Large Subunit Ribosomal RNA Gene Research Articles

Taxonomy and toxicity of Prorocentrum from Perhentian Islands (Malaysia), with a description of a non-toxigenic species Prorocentrum malayense sp. nov. (Dinophyceae)

Thirteen isolates of Prorocentrum species were established from the coral reefs of Perhentian Islands Marine Park, Malaysia and underwent morphological observations and molecular characterization. Six species were found: P. caipirignum, P. concavum, P. cf. emarginatum, P. lima, P. mexicanum and a new morphotype, herein designated as P. malayense sp. nov. Prorocentrum malayense, a species closely related to P. leve, P. cf. foraminosum, P. sp. aff. foraminossum, and P. concavum (Clade A sensu Chomérat et al. 2018), is distinguished from its congeners as having larger thecal pore size and a more deeply excavated V-shaped periflagellar area. Platelet arrangement in the periflagellar area of P. malayense is unique, with the presence of platelet 1a and 1b, platelet 2 being the most anterior platelet, and a broad calabash-shaped platelet 3. The species exhibits consistent genetic sequence divergences for the nuclear-encoded large subunit ribosomal RNA gene (LSU rDNA) and the second internal transcribed spacer (ITS2). The phylogenetic inferences further confirmed that it represents an independent lineage, closely related to species in Clade A sensu Chomérat et al. Pairwise comparison of ITS2 transcripts with its closest relatives revealed the presence of compensatory base changes (CBCs). Toxicity analysis showed detectable levels of okadaic acid in P. lima (1.0–1.6 pg cell˗1) and P. caipirignum (3.1 pg cell˗1); this is the first report of toxigenic P. caipirignum in the Southeast Asian region. Other Prorocentrum species tested, including the new species, however, were below the detection limit.

Next generation sequencing from Hepatozoon canis (Apicomplexa: Coccidia: Adeleorina): Complete apicoplast genome and multiple mitochondrion-associated sequences

Extrachromosomal genomes of the adeleorinid parasite Hepatozoon canis infecting an Israeli dog were investigated using next-generation and standard sequencing technologies. A complete apicoplast genome and several mitochondrion-associated sequences were generated. The apicoplast genome (31,869 bp) possessed two copies of both large subunit (23S) and small subunit (16S) ribosomal RNA genes (rDNA) within an inverted repeat region, as well as 22 protein-coding sequences, 25 transfer RNA genes (tDNA) and seven open reading frames of unknown function. Although circular-mapping, the apicoplast genome was physically linear according to next-generation data. Unlike other apicoplast genomes, genes encoding ribosomal protein S19 and tDNAs for alanine, aspartic acid, histidine, threonine and valine were not identified. No complete mitochondrial genome was recovered using next-generation data or directed PCR amplifications. Eight mitochondrion-associated (215–3523 bp) contigs assembled from next-generation data encoded a complete cytochrome c oxidase subunit I coding sequence, a complete cytochrome c oxidase subunit III coding sequence, two complete cytochrome B coding sequences, a non-coding, pseudogene for cytochrome B and multiple fragmented mitochondrial rDNA genes (SSUA, SSUB, SSUD, LSUC, LSUG, RNA6, RNA10, RNA14, RNA18). The paucity of NGS reads generating each of the mitochondrion-like sequences suggested that a complete mitochondrial genome at typically high copy number was absent in H. canis. In contrast, the complete nuclear rDNA unit sequence of H. canis (18S rDNA to 28S rDNA, 6977 bp) had >1000-fold next-generation coverage. Multiple divergent (from 93.6% to 99.9% pairwise identities) nuclear 18S rDNA contigs were generated (three types with 10 subtypes total). To our knowledge this is the first apicoplast genome sequenced from any adeleorinid coccidium and the first mitochondrion-associated sequences from this serious pathogen of wild and domestic canids. These newly generated sequences may provide useful genetic loci for high-resolution species-level genotyping that is currently impossible using existing nuclear rDNA targets.

