The phylogeographic importance of the Strait of Gibraltar as a gene flow barrier in terrestrial arthropods: a case study with the scorpion Buthus occitanus as model organism

The phylogenetic relationship between Buthus occitanus populations across the Strait of Gibraltar was investigated using nuclear 18S/ITS-1 DNA sequences and mitochondrial 16S and COI DNA sequences. All analyses showed that the European samples are highly separated from North African samples, and also suggest the existence of three main groups within this species complex, i.e., an European, an Atlas (=Moroccan samples) and a Tell-Atlas group (=Tunisian samples). The European clade was subdivided into three distinct subclades. The application of a previous calibration of the molecular clock of another buthid species suggested that most of the detected mitochondrial DNA lineages including the European lineages are about three times older than the re-opening of the Gibraltar Strait, and consequently, that other and older vicariant events are responsible for the observed phylogeographic structure of this species complex. Concerning the Moroccan samples, a discordance between nuclear and mitochondrial gene markers was observed. The 18S/ITS-1 gene tree could not resolve the phylogenetic relationships among the Moroccan B. occitanus subspecies and the closely related species B. atlantis, whereas mitochondrial genes suggested the co-existence of several old phylogenetic lineages in Morocco. We hypothesized that this difference may be explained by male-biased gene flow and gene conversion at the tandemly repeated 18S/ITS-1 gene regions.     

Molecular phylogeny of Babesia poelea from brown boobies (Sula leucogaster) from Johnston Atoll, central Pacific

The phylogenetic relationship of avian Babesia with other piroplasms remains unclear, mainly because of a lack of objective criteria such as molecular phylogenetics. In this study, our objective was to sequence the entire 18S, ITS-1, 5.8S, and ITS-2 regions of the rRNA gene and partial beta-tubulin gene of B. poelea, first described from brown boobies (Sula leucogaster) from the central Pacific, and compare them to those of other piroplasms. Phylogenetic analyses of the entire 18S rRNA gene sequence revealed that B. poelea belonged to the clade of piroplasms previously detected in humans, domestic dogs, and wild ungulates in the western United States. The entire ITS-1, 5.8S, ITS-2, and partial beta-tubulin gene sequence shared conserved regions with previously described Babesia and Theileria species. The intron of the beta-tubulin gene was 45 bp. This is the first molecular characterization of an avian piroplasm.     

Cladogram of Panamanian Clusia Based on Nuclear DNA: Implications for the Origins of Crassulacean Acid Metabolism

Abstract: The internal transcribed spacer (ITS) region of 18-26S nuclear ribosomal DNA repeat was sequenced for 31 of the approximately 40 species of Clusia known to occur in Panama. Several species from other genera of the Clusiaceae were used as outgroups in the phylogenetic calculation. High sequence alignment and minimal length variation among ITS-1, 5.8S and ITS-2 sequences facilitated determination of positional homology of nucleotide sizes. Sequence alignment was evaluated with character state (Maximum Parsimony) and distance methods (Neighbour Joining). Phylogenetic trees obtained with the two methods were largely concordant and revealed three main groups that roughly correspond to previous arrangements of species into three large morphological groups, the C. flava group, the C. minor group and the C. multiflora group. Because species of Clusia are either regular C₃ plants or exhibit crassulacean acid metabolism (CAM) involving varying proportions of CO₂ fixation in the dark versus the light, we mapped photosynthetic pathways onto the cladograms. Photosynthetic pathway classification was based on measurements of ¹³C/¹²C ratios of plant carbon and also on information available from the literature. Both the C. flava and C. minor group contained species exhibiting CAM, distributed on distinct branches of the cladograms, whereas the third group ( C. multiflora group) was composed of species which are not known to use CAM.     

