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While the central dogma of molecular biology describes how genetic information flows, gene expression is also affected by epigenetic and epitranscriptomic processes. A recent report by Rajan et al. demonstrates how pseudouridylation of a Leishmania ribosomal rRNA affects the expression of particular proteins: an example of epitranslatomic control. [ABSTRACT FROM AUTHOR]

Summary: A new species of Aporcelinus , collected in a natural habitat of central Mexico, is described and illustrated, including molecular (D2-D3 28 S -rRNA) data. Aporcelinus zapotitlanensis sp. n. is characterised by its 1.19-1.43 mm long body, lip region offset by a deep constriction and 16.5-18.5 μ m broad, odontostyle 19.5-22 long, neck 325-368 μ m long, pharyngeal expansion 150-201 μ m long or 46-55% of the total neck length, uterus 130-167 μ m long and tripartite, V = 54-56, tail conical with finely rounded tip (30-37 μ m, c = 34-43, c′ = 1.2-1.4) lacking a distinct dorsal concavity, spicules 48-51 μ m long, and 11-12 spaced ventromedian supplements with one or two of them located within the range of the spicules. Molecular analyses confirm the monophyly of Aporcelinus and places the new species within a clade shared with A. infundibulicaudatus and A. amazonicus. [ABSTRACT FROM AUTHOR]

Despite of the integrity of their RNA, some desert truffles present a non-canonical profile of rRNA where 3.3 kb is absent, 1.8 kb is clear and a band of 1.6 kb is observed. A similar rRNA profile was identified in organisms belonging to different life kingdoms, with the exception of the Kingdom Fungi, as a result of a split LSU rRNA called hidden gap. rRNA profiles of desert truffles were analyzed to verify the presence of the non-canonical profile. The RNA of desert truffles and yeast were blotted and hybridized with probes complementary to LSU extremes. RACE of LSU rRNA was carried out to determine the LSU rRNA breakage point. LSU rRNA of desert truffles presents a post-transcriptional cleavage of five nucleotides that generates a hidden gap located in domain D7. LSU splits into two molecules of 1.6 and 1.8 kb. Similar to other organisms, a UAAU tract, downstream of the breakage point, was identified. Phylogenetic comparison suggests that during fungi evolution mutations were introduced in the hypervariable D7 domain, resulting in a sequence that is specifically post-transcriptionally cleaved in some desert truffles.

The most studied digenean of marine organisms in Chile is by far Proctoeces humboldti , a parasite of the intestine of the clingfish Sicyases sanguineus and gonad of the keyhole limpet Fissurella spp. (progenetic metacercariae). The mussel Perumytilus purpuratus has been suggested as the first intermediate host for this digenean. In a study examining the parasites of S . sanguineus from central Chile, we found specimens of Proctoeces showing significant morphological differences with P . humboldti . To assist in the resolution of the taxonomic identification of these specimens, as well sporocysts obtained from the mussel P . purpuratus from central and northern Chile, phylogenetic studies using DNA sequences from the SSU rRNA, as well the LSU rRNA and Cox 1 gene were performed. Results showed that the clingfish S . sanguineus is a host for two species of Proctoeces ( P . humboldti and P . syciases n. sp.) along the northern and central Chilean coast, without geographic separation; the mussel P . purpuratus is the first intermediate host for P . syciases n. sp. but not for P . humboldti in central and northern Chile. Fissurellids (Archaeogastropoda) along the Chilean coast harbor only progenetic stages of P . humboldti , but there is no evidence of progenesis for P . syciases . The reinstatement of Proctoeces humboldti is strongly suggested. [ABSTRACT FROM AUTHOR]

