Almyronema epifaneia gen. & sp. nov. (Cyanobacteria, Nodosilineaceae) isolated from an Indian mangrove forest

A total of 128 strains was isolated from more than 23 legume hosts in Korea. Phylogenetic relationships between these Korean isolates and reference strains of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium were analysed using their 16S rRNA gene and internally transcribed spacer (ITS) region sequences. Among the Bradyrhizobium strains, dendrograms based on both the 16S rRNA gene and ITS region sequences produced two main groups. The ITS tree yielded at least two new clusters that were discernable from the seven previously delineated genospecies. Large discrepancies were revealed between phylogenetic dendrograms based on 16S rRNA gene and ITS region sequences for members of the genus Rhizobium, reflecting their taxonomic heterogeneity. The amalgamation of Rhizobium and former members of Agrobacterium was confirmed using the 16S rRNA tree. Phylogenetic analysis of ITS region sequences showed that the Rhizobium giardinii clade (group II) and the Rhizobium radiobacter/Rhizobium rubi clade (group III) could be tentatively recognized as groups that are separable from the core group (group I), which includes Rhizobium leguminosarum. Dendrograms based on the 16S rRNA gene and ITS region sequences of Mesorhizobium strains were highly conflicting due to the poor taxonomic resolution of the 16S rRNA gene sequences and the low confidence in the ITS dendrogram. Several Korean isolates within the genus Mesorhizobium are thought to represent novel taxa when considering their relatively low ITS region sequence similarities (<80 %) to the reference strains.

The Campylobacter genus consists of a number of important human and animal pathogens. Although the 16S rRNA gene has been used extensively for detection and identification of Campylobacter species, there is currently limited information on the 23S rRNA gene and the internal transcribed spacer (ITS) region that lies between the 16S and 23S rRNA genes. We examined the potential of the 23S rRNA gene and the ITS region to be used in species differentiation and delineation of systematic relationships for 30 taxa within the Campylobacter genus. The ITS region produced the highest mean pairwise percentage difference (35.94%) compared to the 16S (5.34%) and 23S (7.29%) rRNA genes. The discriminatory power for each region was further validated using Simpson's index of diversity (D value). The D values were 0.968, 0.995, and 0.766 for the ITS region and the 23S and 16S rRNA genes, respectively. A closer examination of the ITS region revealed that Campylobacter concisus, Campylobacter showae, and Campylobacter fetus subsp. fetus harbored tRNA configurations not previously reported for other members of the Campylobacter genus. We also observed the presence of strain-dependent intervening sequences in the 23S rRNA genes. Neighbor-joining trees using the ITS region revealed that Campylobacter jejuni and Campylobacter coli strains clustered in subgroups, which was not observed in trees derived from the 16S or 23S rRNA gene. Of the three regions examined, the ITS region is by far the most cost-effective region for the differentiation and delineation of systematic relationships within the Campylobacter genus.

The 16S rRNA gene sequences for 34 strains, including 11 isolates, were determined to classify scab-causing Streptomyces spp. and relatives isolated from potato scab lesions collected in Jeju, Korea. The 16S-23S rDNA internally transcribed spacer (ITS) sequences were determined to investigate whether the 16S-23S ITS region is useful for analysing intra- and interspecific relationships in these bacteria. On the basis of phylogenetic analysis of 16S rRNA gene sequences, most of the isolates were classified as Streptomyces scabiei and Streptomyces acidiscabies. Isolate KJO61 was placed in an ambiguous taxonomic position between Streptomyces reticuliscabiei and Streptomyces turgidiscabies. 16S-23S ITS region sequence analysis showed that tRNA genes were not found in this region of Streptomyces spp. The 16S-23S ITS regions of Streptomyces spp. exhibited various lengths and highly variable sequence similarities (35-100%) within strains as well as intra- and interspecies. It was revealed that Streptomyces europaeiscabiei could be clearly differentiated from Streptomyces scabiei. However, it was clarified that ITS regions are not useful in phylogenetic analysis of Streptomyces spp.