Prototheca paracutis sp. nov., a novel oleaginous achlorophyllous microalga isolated from a mangrove forest

Abstract Two strains of yeast-like achlorophyllous alga belonging to the genus Prototheca were isolated from the water and soil, respectively, from a mangrove forest in Thailand. Cultures of both strains were achlorophyllous when grown under light and dark conditions, and they reproduced by release of sporangiospores. Phylogenetic analyses, based on the D1/D2 region of the large subunit ribosomal RNA gene and the small subunit ribosomal RNA gene nucleotide sequences, revealed these two strains were closely related to Prototheca cutis. The strains grew well at 25 °C, weakly or slowly at 30 °C, but not at 35 °C and higher, and were found to be susceptible to 50 μg/disk clotrimazole, as determined by the disk diffusion test. They assimilated a limited number of carbon/nitrogen compounds; glucose, galactose, trehalose, ethanol, glycerol, propylene glycol, lactic acid, fructose and mannose as sole sources of carbon, and ammonium, lysine and cadaverine as sole sources of nitrogen. The two strains are clearly distinguished from P. cutis by the abilities to assimilate polypropylene glycol and the inability to grow at 35 °C and higher. In this study, the ninth member of the genus Prototheca, Prototheca paracutis sp. nov. (ex-type strain YMTW3-1T = JCM 32112T = TBRC8745T), is proposed. The MycoBank number is MB 821626. In addition, P. paracutis sp. nov. was observed to accumulate lipid at up to 21% of the cell dry weight, characterizing it as an oleaginous microorganism.

Basilaphelenchus gorganensis n. sp. (Aphelenchoidea, Tylaphelenchinae) from wood from northern Iran

AbstractBasilaphelenchus gorganensis n. sp. is described and illustrated from wood and bark of a dead tree from northern Iran. The new species is characterized by female body length (415–559 µm), three‐lined lateral fields, a sclerotized cephalic vestibule and cephalic framework, thin stylet with three elongate backwardly directed knobs, small spherical to oval metacorpus, with small and posteriorly located valve, simple vulva without any flap apparatus, 59‐ to 79‐µm‐long post‐vulval uterine sac, functional rectum and anus and dorsally convex, ventrally concave, usually ventrally bent conical female tail with a sharp tip. Males are common, apparently functional and characterized by well‐curved spicules, three pairs of small caudal papillae and no bursa at tail tip. Molecular phylogenetic inferences using partial sequences of small and large subunit ribosomal RNA genes (SSU and LSU rDNA) from different isolates of the new species revealed it differs from currently sequenced species and belongs to the Tylaphelenchinae clade.

New observations on Miculenchus Andrássy, 1959 (Nematoda: Tylenchidae) with descriptions of two new and one known species from Iran

Summary During nematological surveys in grasslands and natural forests of north and north-western Iran, three species of Miculenchus, including two new and one known species, were recovered and characterised based upon morphological and molecular approaches. Miculenchus brevisalvus n. sp., the first new species, is mainly characterised by its short females 334-388 μm long and with a short 6.0-7.5 μm long stylet, pyriform to pyriform-elongate pharyngeal bulb, 4-8 μm long post-uterine sac (PUS), offset rounded spermatheca filled with small spheroid sperm, elongate conoid tail 62-83 μm long with a sharp tip, and males with simple cloacal lips. Miculenchus muscus n. sp., the second new species, is characterised by a combination of the following features: body 401-467 μm long, well-developed protuberant labial plate at the anterior end under light microscopy, stylet 7-9 μm long, pyriform pharyngeal bulb, PUS 4-9 μm long, gradually narrowing conical tail 62-74 μm long with a finely pointed or sharp end and bearing several fine bristles at tip, and a male with projecting cloacal lips. Both newly described species were morphologically compared with four currently known species of the genus, viz., M. elegans, M. salmae, M. salvus, and M. tesselatus. Miculenchus salmae was also recovered and reported from Iran for the first time. It is mainly characterised by lacking a PUS and the characteristic vagina shape. Miculenchus muscus n. sp. and M. salmae were both characterised using scanning electron microscopic images, yielding new morphological observations for the genus. All three species are studied for their molecular phylogenetic characters using sequences of near-full length fragments of the small subunit ribosomal RNA gene (SSU rDNA) and the D2-D3 expansion segments of the large subunit ribosomal RNA gene (LSU rDNA D2-D3). In both SSU and LSU phylogenies, all currently sequenced species of Miculenchus formed a monophyletic group with maximal clade support in both Bayesian inference and maximum likelihood analysis.

