Molecular Diversity among Communities of Freshwater Microchlorophytes

Current hypotheses on the distribution of freshwater microchlorophytes lead to predictions of low diversity and wide environmental tolerances. Thus, the same few species should be found worldwide in many different habitats. However, these hypotheses are based on a morphospecies concept, which precludes the possibility of numerous cryptic species among these organisms. In this study, we examined the diversity of coccoid green microalgae and chlamydomonads (Chlorophyta) isolated from sites in Minnesota and North Dakota (USA) using techniques of 18S rDNA sequence analysis. Of 93 distinct 18S rDNA sequences identified from among 273 isolates examined by molecular techniques, all but four are new to science. The spatial distribution of organisms represented by these 18S rDNA sequences was not uniform, because some lakes and ponds yielded distinct 18S rDNA types not found at other sites. In addition, organisms generally considered to be cosmopolitan, such as Chlamydomonas reinhardtii and Chlorella vulgaris, were not found. These results challenge predictions of low species number and wide environmental tolerances among these eukaryotic microorganisms.     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

Comprehensive comparisons of three pennate diatoms, Diatoma tenuae, Fragilaria vaucheriae, and Navicula pelliculosa, isolated from summer Arctic reservoirs (Svalbard 79°N), by fine-scale morphology and nuclear 18S ribosomal DNA

Here we report morphological and molecular characteristics of dominant freshwater diatoms in summer Arctic reservoirs of Svalbard (Norway), using four culture isolates, when we collected the samples in the field on 15 August 2005. Analyses of morphology and BLAST searches with 18S rDNA sequences identified them to Diatoma tenue (HYNP006, HYNP013), Navicula pelliculosa (HYNP021), and Fragilaria vaucheriae (HYNP022), respectively. Comparative studies of morphology revealed that the body shapes of the three polar diatoms were nearly identical to the known morphology of each species; however, they were considerably shorter in body length than previously described identical species from other locations. The 18S rDNA sequences of the diatoms were nearly identical to the same species from temperate and other regions. Phylogenetic analysis showed that the polar diatoms each formed a clade with their identical species and genera according to their taxonomic positions. This suggests that the polar diatoms may possess little or no genetic or morphological variation compared to more temperate strains.     

Assessing the Biodiversity of Monoraphidium Using 18S rDNA Sequences

The taxonomy of the genus Monoraphidium is unclear due in part to the absence of morphological features to clearly distinguish one species from another. Phytoplankton samples collected from lakes in the Arrowwood National Refuge in eastern North Dakota were found to contain several morphological species of Monoraphidium. Eighteen Monoraphidium isolates were examined with light microscopy and six morphological species were identified. PCR–RFLP of the 18S rDNA was used to type the isolates. Following digestion by Hae III and Taq I, the 18S rDNA PCR–RFLP patterns indicated 10 different types. Presently, the 18S rDNA product is being sequenced for each of the 10 types. By examining morphological characters and 18S rDNA sequences, congruence between morphology and sequence data may be compared. Also, because there is a lack of morphological characters defining Monoraphidium species, diversity within the 18S rDNA sequences may aid in the taxonomy of the genus and its place within the Chlorococcales. Supported by National Science Foundation Grants MCB‐0084188 and DBI‐0070387.     

Evidence for Local Ciliate Endemism in an Alpine Anoxic Lake

Despite its long history, biogeography has received relatively little attention within the field of microbial ecology. Consequently, a fierce debate rages whether protists inhabit restricted geographic areas (endemism hypothesis) or are globally dispersed (ubiquitous dispersal hypothesis). The data presented in this article support the endemism hypothesis. We succeeded in isolating an oligohymenophorean ciliate from a microbial mat in a meromictic anoxic alpine lake (Alatsee) in Germany. The ciliary pattern and the morphometry of this isolate are remarkably similar to Urocentrum turbo (Mueller, 1786) Nitzsch, 1827. However, the organism does not possess trichocysts, a conspicuous and characteristic feature of U. turbo. Instead, the U. turbo-like isolate from lake Alatsee displays merely trichocyst anlagen (“ghosts”) in the cytoplasm that are only visible after protargol impregnation and which become never attached to the cell’s cortex. Despite the distinctness of this difference, such a morphospecies has not been described from any other environment. Thus, we suggest that the U. turbo-like isolate from lake Alatsee is a local endemic ecotype, although the sequences of the 18S rRNA, ITS1, 5.8S rRNA, and ITS2 genes are nearly identical to those of U. turbo (Mueller, 1786) Nitzsch, 1827. This indicates that neither 18S rDNA nor ITS1, ITS2, and 5.8S rDNA sequences are reliable means to conclusively resolve different morphospecies or ecotypes of ciliates. As a consequence, we argue that protist species richness can only be reliably accounted for by considering both molecular and morphological data.     