A new nontoxic Pseudo-nitzschia species belonging to the P. pseudodelicatissima complex, P. arctica, was isolated from different areas of the Arctic. The erection of P. arctica is mainly supported by molecular data, since the species shares identical ultrastructure with another species in the complex, P. fryxelliana, and represents a new case of crypticity within the genus. Despite their morphological similarity, the two species are not closely related in phylogenies based on LSU, ITS and rbcL. Interestingly, P. arctica is phylogenetically most closely related to P. granii and P. subcurvata, from which the species is, however, morphologically different. P. granii and P. subcurvata lack the central larger interspace which is one of the defining features of the P. pseudodelicatissima complex. The close genetic relationship between P. arctica and the two species P. granii and P. subcurvata is demonstrated by analysis of the secondary structure of ITS2 which revealed no compensatory base changes, two hemi-compensatory base changes, and two deletions in P. arctica with respect to the other two species. These findings emphasize that rates of morphological differentiation, molecular evolution and speciation are often incongruent for Pseudo-nitzschia species, resulting in a restricted phylogenetic value for taxonomic characters used to discriminate species. The description of a new cryptic species, widely distributed in the Arctic and potentially representing an endemic component of the Arctic diatom flora, reinforces the idea of the existence of noncosmopolitan Pseudo-nitzschia species and highlights the need for combined morphological and molecular analyses to assess the distributional patterns of phytoplankton species. [ABSTRACT FROM AUTHOR]

Halicephalobus gingivalis is an opportunistic parasite which is known to cause fatal meningoencephalomyelitis primarily in equines but sporadically also in humans. In April 2014, laboratory examination of the head of a young dairy calf, euthanized due to severe central nervous system symptoms, revealed the presence of granulomatous to necrotizing encephalitis and myriads of nematodes in the brain lesion. Morphologically the parasites were identified as H. gingivalis. The diagnosis was confirmed by molecular analysis of the large subunit (LSU) rRNA and the small subunit (SSU) rRNA genes, revealing genetic variations of 0.5–4.4% and 0.7–8.6%, respectively, between the H. gingivalis isolated from the Danish calf and published isolates, collected worldwide from free-living and parasitic stages of the nematode. Clinical symptoms and histological changes indicated infection with H. gingivalis from another three calves in the herd. This is the first scientific publication of H. gingivalis induced meningoencephalomyelitis in ruminants. As ante mortem diagnosis is a major challenge, the infection may easily remain undiagnosed in cattle. [ABSTRACT FROM AUTHOR]

Calcareous sponges are taxonomically difficult, and their morpho-systematic classification often conflicts with molecular phylogenies. Consequently, species descriptions that rely solely on morphological characters,and taxonomic revisions appear to provide little to no information about phylogenetic affiliations and integrative approaches, combining DNA and morphological data, are applied more frequently. However, a standardized database that combines DNA sequence and morphological specimen information is still missing for calcareous sponges. The mitochondrial cytochrome oxidase subunit 1 gene (COI) is the marker of choice for rapid species identification in many other animal taxa, including demosponges, for which COI sequences and morphological information have been compiled in the sponge barcoding database (). But due to the peculiarities of calcarean mitochondrial genomes, sequencing COI in Calcarea is methodologically challenging. We here propose the use of one more commonly used DNA marker, the C-region of the 28S gene (LSU), as standard barcoding marker for Calcarea, after also considering the internal transcribed spacer (ITS) region for such proposes. Especially in the subclass Calcaronea, we observed severe problems of high intra- and intergenomic variation that impedes pan-calcarean ITS alignments. In contrast, the C-region of LSU provides a short but phylogenetically informative DNA sequence, alignable across both subclasses with the help of a newly developed secondary structure and which also can be used to address exemplary taxonomic questions. With our work, we start to close the gap of Calcarea in the sponge barcoding project () and provide a resource for biodiversity studies and potentially for DNA-guided species identification. [ABSTRACT FROM AUTHOR]

The Saprolegnia-Achlya clade comprises species of major environmental and economic importance due to their negative impact on aquaculture and aquatic ecosystems by threatening fishes, amphibians, and crustaceans. However, their taxonomy and phylogenetic relationships remain unresolved and suffer from many inconsistencies, which is a major obstacle to the widespread application of molecular barcoding to identify pathogenic strains with quarantine implications. We assessed phylogenetic relationships of major genera using three commonly used markers (ITS, SSU rRNA, and LSU rRNA). A consensus tree of the three genes provided support for nine clades encompassing eleven documented genera and a new clade (SAP1) that has not been described morphologically. In the course of this study, we isolated a new species, Newbya dichotoma sp. nov., which provided the only culture available for this genus. In parallel, we attempted to summarize the evolution of traits in the different genera, but their successive reversals rendered the inference of ancestral states impossible. This highlights even more the importance of a bar-coding strategy for saprolegniacean parasite detection and monitoring. [ABSTRACT FROM AUTHOR]