Description of Basilaphelenchus brevicaudatus n. sp. (Aphelenchoidea: Tylaphelenchinae) from a dead forest tree in northern Iran

Summary Basilaphelenchus brevicaudatus n. sp., the third species of this apparently rare genus, is described and illustrated. It was recovered from wood and bark samples from a dead forest tree in the Golestan province of northern Iran. It is typologically characterised by female body length (448 (365-492) μm), three lines in the lateral fields, raised cephalic region having a sclerotised vestibule and cephalic framework, stylet thin with delicate conus and thicker shaft, both parts lacking a visible lumen, and with three elongate, backwardly directed knobs, small, spherical to spade-shaped metacorpus with small, posteriorly located valve (at 72 (58-74)% of metacorpus length), simple vulva without flap at 72.5 (69-78)% of body length, post-vulval uterine sac 32.4 (29.0-37.0) μm long, functional rectum and anus, female tail conical, short (c′ = 2.6 (1.9-3.3) in female, and 2.5 (2.3-2.8) in male), dorsally convex and ventrally concave with blunt end or having a small mucron, both forms with a hyaline-like tip. Males common, with well-curved 9.2 (9.0-10.5) μm long spicules measured along the mid-line, three pairs of small caudal papillae (lacking the single P1 ventral papilla) and no bursa at tail tip, but with hyaline region, similar to females. Basilaphelenchus brevicaudatus n. sp. is compared with the two currently known species of the genus, the type species, B. persicus, and B. grosmannae. Molecular phylogenetic inferences using partial sequences of small and large subunit ribosomal RNA genes (SSU and LSU) from different isolates of the new species revealed that it belongs to the Tylaphelenchinae clade.

Seinura caverna n. sp. (Tylenchomorpha: Aphelenchoididae), an androdioecious species isolated from bat guano in a calcareous cave

SummaryA new species of a predator aphelenchoidid,Seinura cavernan. sp., is described and illustrated. The new species was isolated from bat guano collected from a calcareous cave in Japan. The new species is characterised by its three-lined lateral field, secretory-excretory pore at the level of the posterior two-thirds of the metacorpus, a long post-uterine sac, hermaphrodite tail shape elongate conoid with a filiform tip, and an androdioecious reproductive mode. The new species is typologically and biologically close toS. steineri, but is distinguished by its longer post-uterine sac, slightly longer stylet and slightly larger median bulb. The comparisons with other morphologically similar species,i.e.,S. chertkovi,S. tenuicaudataandS. steineri, are discussed. A molecular phylogenetic analysis based on two small and large subunit ribosomal RNA genes revealed that the new species is located at the basal position of clade 3 of the Aphelenchoididae, clearly separate fromS. demani, its congener with unclear rectum and anus, which is located at the derived position, suggesting that further generic revision is necessary for the genus.Aphelenchoides liin. comb. (=Seinura lii) is proposed.

Molecular detection of Pneumocystis in the lungs of cats.

The genus Pneumocystis comprises potential pathogens that reside normally in the lungs of a wide range of mammals. Although they generally behave as transient or permanent commensals, they can occasionally cause life-threatening pneumonia (Pneumocystis pneumonia; PCP) in immunosuppressed individuals. Several decades ago, the presence of Pneumocystis morphotypes (trophic forms and cysts) was described in the lungs of normal cats and cats with experimentally induced symptomatic PCP (after immunosuppression by corticosteroids); yet to date spontaneous or drug-induced PCP has not been described in the clinical feline literature, despite immunosuppression of cats by long-standing retrovirus infections or after kidney transplantation. In this study, we describe the presence of Pneumocystis DNA in the lungs of normal cats (that died of various unrelated causes; n = 84) using polymerase chain reactions (PCRs) targeting the mitochondrial small and large subunit ribosomal RNA gene (mtSSU rRNA and mtLSU rRNA). The presence of Pneumocystis DNA was confirmed by sequencing in 24/84 (29%) cats, with evidence of two different sequence types (or lineages). Phylogenetically, lineage1 (L1; 19 cats) and lineage 2 (L2; 5 cats) formed separate clades, clustering with Pneumocystis from domestic pigs (L1) and carnivores (L2), respectively. Results of the present study support the notion that cats can be colonized or subclinically infected by Pneumocystis, without histological evidence of damage to the pulmonary parenchyma referable to pneumocystosis. Pneumocystis seems most likely an innocuous pathogen of cats’ lungs, but its possible role in the exacerbation of chronic pulmonary disorders or viral/bacterial coinfections should be considered further in a clinical setting.