Novel Endophytes of Rice form a Taxonomically Distinct Subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens(more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens(≥86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

A novel clade of sporocarp-forming species of glomeromycotan fungi in the <Emphasis Type="Italic">Diversisporales</Emphasis> lineage

In the early times of taxonomy of arbuscular mycorrhizal fungi (Glomeromycota), exclusively sporocarpic species were described. Since then the focus has mainly shifted to species forming spores singly. For many of the sporocarpic species, no molecular data have been made available, and their phylogenetic position has remained unclear. We obtained small subunit ribosomal rDNA and internal transcribed spacer data from specimens of glomeromycotan sporocarps from tropical areas that were assigned to three morphospecies. The complete sequence of the 18S small rDNA subunit sequence, internal transcribed spacers (ITS) 1 and 2 and 5.8S rDNA subunit, was determined from a sporocarp of Glomus fulvum. Partial sequences of the small subunit and the other regions were obtained from Glomus pulvinatum and the newly described species Glomus megalocarpum. Molecular phylogenetic analyses placed all species analyzed as a monophyletic sister group to the Diversispora spurca/Glomus versiforme clade group (“Glomus group C”) within the Diversisporales. The phylogenetic divergence from other known species suggests that this clade may constitute a new genus. These findings will have important consequences for taxon definition in the Diversisporales. They will facilitate identification of these fungi using rDNA sequences within colonized roots or the environment. Taxonomic novelties: Glomus megalocarpum D. Redecker     

Biochemical and phylogenetic characterization of two novel deep-sea Thermococcus isolates with potentially biotechnological applications

The partial 16S rDNA gene sequences of two thermophilic archaeal strains, TY and TYS, previously isolated from the Guaymas Basin hydrothermal vent site were determined. Lipid analyses and a comparative analysis performed with 16S rDNA sequences of similar thermophilic species showed that the strains isolated from deep-sea vents were not identical to the other species belonging to the genus Thermococcus. On the basis of the results of the phylogenetic analyses, lipid analyses, and previously reported physiological data, we believe that strains TY and TYS are significantly different from the previously described Thermococcus species. According to specific physiological and molecular features, we propose the use of these isolates as potential tools for the development of biotechnological applications in the field of starch processing and DNA technology. Received: 19 August 1996 / Accepted: 6 November 1996     

THE CONTRIBUTION OF SUB‐SAHARAN AFRICAN STRAINS TO THE PHYLOGENY OF CYANOBACTERIA: FOCUSING ON THE NOSTOCACEAE (NOSTOCALES,CYANOBACTERIA)1

To date, phylogenies have been based on known gene sequences accessible at GenBank, and the absence of many cyanobacterial lineages from collections and sequence databases has hampered their classification. Investigating new biotopes to isolate more genera and species is one way to enrich strain collections and subsequently enhance gene sequence databases. A polyphasic approach is another way of improving our understanding of the details of cyanobacterial classification. In this work, we have studied phylogenetic relationships in strains isolated from freshwater bodies in Senegal and Burkina Faso to complement existing morphological and genetic databases. By comparing 16S rDNA sequences of African strains to those of other cyanobacteria lineages, we placed them in the cyanobacterial phylogeny and confirmed their genus membership. We then focused on the Nostocaceae family by concatenated analysis of four genes (16S rDNA, hetR, nifH, and rpoC1 genes) to characterize relationships among Anabaena morphospecies, in particular, Anabaena sphaerica var. tenuis G. S. West. Using a polyphasic approach to the Nostocaceae family, we demonstrate that A. sphaerica var. tenuis is more closely related to Cylindrospermospsis/Raphidiopsis than to other planktonic Anabaena/Aphanizomenon. On the basis of phylogeny and morphological data, we propose that these three significantly different clusters should be assigned to three genera.     