The Systematic Position of Beesia: Evidence from ITS (nrDNA) Sequence Analysis

Discussed in the present paper is the systematic position of Beesia, a small ranunculaceous genus mainly distributed in SW China. Sequences of the internal transcribed spacers (ITS) and 3’ end of 5.8 S rRNA gene of Beesia calthifolia, Trollius chinensis, Cimicifuga acerina, C. brachycarpa and Actaea asiatica were determined by direct PCR sequencing method. The sequences of ITS-1 in the five species range from 225 bp to 232 bp in size and those of ITS-2 from 201 bp to 217 bp. Beesia is very similar to Cimicifuga and Actaea not only in size, sequence and G+C content of ITS, but also in sequence of 3’ end of 5.8 S rRNA gene. In PAUP analysis, Ranunculus enysii was used as outgroup, and the most parsimonious tree was obtained through exhaustive search. The gaps were treated respectively as missing characters and the fifth base in two analyses. The two analyses show that a monophyletic group comprising Beesia calthifolia, Cimicifuga acerina, C. brachycarpa and Actaea asiatica is strongly supported by the bootstrap value. In the monophyletic group, Beesia calthifolia is basal to the other three species. The present DNA sequence analysis demonstrates that the genus Beesia should be placed in the tribe Cimicifugeae, which is consistent with the results from phytochemistry, palynology and cytology. In addition, Beesia may be the most original genus in the tribe Cimicifugeae in view of simple leaf,apetalous flower and molecular evidence.     

Taxonomy and molecular phylogeny of three freshwater scuticociliates,with descriptions of one new genus and two new species (Protista,Ciliophora, Oligohymenophorea)

Three freshwater scuticociliates, Apouronema harbinensis gen. nov. spec. nov., Cyclidium vorax spec. nov., and C. glaucoma Müller, 1773, collected from rivers in Hulan District, Harbin, northeastern China, were investigated using morphological and phylogenetic criteria. Apouronema gen. nov., assigned to the family Uronematidae, is mainly distinguished from the other genera of the family by its paroral membrane extending anteriorly to the middle of membranelle 1. Apouronema harbinensis spec. nov. is defined by body size in vivo about 45–55 × 20–25 μm, buccal field about 70–80% of cell length; 12 or 13 somatic kineties; membranelle 1 having two rows, with 16–18 basal bodies in each kinety; membranelle 2 and membranelle 3 both having two rows each; scutica X-shaped with five pairs of basal bodies. Cyclidium vorax spec. nov. is characterized by the following features: body size 35–40 × 18–20 μm in vivo; 9 or 10 somatic kineties; membranelle 1 having two longitudinal rows, much shorter than M2; M2 triangle-shaped. The phylogenetic analyses show that: (1) Apouronema clustered in the Uronematidae clade, and grouped with genera Uronemita and Uronema; (2) Cyclidium vorax spec. nov. grouped with C. glaucoma and C. sinicum, which supports the assignment of the new species to the genus Cyclidium; (3) Cyclidium remains non-monophyletic with the addition of the new sequence.     

Nuclear rDNA-based molecular clock of the evolution of triatominae (Hemiptera: reduviidae), vectors of Chagas disease

The evolutionary history and times of divergence of triatomine bug lineages are estimated from molecular clocks inferred from nucleotide sequences of the small subunit SSU (18S) and the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA of these reduviids. The 18S rDNA molecular clock rate in Triatominae, and Prosorrhynchan Hemiptera in general, appears to be of 1.8% per 100 million years (my). The ITS-2 molecular clock rate in Triatominae is estimated to be around 0.4-1% per 1 my, indicating that ITS-2 evolves 23-55 times faster than 18S rDNA. Inferred chronological data about the evolution of Triatominae fit well with current hypotheses on their evolutionary histories, but suggest reconsideration of the current taxonomy of North American species complexes.     