Taxonomic revision of Spiniferites elongatus (the resting stage of Gonyaulax elongata) based on morphological and molecular analyses

We restudied the morphological complex comprising the cyst-based species Spiniferites elongatus/Spiniferites frigidus/Rottnestia amphicavata. We reviewed existing studies, and acquired new morphometric measurements of recent cysts from across the Northern Hemisphere, scanning electron microscopy (SEM) observations of cysts from Barents Sea surface sediments, and genetic analyses of cysts from the Beaufort Sea. The measurements suggest that populations and morphospecies cannot be distinguished based on morphometric criteria. Furthermore, sequential sediment trap samples from Hudson Bay reveal that morphological variation can occur at the same location over a few weeks, arguing against a uniform morphological response to environmental parameters. The SEM observations reveal a consistent gonyaulacacean tabulation (Po, 4′, 6′′, 6c, 5 s, *6′′′, 1p, 1′′′′). The small and large subunit ribosomal RNA genes and the internal transcribed spacer sequences obtained from differing cysts from the Beaufort Sea with morphologies attributable to the complex, including forms that correspond to Rottnestia amphicavata, were all identical and conform to those of Gonyaulax elongata from the Orkney Islands. The molecular analyses thus support the conclusion that the morphological variability is not reflected genetically and occurs within one species. Based on arguments against the generic attribution of amphicavata to Rottnestia, the continuum between the extreme ends of the morphological range, and the molecular data, we suggest Rottnestia amphicavata to be conspecific with Spiniferites frigidus, and both to be junior synonyms of Spiniferites elongatus. The morphometric data further indicate that Spiniferites ellipsoideus, an elongate cyst from the Miocene, can also be considered a junior synonym of Spiniferites elongatus. It is recommended to use two informal types in census work (i.e. Spiniferites elongatus – Beaufort morphotype for morphologies formerly assignable to Spiniferites frigidus/Rottnestia amphicavata, and Spiniferites elongatus – Norwegian morphotype for cysts with strongly reduced processes) to separate specimens at both extreme ends of the morphological spectrum from typical specimens of Spiniferites elongatus.

Preservation of diversity and oenological properties of wine yeasts during long-term laboratory maintenance: A study of strains of a century-old Tokaj wine yeast collection

The effect of long-term laboratory maintenance on the biological diversity and certain oenolological properties of wine yeasts was investigated by the analysis of 17 strains isolated from Tokaj (Hungary) wines more than a century ago and maintained since then in culture collections. The analysis of the D1/D2 domains of the large subunit ribosomal RNA genes and the ITS sequences assigned them all to S. cerevisiae but divided them into two groups. One group showed sequence identity with wine strains occurring mainly in countries of the region. The combined results of the interdelta, RAPD, microsatellite-primed PCR (MSP), mtDNA and karyotype analyses found both groups diverse and defined each strain as unique, demonstrating that the long period of maintenance under identical conditions has not resulted in convergent evolution in their genomes. High diversity was detected also in certain phenotypic traits of oenological relevance (osmotolerance, killer phenotype, production of glycerol, ethanol, H2S and volatile acids). In the microvinification experiments all strains performed better than the laboratory strain S288c and were comparable to the industrial strain used as a control or even surpassed it in certain parameters. Thus, the oenological abilities of wine yeasts can be preserved over long periods of maintenance under laboratory conditions.

Spencerozyma siamensis sp. nov., a novel anamorphic basidiomycetous yeast species in Puccinomycotina isolated from coral in Thailand

Strain DMKU13-2T, representing a novel anamorphic yeast species in the class Microbotryomycetes, subphylum Puccinomycotina, phylum Basidiomycota, was isolated from a soft coral collected in the sea off Ko Mu island, Thailand. The phylogenetic analysis based on the concatenated sequences of the internal transcribed spacer (ITS) region and the D1/D2 region of the large subunit ribosomal RNA (LSU rRNA) gene indicated that this strain was a novel species in the genus Spencerozyma and distant from Spencerozyma crocea, the only species of the genus. The novel species differed from the type of S. crocea (CBS 2029T) by 5.2 % nucleotides (31 nucleotide substitutions out of 594 bp) in the D1/D2 region of the LSU rRNA gene and 9.0 % nucleotides (66 nucleotide substitutions out of 735 bp) in the ITS region. The name Spencerozyma siamensis sp. nov. is proposed. The type is DMKU13-2T (=CBS 14683=TBRC 7039). The MycoBank number is MB 824889.