CHARACTERIZATION OF OSTREOPSIS AND COOLIA (DINOPHYCEAE) ISOLATES IN THE WESTERN MEDITERRANEAN SEA BASED ON MORPHOLOGY,TOXICITY AND INTERNAL TRANSCRIBED SPACER 5.8S rDNA SEQUENCES1

Several isolates of epiphytic dinoflagellates belonging to the genera Ostreopsis Schmidt and Coolia Meunier from the western Mediterranean Sea were examined by LM and EM, toxicity assays, and internal transcribed spacer (ITS) regions of nuclear rDNA, and 5.8S rDNA were sequenced. Morphological comparisons based on the analyses of cell shape, size, thecal plates, and surface ornamentation revealed two distinct species in the western Mediterranean: O. cf. siamensis Schmidt from the Catalan, Andalusian, and Sicilian coasts and O. ovata Fukuyo from the Ligurian coast, southern Tyrrhenian Sea, and Balearic Islands. Both Ostreopsis species were toxic; however, no differences in toxicity were detected between the two Ostreopsis species. Coolia monotis Meunier was nontoxic. The morphological studies were supported by phylogenetic analyses; all western Mediterranean isolates of O. cf. siamensis showed ITS and 5.8S rDNA sequences identical to each other and so did those of O. ovata, whereas high genetic diversity was detected between the western Mediterranean and Asian isolates of O. ovata. The nucleotide sequence analyses of the C. monotis strains showed that all C. monotis isolates from Europe formed a homogeneous clade. Further, the genetic diversity was high between the European and Asian C. monotis isolates. In this study, genetic markers combined with morphology and toxicity analyses was useful in the taxonomic and phylogenetic studies of the Ostreopsidaceae in a temperate area.     

Molecular genetic divergence of the centric diatom Cyclotella and Discostella (Bacillariophyceae) revealed by nuclear ribosomal DNA comparisons

The centric diatom Cyclotella, including the recently separated Discostella, is commonly present in freshwater and several species are important bio-indicators. Here, we describe molecular characteristics of the nuclear rDNA, spanning 18S to D1/D2 region of the 28S rDNA, of two genera Cyclotella and Discostella, particularly using Korean isolates of C. meneghiniana, Discostella sp. c.f. D. pseudostelligera. Molecular and morphological analyses showed that our isolates had nearly identical genotypes in rDNA and similar morphology as compared to presumably the same species from other geographical areas. Phylogenetic analyses of individual 18S and partial 28S rDNAs of Cyclotella sensu lato showed that all sequences were separated into two clades: one containing Cyclotella, the other Discostella including C. ocellata and C. bodanica. Statistical tests with pairwise genetic distance scores showed that the two genera were significantly different (one-factor ANOVA, p?<?0.01). In addition, divergence in the partial 28S rDNA was significantly high (p?<?0.01) as compared to 18S rDNA. This provides evidence that the two genera, Cyclotella and Discostella, belong to genetically well-separated groups. In addition, 28S rDNAs is a more suitable molecular marker for the discrimination of Cyclotella sensu lato.     

Morphological and Molecular Characterization of the Causal Agent of Downy Mildew on Quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis>)

Downy mildew is an economically important and widespread disease in quinoa (Chenopodium quinoa) growing areas. Although in many studies Peronospora farinosa is most commonly regarded as the causal agent of the disease, identification and classification of the pathogen remain still uncertain due to its taxonomic confusion. Thirty-six Peronospora isolates from quinoa with different geographic origins including Argentina, Bolivia, Denmark, Ecuador, and Peru were morphologically and molecularly compared with Peronospora species from other Chenopodium species. The morphology of three herbarium specimens was similar to that of P. variabilis, which originated from C. album, characterized by flexuous to curved ultimate branchlets and pedicellated conidia. Phylogenetic analysis based on ITS rDNA sequences also placed the quinoa pathogen within the same clade as P. variabilis. Within the ITS rDNA sequences of the quinoa pathogens, two base substitutions were found, which separated the majority of the Danish isolates from isolates from South America, but no sequence difference was found among the isolates from different cultivars of quinoa. The present results indicate that the pathogen responsible for the quinoa downy mildew is identical to Peronospora variabilis and that it should not be lumped with P. farinosa as claimed previously by most studies.     

Diversity and Evolution of Paramoeba spp. and their Kinetoplastid Endosymbionts

Members of the genus Paramoeba (including Neoparamoeba) (Amoebozoa) are single‐celled eukaryotes of economic and ecological importance because of their association with disease in a variety of marine animals including fish, sea urchins, and lobster. Interestingly, they harbor a eukaryotic endosymbiont of kinetoplastid ancestry, Perkinsela sp. To investigate the complex relationship between Paramoeba spp. and Perkinsela sp., as well as the relationships between different Paramoeba species, molecular data was obtained for four novel isolates. We also acquired new data from the urchin pathogen P. invadens. Comprehensive molecular phylogenetic analyses were carried out using 33 newly obtained 18S rDNA sequences from the host amoebae and 16 new 18S rDNA sequences from their corresponding Perkinsela sp., together with all publicly available 18S molecular data. Intra‐isolate 18S rDNA nucleotide diversity was found to be surprisingly high within the various species of Paramoeba, but relatively low within their Perkinsela sp. endosymbionts. 18S rDNA phylogenies and ParaFit co‐evolution analysis revealed a high degree of congruence between the Paramoeba and Perkinsela sp. tree topologies, strongly suggesting that a single endosymbiotic event occurred in the common ancestor of known Paramoeba species, and that the endosymbionts have been inherited vertically ever since.     