The Application of Sequence Analysis of rDNA Fragment to the Systematic Study of the Subfamily Cyrtandroideae (Gesneriaceae)

A molecular phylogeny of the subfamily Cyrtandroideae represented by five genera of four tribes was constructed using sequence analysis of the internal transcribed spacers (ITS) and partial 5.8 S rRNA gene (3’end) of nuclear ribosomal DNA. Direct PCR sequencing method was used in the study. The sequences of ITS-1 in the five species range from 234 bp to 258 bp in size and those of ITS-2 from 218 bp to 246 bp. The ITS-1 (258 bp) and ITS-2 (218 bp) of Whytockia bijieensis differ greatly from those of the other species in size, sequence and G + C content, and therefore the tribe Klugieae represented by W. bijieensis may have diverged from the ancestor of the subfamily Cyrtandroideae at a very early time. In PAUP analysis, W. bijieensis was used as the functional outgroup, and only one most parsimonious Fitch tree was obtained through exhaustive search. The tree has 353 steps, with CI = 0. 932 and RI = 0. 529. In the tree, Chirita crassifolia is basal to a monophyletic group comprising Cyrtandra umbellifera, Briggisia longipes and Anna mollifolia, and the monophyletic group is strongly supported by the bootstrap value (97). The tribes Trichosporeae and Cyrtandreae represented respectively by Anna mollifolia and Cyrtandra urnbellifera both evolved from the tribe Didymorcarpeae, which can explain why many intermediate taxa exist among the three tribes. According to this, the present authors suggest that the tribe Trichosporeae and the tribe Cyrtandreae be merged with the tribeDidymocarpeae.     

A comparison of five methods for DNA isolation from liver and rumen flukes to perform ITS-2+ amplification

Five different DNA isolation methods (4 commercial kits and a modification of phenol-chloroform method) were compared for the discrimination of adults of Fasciola hepatica and Dicrocoelium dendriticum (liver flukes), and Calicophoron daubneyi (rumen fluke) collected from sheep in southern Italy. The second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) plus flanking 5.8S and 28S sequence (ITS-2+) was amplified by polymerase chain reaction (PCR) from serial diluted DNA templates (6 ng - 60 fg) of each fluke species. Overall, in terms of efficiency in detection limit, the best results were obtained either with phenol-chloroform purification or with QIAamp DNA Mini Kit (Qiagen), but using this latter method, rapid, safe and not expensive, an increased level of sensitivity sufficient to detect small amounts of target-DNA was achieved. In addition, electrophoresis analysis following PCR also showed that ITS-2+ could be useful as a genetic marker for the molecular identification of F. hepatica, D. dendriticum and C. daubneyi in definitive and intermediate hosts. Furthermore, for the first time, the ITS-2 sequence of D. dendriticum was defined.     

Molecular phylogeny of Trichomonadidae family inferred from ITS-1, 5.8S rRNA and ITS-2 sequences

The Trichomonads have been the subject of several molecular studies that reported some discrepancies both at the lower and higher taxonomic levels. The purpose of this study was to make an extensive phylogenetic analysis of the Trichomonadidae using ITS-1/5.8S/ITS-2 sequences, to better understand its phylogeny and the usefulness of this marker. ITS-1/5.8S/ITS-2 sequences of 36 strains from 14 species belonging to Trichomonadidae and Monocercomonadidae were analysed, in which 20 were newly determined. Maximum likelihood, maximum parsimony, neighbour joining, and Bayesian phylogenetic methods were employed in order to reconstruct and compare the evolutionary history of this group. Tetratrichomonas gallinarum and four strains of Tetratrichomonas sp. isolated from bull genital organs were found closely related, confirming the classification of the latter, probably as a new species. The monophyly of Tritrichomonadinae and Trichomonadinae subfamilies were corroborated, with the exclusion of Trichomitus batrachorum from the latter since it grouped consistently with Hypotrichomonas acosta. Tritrichomonas foetus, Tritrichomonas suis and potentially also Tritrichomonas mobilensis seemed to correspond to the same species. Monocercomonas sp. and Ditrichomonas honigbergii emerged as independent lineages, with their phylogenetic positions undetermined. Neither Trichomonadidae nor Monocercomonadidae were supported as monophyletic groups. The ITS-1/5.8S/ITS-2 seems to be a reliable locus for phylogenetic studies in the Trichomonadida, mainly at lower taxonomic levels, and at least up to the family level.     