ITSxpress: Software to rapidly trim internally transcribed spacer sequences with quality scores for marker gene analysis

The internally transcribed spacer (ITS) region between the small subunit ribosomal RNA gene and large subunit ribosomal RNA gene is a widely used phylogenetic marker for fungi and other taxa. The eukaryotic ITS contains the conserved 5.8S rRNA and is divided into the ITS1 and ITS2 hypervariable regions. These regions are variable in length and are amplified using primers complementary to the conserved regions of their flanking genes. Previous work has shown that removing the conserved regions results in more accurate taxonomic classification. An existing software program, ITSx, is capable of trimming FASTA sequences by matching hidden Markov model profiles to the ends of the conserved genes using the software suite HMMER. ITSxpress was developed to extend this technique from marker gene studies using Operational Taxonomic Units (OTU’s) to studies using exact sequence variants; a method used by the software packages Dada2, Deblur, QIIME 2, and Unoise. The sequence variant approach uses the quality scores of each read to identify sequences that are statistically likely to represent real sequences. ITSxpress enables this by processing FASTQ rather than FASTA files. The software also speeds up the trimming of reads by a factor of 14-23 times on a 4-core computer by temporarily clustering highly similar sequences that are common in amplicon data and utilizing optimized parameters for Hmmsearch. ITSxpress is available as a QIIME 2 plugin and a stand-alone application installable from the Python package index, Bioconda, and Github.

ITSxpress: Software to rapidly trim internally transcribed spacer sequences with quality scores for marker gene analysis.

The internally transcribed spacer (ITS) region between the small subunit ribosomal RNA gene and large subunit ribosomal RNA gene is a widely used phylogenetic marker for fungi and other taxa. The eukaryotic ITS contains the conserved 5.8S rRNA and is divided into the ITS1 and ITS2 hypervariable regions. These regions are variable in length and are amplified using primers complementary to the conserved regions of their flanking genes. Previous work has shown that removing the conserved regions results in more accurate taxonomic classification. An existing software program, ITSx, is capable of trimming FASTA sequences by matching hidden Markov model profiles to the ends of the conserved genes using the software suite HMMER. ITSxpress was developed to extend this technique from marker gene studies using Operational Taxonomic Units (OTU's) to studies using exact sequence variants; a method used by the software packages Dada2, Deblur, QIIME 2, and Unoise. The sequence variant approach uses the quality scores of each read to identify sequences that are statistically likely to represent real sequences. ITSxpress enables this by processing FASTQ rather than FASTA files. The software also speeds up the trimming of reads by a factor of 14-23 times on a 4-core computer by temporarily clustering highly similar sequences that are common in amplicon data and utilizing optimized parameters for Hmmsearch. ITSxpress is available as a QIIME 2 plugin and a stand-alone application installable from the Python package index, Bioconda, and Github.

The complete mitochondrial genome of the stalk-forming diatom Didymosphenia Geminata

The complete mitogenome of the stalk-forming diatom Didymosphenia geminata collected from Mineral County, WV, USA was sequenced on the Ion Torrent PGM and Proton sequencers. The D. geminata mitogenome is 37,765 bp and encodes 35 protein coding genes, 25 tRNAs, and both large and small subunit ribosomal RNA genes. The nad11 gene is split into two domains as observed in Phaeodactylum tricornutum, and D. geminata also lacks the large repeat region found in the P. tricornutum mitogenome. Gene order and content within the D. geminata mitogenome is similar to the diatom Berkeleya fennica.

Development and Validation of PCR-RFLP Assay for Identification of Gambierdiscus species in the Greater Caribbean region.