About 200 individual sarcocysts were excised from 12 samples of cattle beef from five countries (Argentina, Brazil, Germany, New Zealand, Uruguay) and tentatively identified to species or cyst type on the basis of their size and shape and cyst wall morphology. Genomic DNA was extracted from 147 of these sarcocysts and used initially for PCR amplification and sequencing of the partial mitochondrial cytochrome c oxidase subunit I gene (cox1) in order to identify the sarcocysts to species and/or sequence type. In addition, seven Sarcocystis sinensis-like sarcocysts collected from the oesophagus of water buffaloes in Egypt were examined at cox1 for comparative purposes. Based on the results from the cox1 marker, selected sarcocyst isolates from both hosts were further characterised at one to three regions of the nuclear ribosomal (r) DNA unit, i.e. the complete 18S rRNA gene, the complete internal transcribed spacer 1 (ITS1) region and the partial 28S rRNA gene. This was done in order to compare the results with previous molecular identifications based on 18S rRNA gene sequences and to evaluate the utility of these regions for species delimitations and phylogenetic inferences. On the basis of sarcocyst morphology and molecular data, primarily the cox1 sequences, four Sarcocystis spp. were identified in the samples of cattle beef. Twenty-two microscopic sarcocysts (1 × 0.1 mm) with hair-like protrusions were assigned to Sarcocystis cruzi, 56 macroscopic sarcocysts (3-8 × 0.5 mm) with finger-like protrusions were assigned to Sarcocystis hirsuta and 45 and 24 microscopic sarcocysts (1-3 × 0.1-0.2 mm) with finger-like protrusions were assigned to Sarcocystis bovifelis and Sarcocystis bovini n. sp., respectively. Sarcocysts of S. cruzi were identified in samples of beef from Argentina and Uruguay; sarcocysts of S. hirsuta in samples from Argentina, Brazil, Germany and New Zealand; sarcocysts of S. bovifelis in samples from Argentina and Germany; and sarcocysts of S. bovini in samples from Argentina and New Zealand. The microscopic sarcocysts from water buffaloes were confirmed to belong to S. sinensis. The cox1 sequences of S. bovifelis and S. bovini, respectively, shared an identity of 93-94 % with each other, and these sequences shared an identity of 89-90 % with cox1 of S. sinensis. In contrast, the intraspecific sequence identity was 98.4-100 % (n = 45), 99.3-100 % (n = 24) and 99.5-100 % (n = 7) for sequences of S. bovifelis, S. bovini and S. sinensis, respectively. In each of the latter three species, an aberrant type of cox1 sequences was also identified, which was only 91-92 % identical with the predominant cox1 type of the same species and about 98 % identical with the aberrant types of the two other species. These aberrant cox1 sequences are believed to represent non-functional nuclear copies of the mitochondrial genes (numts or pseudogenes). They might be used as additional markers to separate the three species from each other. Sequencing of a considerable number of clones of S. bovifelis, S. bovini and S. sinensis from each of the three regions of the rDNA unit revealed intraspecific sequence variation in all loci in all species and particularly in the ITS1 locus (78-100 % identity). As regards the 18S rRNA gene, it was possible to separate the three species from each other on the basis of a few consistent nucleotide differences in the less variable 3' end half of the gene. A comparison of the new sequences with GenBank sequences obtained from S. sinensis-like sarcocysts in cattle in other studies indicated that previous sequences derived from cattle in Germany and Austria belonged to S. bovifelis, whereas those derived from cattle in China belonged to S. bovini. On the basis of the new 28S rRNA sequences, it was possible to separate S. sinensis from S. bovifelis and S. bovini, whereas the latter two species could not be separated from each other. Based on ITS1 sequences, the three species were indistinguishable. Phylogenetic analysis using maximum parsimony placed with fairly high support cox1 sequences of S. bovifelis, S. bovini and S. sinensis, respectively, into three monophyletic clusters, with S. bovifelis and S. bovini being a sister group to S. sinensis. In contrast, phylogenies based on each of the three regions of the rDNA unit did not separate sequences of the three species completely from each other. Characterisation of cox1 of 56 isolates of S. hirsuta from four countries revealed only 13 haplotypes and an intraspecific sequence identity of 99.3-100 %. In the three regions of the rDNA unit, there was more extensive sequence variation, particularly in the ITS1 region. The 22 cox1 sequences of S. cruzi displayed a moderate intraspecific variation (98.6-100 %), whereas there was no variation at the 18S rRNA gene among 10 sequenced isolates. Sequencing of 16 clones of the partial 28S rRNA gene of S. cruzi yielded two markedly different sequence types, having an overall sequence identity of 95-100 %.