Combining DNA sequences and morphology in systematics: testing the validity of the dragonfly species Cordulegaster bilineata

Morphological and molecular techniques are rarely combined when answering questions of taxonomic validity. In this study, we combine morphological techniques with DNA sequences to determine the validity of the dragonfly species Cordulegaster bilineata. The two dragonfly species C. bilineata and C. diastatops are very similar in size, body color, and morphological characters, and due to these similarities, the status of C. bilineata as a valid species is in question. In this study we compare morphological measurements of males and internal transcribed spacer 1 (ITS-1) sequences of rDNA between the two taxa. The hamule measurements (where copulation occurs) of males show little difference between the taxa in question, but the anal appendage measurements (where the male first contacts the female) show marked divergence between the two taxa. Cluster analysis with these anal appendage measurements correctly assigns almost all individuals measured into their respective taxon. PCR amplification products of ITS-1 display a approximately 50 bp size difference between C. bilineata (n = 4) and C. diastatops (n = 5) regardless of collection site. Sequence data for these amplifications show 51 bp missing in one locus in the ITS-1 of C. bilineatarelative to C. diastatops. A lone population of C. diastatops from Wisconsin has three individuals with ITS-1 products that match the size of both C. bilineata and C. diastatops. One individual from this population appears to yield two ITS-1 amplification products that match both C. bilineata and C. diastatops. Although this population may be evidence for hybridization between the two taxa, such hybridization is not necessarily sufficient to disqualify the validity of a separate species designation for C. bilineata. Morphology and ITS-1 sequences depict a high degree of divergence that is consistent with species-level differences.     

Direct comparison of selected methods for genetic categorisation of Cryptosporidium parvum and Cryptosporidium hominis species

A study was undertaken to compare the performance of five different molecular methods (available in four different laboratories) for the identification of Cryptosporidium parvum and Cryptosporidium hominis and the detection of genetic variation within each of these species. The same panel of oocyst DNA samples derived from faeces (n=54; coded blindly) was sent for analysis by: (i) DNA sequence analysis of a fragment of the HSP70 gene; (ii) DNA sequence analysis and the ssrRNA gene in laboratory 1; (iii) single-strand conformation polymorphism analysis of part of the ssrRNA; (iv) SSCP analysis of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA region in laboratory 2; (v) 60 kDa glycoprotein (gp60) gene sequencing with prior species determination using PCR with restriction fragment length polymorphism analysis of the ssrRNA gene in laboratory 3; and (vi) multilocus genotyping at three microsatellite markers in laboratory 4. For detecting variation within C. parvum and C. hominis, SSCP analysis of ITS-2 was considered to have superior utility and determined 'subgenotypes' in samples containing DNA from both species. SSCP was also most cost effective in terms of time, cost and consumables. Sequence analysis of gp60 and microsatellite markers ML1, ML2 and 'gp15' provided good comparators for the SSCP of ITS-2. However, applicability of these methods to other Cryptosporidium species or genotypes and to environmental samples needs to be evaluated. This trial provided, for the first time, a direct comparison of multiple methods for the genetic characterisation of C. parvum and C. hominis samples. A protocol has been established for the international distribution of samples for the characterisation of Cryptosporidium. This can be applied in further evaluation of molecular methods by investigation of a larger number of unrelated samples to establish sensitivity, typability, reproducibility and discriminatory power based on internationally accepted methods for evaluation of microbial typing schemes.     