Haptophytes are a key phylum of marine protists, including ~300 described morphospecies and 80 morphogenera. We used 454 pyrosequencing on large subunit ribosomal DNA (LSU rDNA) fragments to assess the diversity from size-fractioned plankton samples collected in the Bay of Naples. One group-specific primer set targeting the LSU rDNA D1/D2 region was designed to amplify Haptophyte sequences from nucleic acid extracts (total DNA or RNA) of two size fractions (0.8-3 or 3-20 μm) and two sampling depths [subsurface, at 1 m, or deep chlorophyll maximum (DCM) at 23 m]. 454 reads were identified using a database covering the entire Haptophyta diversity currently sequenced. Our data set revealed several hundreds of Haptophyte clusters. However, most of these clusters could not be linked to taxonomically known sequences: considering OTUs97% (clusters build at a sequence identity level of 97%) on our global data set, less than 1% of the reads clustered with sequences from cultures, and less than 12% clustered with reference sequences obtained previously from cloning and Sanger sequencing of environmental samples. Thus, we highlighted a large uncharacterized environmental genetic diversity, which clearly shows that currently cultivated species poorly reflect the actual diversity present in the natural environment. Haptophyte community appeared to be significantly structured according to the depth. The highest diversity and evenness were obtained in samples from the DCM, and samples from the large size fraction (3-20 μm) taken at the DCM shared a lower proportion of common OTUs97% with the other samples. Reads from the species Chrysoculter romboideus were notably found at the DCM, while they could be detected at the subsurface. The highest proportion of totally unknown OTUs97% was collected at the DCM in the smallest size fraction (0.8-3 μm). Overall, this study emphasized several technical and theoretical barriers inherent to the exploration of the large and largely unknown diversity of unicellular eukaryotes. [ABSTRACT FROM AUTHOR]

Detailed morphological studies and molecular analyses based on nuclear-encoded large subunit ribosomal RNA (LSU rRNA) gene sequences were undertaken on Gloiocladia leptophylla, a poorly known species from Japan. Both newly collected samples and the holotype were examined. Herein we describe for the first time the structure of tetrasporangial nemathecia, decussately dividing tetrasporangia and male gametophytes. We show that this species has a thin cortex, decussately divided tetrasporangia formed in nemathecia and a tela arachnoidea (network of inner pericarp filaments surrounding mature carposporophytes), as in the genus Leptofauchea. LSU rRNA analyses resolved G. leptophylla in the Faucheaceae (in which it was robustly separated from Gloiocladia), forming a well-supported clade with five other species of Leptofauchea and two species of Webervanbossea. We therefore propose the new combination Leptofauchea leptophylla comb. nov. Iridescence in water, a distinct short stipe and a medulla composed of three to four cell layers are conspicuous diagnostic characters of L. leptophylla within the genus. [ABSTRACT FROM AUTHOR]