AimCryoconite, a microbe‐mineral aggregate found on glaciers worldwide, is formed by microbial phototrophs, principally cyanobacteria. Despite their ecological importance in supraglacial environments, the phylogeographical distributions of supraglacial cyanobacteria are poorly understood. Here, we investigate the biogeographical distribution of cyanobacteria on glaciers in the Antarctic, Arctic and Asia.LocationGlaciers in the Antarctic, Arctic and Asia.MethodsWe analysed contiguous sequences of 16S rRNA genes and 16S–23S internal transcribed spacer (ITS) regions, determined by a long read strategy and single‐filament PCR analysis in 38 glacial samples. We analysed cyanobacterial distribution patterns and genetic differentiation.ResultsThe cyanobacterial 16S rRNA gene sequences were grouped into 20 operational taxonomic units (OTUs), and the six major OTUs that accounted for 88% of sequences were distributed broadly from polar to Asian glaciers, suggesting that they are cosmopolitan at the species level. However, analysis of the more variable ITS region revealed geographical differentiation at the strain level. Nineteen OTUs, including the six major OTUs, showed considerable genetic differentiation among geographical regions; at the population level, they are, thus, geographically restricted. Only one of the phylotype exhibits a population structure which does not show a relationship with geographical distribution, suggesting that is cosmopolitan, even at the strain level.Main conclusionsOur 16S rRNA gene analyses suggest a global distribution of species of cyanobacteria colonizing glacier surfaces; however, the 16S–23S ITS regions revealed that most of the phylotypes are fundamentally endemic to particular areas at the population level and indicate limited migration among regions. Our result suggests that selection pressures among geographical regions are strong driving forces shaping genetic structure in cyanobacteria.

Yacon (Smallanthus sonchifolius) is an Andean crop which is very regarded for its medicinal properties. Yacon genetic profiles have been slightly studied using molecular markers. The aim of this study was to prepare the protocol for direct sequencing of the ITS (Internal Transcribed Spacers) regions of yacon DNA genome. The identified ITS regions in three Bolivian yacon's landraces (BOL 20, BOL 22 and BOL 24) were sequenced and then compared with ITS regions found in NCBI database, previously isolated in yacon and in other plants belonging to the genus Smallanthus. The analysed ITS regions of studied Bolivian landraces showed differences in the nucleotides 163-164 and 235-236 with the sequence previously isolated in yacon. A dendrogram was constructed by comparing the sequence presented in the manuscript and those in the NCBI database for Smallanthus, spp. In the dendrogram, the previously reported ITS sequence of Smallanthus sonchifolius was also used. Specific sites of Smallanthus sonchifolius ITS region gives a promising base for molecular approach identification of this underutilized crop.

The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region was PCR- amplified in 32 genera of non-flowering seed plants. Length of the ITS region was determined by restriction site mapping of PCR products and nucleotide sequences were obtained from the ITS-2 and 5.8S rDNA of selected genera. In contrast to the relatively narrow range of ITS region lengths reported from angiosperms (565-700 base pairs (bp)), substantial length variation (975-3125 bp) is observed in the ITS region of Coniferales, Cycadales, Ginkgoales, and Gnetales. Restriction site analyses indicate that the 5.8S rDNA + ITS-2 ranges from 375-450 bp, while the ITS-1 is responsible for most of the length variation found in gymnosperm ITS regions. The representatives of Pinaceae exhibit the greatest variation in ITS region length (1550-3125 bp), while those of sampled members of Cupressaceae, Taxodiaceae, Cephalotaxaceae, and Taxaceae are relatively stable (975-1125 bp). The observed ITS region lengths in Sciadopityaceae (1250 bp) and Araucariaceae (1325-1350 bp) are somewhat larger than those of Cupressaceae, Taxodiaceae, Cephalotaxa- ceae, and Taxaceae, while those of sampled Podocarpaceae (2000-2100 bp) fall into the range observed in the Pinaceae. Outside of the Coniferales, ITS region length is similar among Cycadales (1150- 1450 bp), Ginkgoales (1200 bp), and two of the three members of Gnetales, Ephedra (1500 bp) and Gnetum (1200 bp). In contrast, the ITS region of Welwitschia is only 750 bp long, ca. 50 bp longer than the longest known angiosperm ITS region. Levels of nucleotide sequence variation as estimated by restriction site mapping suggest that phylogenetic analysis of the ITS region will be informative at the intrageneric level in most non-flowering seed plants. In Cupressaceae, intergeneric comparisons may also be feasible. Because the ITS region is relatively long, it may also permit population-level phylogenetic analysis in many non-flowering seed plants.