Molecular characterization of hard tick <Emphasis Type="Italic">Haemaphysalis longicornis</Emphasis> from China by sequences of the internal transcribed spacers of ribosomal DNA

In the present study, the entire first and second internal transcribed spacer (ITS-1 and ITS-2) regions of nuclear ribosomal DNA (rDNA) of Haemaphysalis longicornis from China were amplified by polymerase chain reaction. The 45 representative amplicons were sequenced, and sequence variation in the ITS was examined. The ITS sequences of H. longicornis were 3644 bp in size, including the part of 18S rDNA, 28S rDNA sequences and the complete ITS-1, 5.8S rDNA and ITS-2 sequences. Sequence analysis revealed that the ITS-1, 5.8S rDNA and ITS-2 of this hard tick were 1582, 152, and 1610 bp in size, respectively. The intra-specific sequence variations of ITS-1 and ITS-2 within H. longicornis were 0–2 and 0–2.2%; however, the inter-specific sequence differences among members of the genus Haemaphysalis were significantly higher, being 35.1–55.2 and 37–52% for ITS-1 and ITS-2, respectively. The molecular approach employed in this study provides the foundation for further studies of the genetic variation of H. longicornis from different hosts and geographical origins in China.     

Two new species of Sarcocystis (Apicomplexa: Sarcocystidae) infecting the wolverine (Gulo gulo) from Nunavut, Canada

Infection with Sarcocystis species is common in many species of animals, but it has not yet been reported in wolverines (Gulo gulo). Histological sections of tongues from 41 wolverines in the Kitikmeot Region, Nunavut, Canada, were examined for sarcocysts. Sarcocysts were found in 33 (80.4%) wolverines. Two structurally distinct types of sarcocysts were found. Type A sarcocysts were thin (<1 μm thick) walled. Ultrastructurally, the parasitophorous vacuolar membrane (Pvm) had minute undulations, but it lacked villar protrusions and was not invaginated into the granular layer. The bradyzoites were slender, about 5 × 1 μm in size. Structurally, these sarcocysts were distinct from known species of Sarcocystis and possessed a novel 18S and ITS-1 sequence, sharing 98% and 78% sequence similarity with Sarcocystis canis . A new species name, Sarcocystis kalvikus, is proposed for type A sarcocysts. In contrast, type B sarcocysts had relatively thicker (about 2 μm) cyst walls and larger bradyzoites, each about 10 × 2-3 μm. Ultrastructurally, the Pvm on the sarcocyst wall had villar protrusions that were either mushroom-like or sloping. Molecular analysis identified a unique 18S and ITS-1 sequence that placed them in a clade within the Sarcocystidae. Based on histology, TEM, and genetic data, the new name, Sarcocystis kitikmeotensis, is proposed. Sarcocystis kalvikus was found in 14 (34.1%), S. kitikmeotensis was found in 7 (17%), and both species were found in 12 (29.2%) of 41 wolverines.     

Molecular characterization of intergenic spacer region of 5S ribosomal RNA genes in subgenus Vigna: extensive hybridization among V. unguiculata subspecies

We report the molecular analysis of the 5S ribosomal RNA intergenic spacer (IGS) region from 57 Vigna species of subgenus Vigna. Sequence analysis revealed that the 5S IGS was highly variable in length (189?C237?bp) and sequence (58% polymorphic sites). Most of the Vigna species analysed harboured a single type of 5S rRNA repeat unit, except V. unguiculata and V. reticulata that showed multiple ??intragenomic?? 5S types. The intragenomic 5S types among the six V. unguiculata subspecies were characterized by PCR-RFLP, genomic RFLP and sequencing. The 5S IGS was phylogenetically informative (comparable to ITS-1 and ITS-2 spacers) in inferring species relationships among the Vigna species analysed. However, due to the presence of multiple intragenomic 5S types and their incomplete homogenization among V. unguiculata subspecies the relationships in section Catiang could not be resolved below species level. The results presented indicate that intraspecies hybridization might have resulted in the ??horizontal transfer?? of 5S types among the V. unguiculata subspecies, while their maintenance could be due to a slow molecular drive.     