Abstract: This study presents a manually constructed alignment of nearly complete rRNA genes from most animal clades (371 taxa from ∼33 of the ∼36 metazoan phyla), expanded from the 197 sequences in a previous study. This thorough, taxon-rich alignment, available at http://www.wsu.edu/~jmallatt/research/rRNAalignment.html and in the Dryad Repository (doi: http://dx.doi.org/10.5061/dryad.1v62kr3q), is based rigidly on the secondary structure of the SSU and LSU rRNA molecules, and is annotated in detail, including labeling of the erroneous sequences (contaminants). The alignment can be used for future studies of the molecular evolution of rRNA. Here, we use it to explore if the larger number of sequences produces an improved phylogenetic tree of animal relationships. Disappointingly, the resolution did not improve, neither when the standard maximum-likelihood method was used, nor with more sophisticated methods that partitioned the rRNA into paired and unpaired sites (stem, loop, bulge, junction), or accounted for the evolution of the paired sites. For example, no doublet model of paired-site substitutions (16-state, 16A and 16B, 7A–F, or 6A–C models) corrected the placement of any rogue taxa or increased resolution. The following findings are from the simplest, standard, ML analysis. The 371-taxon tree only imperfectly supported the bilaterian clades of Lophotrochozoa and Ecdysozoa, and this problem remained after 17 taxa with unstably positioned sequences were omitted from the analysis. The problem seems to stem from base-compositional heterogeneity across taxa and from an overrepresentation of highly divergent sequences among the newly added taxa (e.g., sequences from Cephalopoda, Rotifera, Acoela, and Myxozoa). The rogue taxa continue to concentrate in two locations in the rRNA tree: near the base of Arthropoda and of Bilateria. The approximately uncertain (AU) test refuted the monophyly of Mollusca and of Chordata, probably due to long-branch attraction of the highly divergent cephalopod and urochordate sequences out of those clades. Unlikely to be correct, these refutations show for the first time that rRNA phylogeny can support some ‘wrong’ clades. Along with its weaknesses, the rRNA tree has strengths: It recovers many clades that are supported by independent evidence (e.g., Metazoa, Bilateria, Hexapoda, Nonoculata, Ambulacraria, Syndermata, and Thecostraca with Malacostraca) and shows good resolution within certain groups (e.g., in Platyhelminthes, Insecta, Cnidaria). As another strength, the newly added rRNA sequences yielded the first rRNA-based support for Carnivora and Cetartiodactyla (dolphin+llama) in Mammalia, for basic subdivisions of Bryozoa (‘Gymnolaemata+Stenolaemata’ versus Phylactolaemata), and for Oligostraca (ostracods+branchiurans+pentastomids+mystacocarids). Future improvement could come from better sequence-evolution models that account for base-compositional heterogeneity, and from combining rRNA with protein-coding genes in phylogenetic reconstruction. [Copyright &y& Elsevier]

C onklin K.Y. and S herwood A.R. 2012. Molecular and morphological variation of the red alga Spyridia filamentosa (Ceramiales, Rhodophyta) in the Hawaiian Archipelago. Phycologia 51: 347-357. DOI: 10.2216/10-26.1 Genetic information is proving to be important in conservation and management efforts that involve cryptic species and their role in native ecosystem restoration. Previous molecular studies have demonstrated that the red alga Spyridia filamentosa consists of several cryptic species. Spyridia filamentosa is considered to be a native species in Hawai'i and was recently used in preliminary seagrass meadow restoration efforts in Maunalua Bay, O'ahu. It became necessary to understand the genetic diversity of this species in the Islands and determine if there are relationships between any molecular clades of S. filamentosa and anatomical characters that might be useful (e.g. sediment capture) in habitat remediation. Both molecular and morphological (determinate branch cell dimensions) analyses revealed the presence of multiple S. filamentosa clades in Hawai'i. Nuclear [partial LSU (large subunit)] sequence and determinate branch cell data recovered two broad lineages (I and II), which are known worldwide. The mitochondrial sequence ( cox2-3 spacer) data further separated samples into five distinct clades and indicated that S. filamentosa arrived in Hawai'i on at least six occasions. These results aided decisions made concerning preliminary experiments involved in seagrass habitat remediation and emphasized the importance of genetically evaluating cryptic species that are being considered for use in native ecosystem restoration or conservation. [ABSTRACT FROM AUTHOR]

The aim of this work was to select endophytic fungi from mangrove plants that produced antimicrobial substances. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) or minimal fungicidal concentrations (MFC) of crude extracts from 150 isolates were determined against potential human pathogens by a colorimetric microdilution method. Ninety-two isolates (61.3%) produced inhibitory compounds. Most of the extracts (28-32%) inhibited Staphylococcus aureus (MIC/MBC 4-200/64-200 μg ml). Only two extracts inhibited Pseudomonas aeruginosa (MIC/MBC 200/>200 μg ml). 25.5 and 11.7% inhibited Microsporum gypseum and Cryptococcus neoformans (MIC/MFC 4-200/8-200 μg ml and 8-200/8-200 μg ml, respectively), while 7.5% were active against Candida albicans (MIC/MFC 32-200/32-200 μg ml). None of the extracts inhibited Escherichia coli. The most active fungal extracts were from six genera, Acremonium, Diaporthe, Hypoxylon, Pestalotiopsis, Phomopsis, and Xylaria as identified using morphological and molecular methods. Phomopsis sp. MA194 (GU592007, GU592018) isolated from Rhizophora apiculata showed the broadest antimicrobial spectrum with low MIC values of 8-32 μg mlagainst Gram-positive bacteria, yeasts and M. gypseum. It was concluded that endophytic fungi from mangrove plants are diverse, many produce compounds with antimicrobial activity and could be suitable sources of new antimicrobial natural products. [ABSTRACT FROM AUTHOR]

