizoctonia solani consists of genetically incompatible strain groups (anastomosis groups, AG) distinguished by lack of anastomosis between hyphae of strains. Phylogenetic analysis of internal transcribed spacer (ITS) sequences shows a strong correlation with AG determination. In this study, ITS sequences were reannotated according to the flanking 5.8S and 28S regions which interact during ribogenesis. One or two CBCs were detected between the ITS2 secondary structure of AG-3 potato strains as compared to AG-3 tobacco strains, and between these two strains and all other AGs. When a binucleate Rhizoctonia species related to Ceratobasidiaceae was compared to the AGs of R. solani, which were multinucleate (3–21 nuclei per cell), 1–3 CBCs were detected. The CBCs in potato strains of AG-3 distinguish them from AG-3 tobacco strains and other AGs yielding further evidence that the potato strains of AG-3 originally described as R. solani are a species distinct from other AGs. The ITS1–5.8S–ITS2 sequences were analyzed by direct sequencing of PCR products from 497 strains of AG-3 isolated from potato. The same 10 and 4 positions in ITS1 and ITS2, respectively, contained variability in 425 strains (86%). Nine different unambiguous ITS sequences (haplotypes) could be detected in a single strain by sequencing cloned PCR products indicating that concerted evolution had not homogenized the rRNA cistrons in many AG-3 strains. Importantly, the sequence variability did not affect the secondary structure of ITS2 and CBCs in AG-3. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Species relationships between antifungal chitinase and nuclear rDNA (internal transcribed spacer) sequences in the genus Hordeum.

The sequences of the chitinase gene (Chi-26) and the internal transcribed spacer of 18S - 5.8S - 26S rDNA (ITS1) were determined to analyze the phylogenetic relationships among species representing the four basic genomes of the genus Hordeum. Grouping analysis based on data for Chi-26 gene sequences placed Hordeum secalinum (H genome) near the Hordeum murinum complex (Xu genome), and Hordeum bulbosum distant from the other species that carried the I genome. ITS sequence data showed the expected grouping based on the genome classification of the species studied. Different sequences of ITS were detected even in the genomes of the diploid species. The results are interpreted in terms of defective or unfinished concerted evolution processes in each taxon.     

Reconsideration of systematic relationships within the order Euplotida (Protista, Ciliophora) using new sequences of the gene coding for small-subunit rRNA and testing the use of combined data sets to construct phylogenies of the Diophrys-complex

Comprehensive molecular analyses of phylogenetic relationships within euplotid ciliates are relatively rare, and the relationships among some families remain questionable. We performed phylogenetic analyses of the order Euplotida based on new sequences of the gene coding for small-subunit RNA (SSrRNA) from a variety of taxa across the entire order as well as sequences from some of these taxa of other genes (ITS1-5.8S-ITS2 region and histone H4) that have not been included in previous analyses. Phylogenetic trees based on SSrRNA gene sequences constructed with four different methods had a consistent branching pattern that included the following features: (1) the “typical” euplotids comprised a paraphyletic assemblage composed of two divergent clades (family Uronychiidae and families Euplotidae–Certesiidae–Aspidiscidae–Gastrocirrhidae), (2) in the family Uronychiidae, the genera Uronychia and Paradiophrys formed a clearly outlined, well-supported clade that seemed to be rather divergent from Diophrys and Diophryopsis, suggesting that the Diophrys-complex may have had a longer and more separate evolutionary history than previously supposed, (3) inclusion of 12 new SSrRNA sequences in analyses of Euplotidae revealed two new clades of species within the family and cast additional doubt on the present classification of genera within the family, and (4) the intraspecific divergence among five species of Aspidisca was far greater than those of closely related genera. The ITS1-5.8S-ITS2 coding regions and partial histone H4 genes of six morphospecies in the Diophrys-complex were sequenced along with their SSrRNA genes and used to compare phylogenies constructed from single data sets to those constructed from combined sets. Results indicated that combined analyses could be used to construct more reliable, less ambiguous phylogenies of complex groups like the order Euplotida, because they provide a greater amount and diversity of information.