ITS-2 rDNA sequencing of Gnathostoma species (Nematoda) and elucidation of the species causing human gnathostomiasis in the Americas

From several gnathostome species the complete internal transcribed spacer ITS-2 ribosomal DNA (rDNA) repeat sequence and a fragment of the 5.8S rDNA were obtained by direct polymerase chain reaction cycle-sequencing and silver-staining methods. The size of the complete ITS-1 sequence in agarose gel electrophoresis was also obtained. The ITS-2 enabled the differentiation of Gnathostoma spinigerum from Thailand and Gnathostoma binucleatum from Mexico and Ecuador and confirmed the validity of the latter. Gnathostoma turgidum, Gnathostoma sp. I (=Gnathostoma procyonis sensu Almeyda-Artigas et al., 1994), and Gnathostoma sp. II (=G. turgidum sensu Foster, 1939 pro parte), all from Mexico, proved to be independent species, but Gnathostoma sp. III, also from Mexico, could not be differentiated from G. turgidum. In Mexico and Ecuador, gnathostomes involved in human infection and that had been classified as G. spinigerum belong to G. binucleatum. The 5.8S rDNA sequences of the 6 Gnathostoma species studied were identical. The results of the ITS-1 agreed with those results of ITS-2.     

Sequence variability in the first internal transcribed spacer region within and among Cyclospora species is consistent with polyparasitism

Cyclospora cayetanensis is a coccidian parasite which causes severe gastroenteritis in humans. Molecular information on this newly emerging pathogen is scarce. Our objectives were to assess genetic variation within and between human-associated C. cayetanensis and baboon-associated Cyclospora papionis by examining the internal transcribed spacer (ITS) region of the ribosomal RNA operon, and to develop an efficient polymerase chain reaction- (PCR)-based method to distinguish C. cayetanensis from other closely related organisms. For these purposes, we studied C. cayetanensis ITS-1 nucleotide variability in 24 human faecal samples from five geographic locations and C. papionis ITS-1 variability in four baboon faecal samples from Tanzania. In addition, a continuous sequence encompassing ITS-1, 5.8S rDNA and ITS-2 was determined from two C. cayetanensis samples. The results indicate that C. cayetanensis and C. papionis have distinct ITS-1 sequences, but identical 5.8S rDNA sequences. ITS-1 is highly variable within and between samples, but variability does not correlate with geographic origin of the samples. Despite this variability, conserved species-specific ITS-1 sequences were identified and a single-round, C. cayetanensis-specific PCR-based assay with a sensitivity of one to ten oocysts was developed. This consistent and remarkable diversity among Cyclospora spp. ITS-1 sequences argues for polyparasitism and simultaneous transmission of multiple strains.     

Primary structure of the 5.8S gene of rRNA and internal transcribed spacers of rDNA in diploid wheat Triticum urartu thum. ex. gandil

Nucleotide sequences of 5.8S rRNA gene and rDNA internal transcribed spacers ITS-1 and ITS-2 were determined in diploid wheat Triticum urartu. It was shown that 5.8S rRNA gene of this wheat species consists of 163 base pairs and GC-content is 59.5%. When comparing 5.8S rRNA sequences in diploid wheat, rice and lupine and also 5.8S rRNA in hexaploid wheat and horse beans a high evolutional conservatism of its structure was revealed. The size of ITS-1 and ITS-2 in Tr. urartu is 219 and 225 base pairs long correspondingly. While comparing structures of similar rDNA regions of Tr. urartu, rice and maize a high level of homology was found only between nucleotides adjoining genes of high molecular rRNAs. In ITS-1 of Tr. urartu an insertion of 5'-GACGACGACATTGTCCGTC-3' was found, which is absent in maize and rice.     