Evaluation of PCR Based on Gene <Emphasis Type="Italic">apxIVA</Emphasis> Associated with 16S rDNA Sequencing for the Identification of <Emphasis Type="Italic">Actinobacillus pleuropneumoniae</Emphasis> and Related Species

The pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for App diagnosis and characterization. However, in some isolates, conflicting results are found. The present work focus on the characterization of 29 isolates biochemically classified as A. pleuropneumoniae, collected from swine in herds with or without a clinical history of pleuropneumonia. Sixteen isolates were from healthy swine, initially classified as nonserotypable A. pleuropneumoniae; they displayed differences in the molecular characterization patterns of App (genes cpx and apxI, II, and III). Those bacteria that could not be serotyped were submitted to rDNA 16S sequencing. All 29 isolates were analyzed by PCR for the presence of the apxIVA gene. Thirteen isolates (45%) were confirmed to be A. pleuropneumoniae by PCR, nine being from diseased animals (31%) and four from healthy animals (14%) with conclusive serotyping. The rDNA 16S sequencing was used to classify the other 16 isolates in related species other than A. pleuropneumoniae, resulting in eleven A. minor, three A. porcinus, and two Pasteurella sp. Because of conflicting results between biochemical tests and rDNA 16S sequencing, the biochemical characterization was repeated, and the new results were in agreement with the rDNA 16S sequencing data. Biochemical characterization proved to be efficient for the majority of the A. pleuropneumoniae isolates. Nevertheless, conventional tests can render conflicting results, and other methodologies, such as amplification of A. pleuropneumoniae specific apxIVA gene and rDNA 16S sequencing, are very useful for improved classification. We also observed a great variety in rDNA 16S sequences from different A. minor isolates.     

PHYLOGENY OF THE HYDRODICTYACEAE (CHLOROPHYCEAE): INFERENCES FROM rDNA DATA1

The hydrodictyacean green algal lineage has been the focus of much research due to the fossil record of at least some members, their ornamented cell walls, and their distinctive reproductive strategies. The phylogeny of the family was, until recently, exclusively morphology based. This investigation examines hydrodictyacean isolates from several culture collections, focusing on sequences from ribosomal data: 18S rDNA, 26S rDNA (partial), and internal transcribed spacer (ITS)‐2 data. Results from phylogenetic analyses of independent and combined data matrices support the Hydrodictyaceae as a monophyletic lineage that includes isolates of Chlorotetraedron, Hydrodictyon, Pediastrum, Sorastrum, and Tetraedron. Phylogenetic analyses of rDNA data indicate that the three‐dimensional coenobium of Hydrodictyon is evolutionarily distinct from the three‐dimensional coenobium of Sorastrum. The more robust aspects of the ITS‐2 data corroborate the 18S+26S rDNA topology and provide a structural autapomorphy for the Hydrodictyaceae and Neochloridaceae, that is, an abridgment of helix IV in the secondary structure. The rDNA data do not support monophyly of Pediastrum but rather suggest the existence of four additional hydrodictyacean genera: Monactinus, Parapediastrum, Pseudopediastrum, and Stauridium.     