From sequence divergence of 16S rRNA genes and the internally transcribed spacer (ITS) region it is reported that variation in phylogenetic placement exists among the 17 different serotype strains of Bradyrhizobium that have been isolated from nodules of soybean. Evolutionary relationships among the bradyrhizobia were more resolved using reconstructions derived from ITS than from 16S rRNA gene sequence divergence. Strain USDA 129 was placed together with USDA 62, 110, 122 and 126, but did not cluster with USDA 123 and 127, with which it shares antigenic determinants. The results from the phylogenetic analysis were supported with data from determinations of genetic diversity among additional strains within each of these serogroups using amplified fragment length polymorphism analysis. From these results it was concluded that strains of serogroup 129 were more similar to strains of serogroups 62, 110 and 122 than they were to strains of serogroups 123 and 127. The serotype strain of Bradyrhizobiumjaponicum USDA 135 and the type strain for Bradyrhizobium liaoningense possessed identical 16S rRNA gene and ITS region sequences. Also, the type strain for B. liaoningense cross-reacted with antisera prepared against somatic antigens of USDA 135. Therefore, it was not possible to distinguish B. liaoningense from serogroup 135 in our analysis of B. japonicum and Bradyrhizobium elkanii.

Objective To evaluate the capability of internal transcribed spacer (ITS) region sequencing analysis to identify clinical isolates of filamentous fungi. Methods A total of 267 filamentous fungi isolates collected from clinical specimens were analyzed by ITS region sequencing analysis. Alignment of acquired sequences with known sequences in GenBank and MycoBank was conducted to identify the species of those isolates. Results ITS sequences of the 267 isolates were amplified successfully. Among these isolates, 53.9% (144/267) were identified to species level and 44.2% (118/267) to genus level. Only five isolates were failed to be identified at genus level as they shared >95% homology in ITS sequence with multiple genera. Conclusion ITS region sequencing analysis is preferred for identification of clinical isolates of filamentous fungi at genus level for its high universality and great capability. When species-level identification is required, some informative DNA markers besides ITS region should be included accordingly. Key words: ITS; Sequencing analysis; Filamentous fungi; Identification

Cutaneous Mycobacterium shigaense Infection in Immunocompetent Woman, China

To the Editor: Tortoli et al. (1) reported pulmonary disease caused by M. yongonense strains isolated from patients in Italy; these strains were identified by sequencing the 16S rRNA, hsp65, rpoB, and sodA genes and the internal transcribed spacer 1 (ITS1) region. The 16S rRNA gene sequence of these isolates showed 100% similarity with those of M. yongonense and M. marseillense. The isolates were more closely related to M. yongonense than to M. marseillense in terms of the hsp65 gene and ITS1 region; however, the rpoB gene sequence showed a higher degree of similarity to that of M. intracellulare (99.4%) than to that of M. marseillense (97.4%). The authors did not mention the similarity of the isolates with M. intracellulare in these sequences except for the rpoB gene. However, because these sequences showed high similarity to M. yongonense, a high degree of similarity to M. intracellulare could be inferred. The initial description of M. yongonense highlighted its unique molecular character (2). The 16S rRNA and hsp65 genes and ITS1 region are closely related to those of M. intracellulare ATCC 13950T; however, the rpoB gene is closely related to that of M. parascrofulaceum ATCC BAA-614T (99.4%). No consensus guidelines are available for mycobacterial identification, but the rpoB gene has been used widely as a target gene; multilocus sequence analysis also has been used recently (3,4). Although the authors suggest that a variant of M. yongonense preceded the acquisition of the rpoB gene from M. parascrofulaceum by a lateral gene transfer event (3), the isolates described are more similar to M. intracellulare than to M. yongonense on the basis of the rpoB gene sequence and multilocus sequence analysis. It is also possible that the isolates are a M. yongonense strain that preceded the acquisition of the rpoB gene but that are not the same as the initially described M. yongonense.

A growing body of evidence suggests analysis of 16S rRNA gene sequences provides only a conservative estimate of the actual genetic diversity existing within microbial communities. We examined the less conserved internal transcribed spacer (ITS) region of the ribosomal operon to determine the impact microdiversity may have on our view of marine microbial consortia. Analysis of over 500 ITS sequences and 250 associated 16S rRNA gene sequences from an oceanic time series station in the San Pedro Channel, California, USA, revealed that the community in this region is com- posed of large numbers of distinct lineages, with more than 1000 lineages estimated from 3 clusters alone (the SAR11 clade, the Prochlorococcus low-B/A clade 1, and the Roseobacter NAC11-7 clade). Although we found no instances where divergent ITS sequences were associated with identical 16S rRNA gene sequences, the ITS region showed much greater pairwise divergence between clones. By comparison to our 16S rRNA gene-ITS region linked database, we were able to place all ITS sequences into a phylogenetic framework, allowing them to act as an alternative molecular marker with enhanced resolution. Comparison of SAR11 clade ITS sequences with those available in GenBank indicated phylogenetic groupings based not only on depth but also on geography, potentially indicating localized differentiation or adaptation.