The genus Gambierdiscus is a recognized group of marine epiphytic-benthic dinoflagellates that produce the toxins that cause ciguatera fish poisoning (CFP). To date, thirteen species and six ribotypes of Gambierdiscus have been identified, and multiple species commonly co-occur within a single site or epiphyte community. Toxicity can vary by species, thus it is important to be able to differentiate among species for research and monitoring purposes. Gambierdiscus species have very similar morphological characteristics and are difficult or impossible to distinguish using light microscopy. DNA sequencing has been an important tool in the definition of Gambierdiscus species, but it can be time-consuming and relatively expensive. To provide an alternative approach, a PCR-RFLP protocol was developed for efficient, rapid, and cost-effective identification of Gambierdiscus strains isolated from the Gulf of Mexico and Caribbean Sea, where CFP cases and Gambierdiscus spp. have been reported. The assay targets the D1-D2 hypervariable regions of the large subunit ribosomal RNA gene and uses a single restriction enzyme, BsrI. This method produces distinct RFLP banding patterns for the six species of Gambierdiscus reported from the Gulf of Mexico and Caribbean Sea, and also distinguishes them from four Pacific endemic species. This method was successfully used to type 465 clonal isolates of Gambierdiscus from the U.S. Virgin Islands and Akumal Beach - Mexico This BsrI PCR-RFLP method expands the tools available to researchers and managers engaged in monitoring activities and ecological studies.

Characterisation of the intracellular protozoan MPX in Scottish mussels, Mytilus edulis Linnaeus, 1758.

Ciliates have been reported as pathogens of many species of economically important bivalves. Mussel protozoan X (MPX), is an uncharacterised intracellular ciliate of mussels and has been widely reported in Mytilus spp. around the world. In order to characterise this ciliate, Mytilus edulis samples were collected from a site on the West coast of Scotland, and four different fixatives for histological examination were tested. Fresh preparations of mussel digestive glands were also examined by laser scanning confocal microscopy. Intracellular ciliates were prepared by laser capture microdissection and partial sequences of small subunit ribosomal RNA gene and of large subunit ribosomal RNA gene were generated, using Phyllopharyngea primers. Methacarn solution proved to be the best fixative for both histological and molecular characterisation. The morphological and molecular investigations confirmed that this ciliate belongs to the class Phyllopharyngea, order Rhynchodida. However, this organism does not belong to any known family, genus or species, therefore, a new description is necessary, following further morphological analyses. Most mussel samples containing MPX displayed mild to moderate infections, with no signs of necrosis or haemocytic response, although a single sample displayed a severe infection (∼103 ciliates per section). The localisation of this ciliate in tissues other than the digestive gland, the presence of necrosis in infected tissue of the most severely infected mussel and the binary fission of this ciliate have been observed here for the first time. We also report the first observation of the live ciliate isolated from tissue. Although MPX remains of unknown significance to the mussel industry, tools and protocols described here will be useful in further characterising these and other ciliates (subclass Rhynchodia) known as pathogens for bivalves.

Biological and molecular features of Nosema rachiplusiae sp. n., a microsporidium isolated from the neotropical moth Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae).

Light, electron microscopy and DNA analyses were performed to characterize a microsporidium infecting Rachiplusia nu larvae from a laboratory rearing in Argentina. Diplokaryotic spores were oval and measured 3.61 ± 0.29 × 1.61 ± 0.14μM (fresh). The spore wall was composed of an electron-dense exospore and an electron-lucent endospore, ca. 30nm and 100-120nm thick, respectively. The polar filament was arranged in a single rank of 10-12 coils (typically 11). Microsporidian cells were found in the cytoplasm, next to the endoplasmic reticulum (especially the prespore stages) and generally surrounded by electron-lucent spaces. The infection was polyorganotropic; the fat body appeared as the most heavily invaded tissue, followed by tracheal matrix and epidermis. A molecular phylogeny based on the small (SSU) and large subunit (LSU) ribosomal RNA genes clearly placed the new isolate within the "Nosema bombycis clade". Considering both SSU and LSU concatenated partial sequences, the microsporidium from R. nu showed 99.5% nucleotide similarity with N. bombycis and 99.8% with its closest relative, a microsporidium isolated from Philosamia cynthia. According to its genetic and biological features, the R. nu isolate is proposed as the new species Nosema rachiplusiae sp. n., expanding the limited knowledge on microsporidia associated to endemic South-American moths.