CRYPTIC SPECIES OF SCENEDESMUS (CHLOROPHYTA) FROM DESERT SOIL COMMUNITIES OF WESTERN NORTH AMERICA1

Nine isolates of unicellular green algae were obtained from six geographically separate desert microbiotic crust communities in western North America. Microscopically, eight isolates strongly resembled unicellular forms of Scenedesmus obliquus (Turpin) Kützing. They are oval or crescent shaped, often flattened on one side, with knobby cell apices. SEM indicated a lack of wall ornamentation. Fine filaments connecting cells pole to pole were observed in some isolates, as previously documented in Scenedesmus (Dactylococcus) dissociatus and S. obliquus. The ninth isolate was spherical, without knobby apices or connections between cells, and was similar to unicellular forms that were originally classified as species of Chlorella (Scenedesmus vacuolatus and S. rubescens). None of the isolates formed coenobia in liquid culture. Phylogenetic analysis of the 18S rRNA gene placed all desert isolates in the genus Scenedesmus, separating them into two or three weakly resolved groups along with published sequences of other Scenedesmus isolates. Phylogenetic analyses of the internal transcribed spacer region revealed well‐supported lineages of desert algae that were unsupported with 18S data alone. The eight S. obliquus‐like desert strains formed two distinct clades that excluded the S. obliquus strains from geographically widespread nondesert habitats. The ninth strain was outside of the S. obliquus group, associated with S. raciborskii and S. pectinatus. These results demonstrate three lineages of Scenedesmus from desert soils and provide robust support for the presence of cryptic species in S. obliquus, a morphospecies that is said to have a cosmopolitan distribution. Three new species of Scenedesmus are described.     

DISCRIMINATIVE POWER OF NUCLEAR rDNA SEQUENCES FOR THE DNA TAXONOMY OF THE DINOFLAGELLATE GENUS PERIDINIUM (DINOPHYCEAE)1

The genus Peridinium Ehrenb. comprises a group of highly diversified dinoflagellates. Their morphological taxonomy has been established over the last century. Here, we examined relationships within the genus Peridinium, including Peridinium bipes F. Stein sensu lato, based on a molecular phylogeny derived from nuclear rDNA sequences. Extensive rDNA analyses of nine selected Peridinium species showed that intraspecies genetic variation was considerably low, but interspecies genetic divergence was high (>1.5% dissimilarity in the nearly complete 18S sequence; >4.4% in the 28S rDNA D1/D2). The 18S and 28S rDNA Bayesian tree topologies showed that Peridinium species grouped according to their taxonomic positions and certain morphological characters (e.g., epithecal plate formula). Of these groups, the quinquecorne group (plate formula of 3′, 2a, 7″) diverged first, followed by the umbonatum group (4′, 2a, 7″) and polonicum group (4′, 1a, 7″). Peridinium species with a plate formula of 4′, 3a, 7″ diverged last. Thus, 18S and 28S rDNA D1/D2 sequences are informative about relationships among Peridinium species. Statistical analyses revealed that the 28S rDNA D1/D2 region had a significantly higher genetic divergence than the 18S rDNA region, suggesting that the former as DNA markers may be more suitable for sequence‐based delimitation of Peridinium. The rDNA sequences had sufficient discriminative power to separate P. bipes f. occultaum (Er. Lindem.) M. Lefèvre and P. bipes f. globosum Er. Lindem. into two distinct species, even though these taxa are morphologically only marginally discriminated by spines on antapical plates and the shape of red bodies during the generation of cysts. Our results suggest that 28S rDNA can be used for all Peridinium species to make species‐level taxonomic distinctions, allowing improved taxonomic classification of Peridinium.     

Genetic diversity of Paramecium species on the basis of multiple loci analysis and ITS secondary structure models

Among ciliates, Paramecium has become a privileged model for the study of “species problem” particularly in the case of the “Paramecium aurelia complex” that has been intensely investigated. Despite extensive studies, the taxonomy of Paramecium is still challenging. The major problem is an uneven sampling of Paramecium with relatively few representatives of each species. To investigate species from the less discovered region (Pakistan), 10 isolates of Paramecium species including a standing-alone FT8 strain previously isolated by some of us were subjected to molecular characterization. Fragments of 18S recombinant DNA (rDNA), ITS1-5.8S-ITS2-5′LSU rDNA, cytochrome c oxidase subunit II, and hsp70 genes were used as molecular markers for phylogenetic analysis of particular isolates. The nucleotide sequences of polymerase chain reaction products of all markers were compared with the available sequences of relevant markers of other Paramecium species from GenBank. Phylogenetic trees based on all molecular markers showed that all the nine strains had a very close relationship with Paramecium primaurelia except for the FT8 strain. FT8 consistently showed its unique position in comparison to all other species in the phylogenetic trees. Available sequences of internal transcribed spacer 1 (ITS1) and ITS2 and some other ciliate sequences from GenBank were used for the construction of secondary models. Two highly conserved helices supported by compensatory base changes among all ciliates of ITS2 secondary structures were found similar to other eukaryotes. Therefore, the most conserved 120 to 180 base pairs regions were identified for their comparative studies. We found that out of the three helices in ITS1 structure, helix B was more conserved in Paramecium species. Despite various substitutions in the primary sequence, it was observed that secondary structures of ITS1 and ITS2 could be helpful in interpreting the phylogenetic relationships both at species as well as at generic level.