Group I introns have the ability to catalyse their own excision (self-splice) from pre-RNA, and are found in a wide range of eukaryotic organisms. In fungal nuclear genomes, they have been identified in the small subunit (SSU) and large subunit (LSU) of the ribosomal RNA gene. Sequencing of the 3' region of the LSU rRNA gene of the dermatophyte Trichophyton interdigitale revealed a 393 bp group I intron, Tin.2563, containing the four characteristic conserved motifs (P,Q,R and S) essential for self-splicing. The predicted secondary structure revealed nine sets of conserved paired regions (P1-P9), with most similarity to a subgroup IE intron of the entomopathogenic hyphomycete Beauveria bassiana. Tin.2563 was inserted at a site in the LSU rDNA corresponding to position 2563 of the Escherichia coli 23S rRNA. PCR and sequence analysis showed an intron to be present at an identical location in the LSU rDNA of many dermatophytes, although its distribution was erratic. In contrast, an intron was present at the same location in multiple isolates (n = 20) of the clinically important anthrophilic species Trichophyton rubrum and T. interdigitale. Conservation of intron insertion site, subgroup and P helix sequences showed intron genotyping to be unsuitable for strain identification in dermatophytes. Phylogenetic analysis of intron sequences from different dermatophyte species indicated that lateral transfer of the element was likely to be a rare event. [ABSTRACT FROM AUTHOR]

The toxic dinoflagellate Alexandrium minutum Halim is one of three species that comprise the “ minutum” species complex. This complex is notable due to its role in the etiology of paralytic shellfish poisoning (PSP). Recent increases in PSP incidence and the geographic expansion of toxin-producing Alexandrium dinoflagellates have prompted the intensive examination of genetic relationships among globally distributed strains to address questions regarding their present distribution and reasons for their apparent increase. The biogeography of A. minutum was studied using large subunit ribosomal DNA gene (LSU rRNA) and internal transcribed spacer (ITS) sequences and genotypic data from 12 microsatellite loci. rRNA gene and ITS sequencing data distinguished between two clades, herein termed the “Global” and the “Pacific”; however, little to no resolution was seen within each clade. Genotypic data from 12 microsatellite loci provided additional information regarding genetic relationships within the Global clade, but it was not possible to amplify DNA from the Pacific clade using these markers. With the exception of isolates from Italy and Spain, strains generally clustered according to origin, revealing geographic structuring within the Global clade. Additionally, no evidence supported the separation of A. lusitanicum and A. minutum as different species. With the use of microsatellites, it is now possible to initiate studies on the origin, history, and genetic heterogeneity of A. minutum that were not previously possible using only rRNA gene sequence data. This study demonstrates the power of combining a marker with intermediate resolution (rRNA sequences) with finer-scale markers (microsatellites) to examine intraspecies variability among globally distributed isolates and represents the first effort to employ this technique in A. minutum. [ABSTRACT FROM AUTHOR]

A new photosynthetic dinoflagellate species, Coolia canariensis S. Fraga sp. nov., is described based on samples taken from tidal ponds on the rocky shore of the Canary Islands, northeast Atlantic Ocean. Its morphology was studied by LM and SEM. It is almost spherical and has a thick smooth theca with many scattered pores. Plate 1′ is the biggest of the epithecal plates, and 7″ is twice as wide as it is long. Phylogeny inferred from the D1/D2 regions of the LSU nuclear rDNA of three strains of C. canariensis and several strains of other Coolia species, C. monotis, C. sp., showed that C. canariensis strains clustered in a well-supported clade distinct from the other species. No toxins were detected using mouse bioassay, liquid chromatography with Fluorescence detection (LC-FLD) or liquid chromatography-mass spectrometry (LC-MS). Its pigment composition is of the peridinin type of dinoflagellates. Together with this new species, many other strains of C. monotis from the Atlantic Ocean and Mediterranean Sea have been analyzed for toxin presence, and no evidence of toxin production related to yessotoxins (YTXs) was found, as was previously suggested for C. monotis from Australia. [ABSTRACT FROM AUTHOR]