First record of Trypanosoma dionisii of the T. cruzi clade from the Eastern bent-winged bat (Miniopterus fuliginosus) in the Far East

Chiropteran mammals worldwide harbour trypanosomes (Euglenozoa: Kinetoplastea: Trypanosomatida) of the subgenus 'Schizotrypanum' in the classical sense. Latterly, these trypanosomes have been referred to as members of the 'Trypanosoma cruzi clade' as their phylogenetic relationships, structure and life cycle conform to T. cruzi, parasitising various terrestrial mammals as well as humans in Latin America. Little is known, however, about the trypanosome species in Asian bats. During a survey on Borrelia spp. in the Eastern bent-winged bat (Miniopterus fuliginosus) living in a cave in Wakayama Prefecture, Japan, incidental proliferation of trypanosomes was detected in two of 94 haemocultures. Squat or slender trypanosomes that proliferated in the cultures were 7.5-20.5μm in length between both body ends and 1.0-3.8μm in width with/without free flagella up to 14.5μm (n= 29). The nucleotide sequences of the small subunit ribosomal RNA gene (SSU rDNA; 2176bp), large subunit ribosomal RNA gene (1365bp) and glycosomal glyceraldehyde-3-phosphate dehydrogenase gene (gGAPDH; 843bp) of the present isolates were characterized to clarify their molecular phylogenetic position in T. cruzi-like trypanosomes. The newly obtained SSU rDNA and gGAPDH nucleotide sequences showed the highest identities with Brazilian and European isolates of Trypanosoma dionisii of the T. cruzi clade, ranging between 99.4 and 99.7% or between 95.6 and 99.3% identities, respectively. Although multiple T. dionisii isolates from the North and South American continents showed the closest molecular genetic relatedness to the present Far East isolates, only short SSU rDNA segments of the former isolates were deposited. Therefore, a definitive conclusion cannot be made until full nucleotide sequencing of at least the American isolates' SSU rDNA is available. This is the first confirmation of a Far East distribution of T. dionisii, demonstrating a wide geographical distribution of the species in the Eurasian and American continents with a limited nucleotide variation.

Further insights into the highly derived haptorids (Ciliophora, Litostomatea): Phylogeny based on multigene data

Evolutionary relationships of taxa within the ciliate subclass Haptoria are poorly understood. In this study, we broaden the taxon sampling by adding 14 small subunit ribosomal RNA gene sequences, 13 large subunit ribosomal RNA gene sequences and 13 ITS1‐5.8S‐ITS2 gene sequences of haptorians. This includes the first molecular data from two genera, Pseudotrachelocerca Song, 1990, and Foissnerides Song & Wilbert, 1989. Phylogenies inferred from the three individual genes and concatenated data sets show that: (i) the subclass Haptoria could be a multiphyletic complex with about up to four main clades while “interrupted” by some intermingled with the related subclasses Rhynchostomatia, Trichostomatia and some incertae sedis; (ii) the genus Pseudotrachelocerca Song, 1990, is clearly separated from Litostomatea and clusters within an assemblage comprising the classes Prostomatea, Colpodea and Plagiopylea; (iii) both morphological evidence and molecular evidence indicate that the genus Foissnerides should be transferred from family Trachelophyllidae to Pseudoholophryidae; (iv) the validity of the order Helicoprorodontida Grain, 1994, and its monophyly is strongly supported; (5) the family Chaeneidae does not belong to the order Lacrymarida but represents a distinct clade in the subclass Haptoria.

Rigidotubus tephroleucus gen. et sp. nov. (Cystostereaceae, Agaricales) evidenced by morphological characters and phylogenetic analyses

A new poroid wood-inhabiting fungal genus, Rigidotubus, is proposed to accommodate R. tephroleucus sp. nov. based on morphological characters and molecular evidence. The genus is characterized by tough and poroid basidiomata, white to grayish white pore surface, white sterile margin, a monomitic hyphal system with clamped generative hyphae, ovoid to ellipsoid basidiospores negative in Melzer’s reagent and Cotton Blue. The phylogenetic analyses inferred from the internal transcribed spacer (ITS) and nuclear large subunit (28S) ribosomal RNA gene regions confirmed that Rigidotubus belongs to Cystostereaceae in Agaricales. A key to the accepted genera of Cystostereaceae is provided.