The purpose of the present study was to obtain sarcocysts of Sarcocystis buffalonis and Sarcocystis levinei from water buffaloes and characterize the isolates by molecular methods in order to determine whether the two species were genetically different from Sarcocystis hirsuta and Sarcocystis cruzi, respectively, from cattle, which had been characterized before. About 35 macroscopically visible (3-4 × 1-2mm) and 20 barely visible (1-3 × 0.2mm) sarcocysts were excised from the esophagus of 18 naturally infected and freshly slaughtered adult water buffaloes at three slaughterhouses in Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were first characterized at the mitochondrial cytochrome c oxidase subunit I gene (cox1) gene through PCR amplification and direct sequencing. Selected isolates were subsequently further characterized at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit by direct sequencing or cloning. Only six of the isolated macroscopic sarcocysts belonged to S. buffalonis, whereas the others belonged to Sarcocystis fusiformis. Twelve of the smaller cysts belonged to S. levinei and seven to Sarcocystis sinensis. The characterization of the sarcocysts of S. sinensis and some of the sarcocysts of S. fusiformis have been reported before. Fifteen additional sarcocyst isolates of S. fusiformis were characterized at cox1 in the present study and found to be identical or closely similar to previous isolates. At cox1, the sequence identity between the six isolates of S. buffalonis was 99.8-100% (two haplotypes), whereas the identity between the 12 isolates of S. levinei was 99.0-100% (10 haplotypes). The identity between cox1 sequences of S. buffalonis and S. hirsuta (n = 56) was 92.9-93.6% (on average 93.4%), and the identity between cox1 sequences of S. levinei and S. cruzi (n = 22) was 92.9-94.0% (on average 93.5%). The phylogenetic analyses placed with high support the cox1 sequences of S. buffalonis and S. hirsuta into two monophyletic sister groups, and the same was true for the cox1 sequences of S. levinei and S. cruzi. Hence, the study established that S. buffalonis and S. levinei are distinct species different from S. hirsuta and S. cruzi, respectively. Nucleotide sequences of S. buffalonis could be distinguished from those of S. hirsuta also at the 28S rRNA gene (clearly different) and the ITS1 region (small and uncertain difference) but not at the 18S rRNA gene. Sequences of S. levinei could be distinguished from those of S. cruzi both at the 18S and 28S rRNA genes (ITS1 region not examined). However, the cox1 gene was superior to the 18S and 28S rRNA genes as regards the ability to unambiguously delimit the species within each species pair, since at the latter markers, the number of consistent nucleotide differences between the species was low and there was a slight overlap between the intraspecific and interspecific sequence divergence. Comparison of the newly generated 18S rRNA gene sequences of S. levinei from water buffaloes with similar sequences deposited in GenBank suggested that S. levinei and S. cruzi are not strictly intermediate host specific but might occasionally infect cattle and water buffaloes, respectively.

A new cosmocercid species, Cosmocercoides qingtianensis sp. n., collected from the intestine of the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura) is described using integrated approaches, including light and scanning electron microscopy, and sequencing and analyzing the ribosomal [small ribosomal DNA (18S) and internal transcribed spacer (ITS)] and mitochondrial [cytochrome c oxidase subunit 1 (cox1)] target regions, respectively. The new species can be distinguished from its congeners by the combination of the following morphological characters, including the large body size, the presence of lateral alae and somatic papillae in both sexes, the length of spicules, the particular morphology and length of gubernaculum, the number, arrangement and morphology of caudal rosettes, the presence of large medioventral precloacal papilla and the long tail. Our molecular analysis revealed the level of intraspecific genetic variation of C. qingtianensis sp. n. distinctly lower than that of the interspecific genetic variation in the ITS and cox1 regions. However, there are some overlaps in the range of intra- and interspecific 18S sequence divergence between the new species and some closely related species. The results of molecular analysis supported the validity of the new species based on the morphological observations. The 18S, ITS, and cox1 regions of C. pulcher collected from Bufo japonicus formosus in Japan were also sequenced and analyzed. The results showed a low level of intraspecific genetic variation in 18S and ITS regions (0-0.12% and 0-0.23% nucleotide differences, respectively), but a relatively high level of intraspecific genetic variation in cox1 region (0.78-4.69% nucleotide differences). In addition, it seems more powerful and practical to use the cox1 region as a genetic marker for the accurate identification and differentiation of species of Cosmocercoides than the 18S and ITS regions, especially for the closely related species.

Phylogenetic Relationships of Sorghum and Related Species Inferred from Sequence Analysis of the nrDNA ITS Region