Abstract: The dinoflagellate, Cochlodinium polykrikoides Margalef, has been responsible for mass mortalities of both wild and farmed fish along the Korean coast on virtually an annual basis since 1982. Economic impacts to the fishing and aquaculture industries are extensive, with a loss of USD $95 million reported in 1995 alone. The use of taxon-specific molecular probes for harmful algal species is recognized as a promising approach for the early detection of bloom formation and as part of an effective mitigation strategy. We have developed and successfully applied large subunit ribosomal RNA (LSU rRNA)-targeted probes in both whole cell and sandwich hybridization assay (SHA) formats for the species-specific detection of C. polykrikoides in Korean coastal waters. Sequences of the D1–D3 variable regions used to design probes were identical between five Korean and one Hong Kong C. polykrikoides isolates, while sequences for several N. American Cochlodinium isolates differed to varying degrees from the former. The automated SHA detected C. polykrikoides at levels as low as ∼1–3cells/L in the field, demonstrating its suitability for detecting the target species at pre-bloom concentrations. This method should thus prove valuable to existing monitoring programs aimed at providing aquaculture interests with an early warning of frequently devastating bloom events. [Copyright &y& Elsevier]

Abstract: Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo. [Copyright &y& Elsevier]

It is generally assumed that all mature rRNA molecules assembled into ribosomes within a single cell are identical. However, sequence analysis of Streptomyces coelicolor genome revealed that it harbors six copies of divergent rRNA operons that may express and constitute three and five different kinds of small subunit (SSU) and large subunit (LSU) rRNA molecules, respectively, in a single cell. Phylogenetic analyses of the LSU rRNA genes and the internal transcribed spacer between SSU and LSU genes indicated that the LSU gene of rrnA and rrnE operons might be the result of interspecies recombination between rRNA genes in closely related streptomycetes. Profiling of rRNA species using primer extension analysis showed that heterogeneous rRNA transcripts are expressed and assembled into ribosomes in the cell. As the cells developed from germination to sporulation, the relative amount of LSU rRNA molecules derived from three rRNA operons ( rrnA, D, and E) gradually decreased from ∼85% to ∼60%, whereas the distribution of LSU rRNA molecules from two other operons ( rrnB and F) and rrnC operon gradually increased from ∼10% to ∼20% of the total LSU rRNA. These findings indicate that heterogeneous rRNA molecules are differentially expressed during the life cycle of this developmentally complex microorganism. [ABSTRACT FROM AUTHOR]

Twenty-seven clones were established from elongate Alexandrium sp. cysts collected in six regions along the Russian Pacific coast. All isolates were identified as Alexandrium tamarense via detailed epifluoresence microscopy of thecal plates. Morphological differences of both cultured and wild cells from the study regions mainly occurred in the shape of the cell (length/width ratio), degree of development of the sulcal list, and the shape of the posterior sulcal (S.p.) and second antapical (2″″) plates. Cells were divided into two cell types: ‘short’ (isodiametrical or wide) and ‘tall’. Each cell type exhibits specific features of tabulation, mainly the shape of the S.p. and 2″″ plates and was dominant in each particular region of the study. The short type, with a wide S.p. and reduced length in the dorsoventral 2″″ plates, was characteristic of A. tamarense from Primorye and southern Sakhalin Island. The tall cells, i.e., with cell length exceeding width, and having and elongate S.p. and dorsoventrally elongate 2″″ plates, prevailed in Avachinskaya Guba Inlet and in the Bering Sea. The differences reported here between the two types are within the range of morphological variability of A. tamarense sensu Balech, 1995. The D1-D2 fragment of the large subunit nuclear ribosomal DNA was analyzed for 24 clones. Alexandrium tamarense from the Russian Pacific coast compose three genetically distinct populations that correspond to the Japanese temperate Asian, eastern North American, and western North American ribotypes of the ‘tamarensis’ complex. The presence and distribution of eastern and western North American ribotypes along the Russian Pacific coast suggest that dispersion to the temperate Asian region occurred long ago via natural currents and processes, and not through human-mediated introductions, as has been proposed. No strict correlation was observed between different morphological types of cells and ribotypes. High-performance liquid chromatography toxin analyses showed that all isolates were toxic and demonstrated variability in toxin content and composition among different populations. These data document the significant and previously uncharacterized risk of shellfish contamination with paralytic shellfish poisoning toxins from blooms of A. tamarense in Russian marine waters. [ABSTRACT FROM AUTHOR]