Sequence variation in ITS spacers and 5.8S rDNA and relationship of E, St, P, Ns, Xm, and H genomes in the genera of Agropyron, Elytrigia, Leymus, Pascopyrum, Psathyrostachys, and Hordeum

Understanding species evolution and improvement requires information of their genome origin and differentiation. Among the species in the family Gramineae, genome identities of Agropyron-Elytrigia-Leymus group are still ambiguous. In order to delineate the genome relationship, nucleotide sequence analysis in the rDNA ITS regions was carried out among the species in the genera Elytrigia, Agropyron, Psathyrostachys, Leymus, and Psacopyrum containing E, St, P, Ns, and Xm genomes. The ITS-1 and ITS-2 showed a narrow range of variation in length except for the presence of a pentanucleotide, TGGGG, in/del in some haplotypes, whereas higher numbers of nucleotide substitutions were observed in most genera. There were 187 variable sites in the ITS-1, 5.8S, and ITS-2 regions, in which a few genome specific mutations were observed. While the level of variation was similar between ITS-1 and ITS-2, the rate of transition mutation versus transversion mutations was different among the ITS-1, 5.8S, and ITS-2 segments. GC contents of the ITS regions ranged between 55–65% between genomes and the haplotypes of P and H genomes were slightly higher than others. In phylogenetic analysis, the ITS haplotypes were classified into two groups; one containing H, Ns, NsXm genomes, and another containing P, St, and E genomes, which are congruous to the genome affinities from other studies. Among the four genomes in Pascopyrum smithii (2n=8x=56, StStNsNsHHXmXm), the haplotypes of H and St genomes were identified with the reference diploid species, but the haplotypes having Ns and Xm genomes were not found in the present analysis.     

ITS-1 ribosomal DNA sequence variants are maintained in different species and strains of Echinococcus

This study investigated sequence heterogeneity in the first internal transcribed spacer (ITS-1) of ribosomal DNA within and among species and strains of Echinococcus. Different ITS-1 sequence variants exist in Echinococcus granulosus and Echinococcus multilocularis, which represent at least four evolutionary lineages: (1) a sheep strain-lineage of E. granulosus, (2) a sister lineage of a cervid and camel E. granulosus ITS-1 variants, (3) a lineage including the ITS-1 variants representing horse, bovine and camel strains of E. granulosus, as well as variants from E. multilocularis, Echinococcus oligarthrus and Echinococcus vogeli and (4) a distinct lineage of ITS-1 variants including E. granulosus strains from sheep and cervid, and E. multilocularis. At least two of the species (E. granulosus and E. multilocularis) were paraphyletic for ITS-1. Divergent ITS-1 variants from these two species shared distinct evolutionary lineages. The sequence data provided evidence that at least two turnover mechanisms, namely slippage and unequal crossing over/transposition, have led to the divergence and maintenance of sequence variants in Echinococcus species and strains.     

Variability of Ribosomal DNA ITS-2 and Its Utility in Detecting Genetic Relatedness of Pearl Oyster

The objective of this study was to detect interspecific and intraspecific genetic variations of the second internal transcribed spacer of ribosomal DNA (ITS-2), and explore the feasibility of using it as a molecular marker phylogenetic analyses and species identification among pearl oysters. ITS-2 sequences of 6 pearl oysters were amplified via polymerase chain reaction. The amplified DNA fragments were about 500 bp, spanning the partial sequences of 5.8S and 28S rRNA genes. The GC contents of all species used in this study were higher than the AT contents. The variations of sequences involved substitutions as well as insertions/deletions and were mainly concentrated in spacer regions. Sequences of about 30-bp in spacer regions showed no variations among 5 Pincatda species. Intraindividual and intraspecific polymorphisms of ITS-2 sequences were detected in some species; the interspecific variability was significantly larger than the variability within species, and the variability at the genus level was higher than that at the species level. Both neighbor-joining and parsimony analyses of ITS-2 sequences revealed the distinguishable species boundary of 6 pearl oysters, and indicated that P. chemnitzi and P. nigra were the closely related species, as were P. maxima and P. margaritifera. The findings revealed that ITS-2 sequences could be an appropriate tool for phylogenetic study of pearl oysters.