Abstract: The efficacy of the LSU rDNA PCR primers FLR3 and FLR4 for discrimination of arbuscular mycorrhizal fungi communities via T-RFLP analysis was examined. Analysis of both public database and site-specific derived DNA sequences suggesting LSU rDNA-based T-RFLP analysis represents a valuable alternative for analysis of AMF communities. [Copyright &y& Elsevier]

Abstract: Many Raphidophytes are important algal bloom-forming species. Morphology-based identification of these species is often ambiguous, however, as many species are very similar in shape and size. To accurately detect the presence of these species in pre-bloom conditions, single-cell PCR is probably the most rapid and convenient method. However, direct single-cell PCRs with conserved primers are apparently not effective, probably due to the impermeability of the cell wall. We report here an effective detergent-based pre-PCR cell lysis method, which turned out to be a critical step for effective single-cell PCR of the Raphidophytes. Two PCR-based methods, nested SC-PCR and SC-RAPD, were evaluated. The nested SC-PCR involves two consecutive PCRs, the first of which is performed with the D1 and D2 primers (external primers) resulting in an amplification of a partial LSU rRNA gene. The second amplification is performed with primers targeting the LSU domain and specifically annealing to Chattonella ovata and Chattonella marina only. The SC-RAPD performed with the established random primers, RP1–RP4, produced unique haplotypes that could be exploited to differentiate the two Chattonella species. The assay was demonstrated to be sensitive, with the lowest detection limit of a single Raphidophyceae cell. The method developed is a valuable tool for the study of intra-specific variations of the Raphidophytes and represents a platform for further development of species-specific SC-RAPD for all members of the Raphidophyceae. [Copyright &y& Elsevier]

The evolutionary relationships of 82 species of tylenchid and aphelenchid nematodes were evaluated by use of sequence data of the D2 and D3 expansion fragments of the 28S ribosomal RNA genes. Nine automatic and one culled sequence alignments were analysed using maximum parsimony and Bayesian inference approaches. The molecular data sets showed that the order Tylenchida comprises lineages that largely correspond to two suborders, Hoplolaimina and Criconematina, and other taxonomic divisions as proposed by Siddiqi (2000). Several significant results also derived from our study include: i) the basal position of groups that include entomoparasitic nematodes within tylenchid trees; ii) paraphyly of the superfamily Dolichodoroidea sensu Siddiqi (2000); iii) evidence for a Pratylenchus, Hirschmanniella and Meloidogyne clade; and iv) lack of support for widely held traditional placement of Radopholus within Pratylenchidae and placement of this genus within Hoplolaimidae or Heteroderidae. Congruence and incongruence of molecular phylogeny and traditional classifications and morphological-based hypotheses of phylogeny of tylenchids are discussed. [ABSTRACT FROM AUTHOR]

The toxic marine dinoflagellates Alexandrium tamarense (Lebor) Balech and A. catenella (Whedon and Kofoid) Taylor have been mainly responsible for paralytic shellfish poisoning in Japan. Rapid and precise identification of these algae has been difficult because this genus contains many morphologically similar toxic and nontoxic species. Here, we report a rapid, precise, and quantitative identification method using three fluorescent, rRNA-targeted, oligonucleotide probes for A. tamarense (Atm1), A. catenella (Act1), and the nontoxic A. affine (Inoue et Fukuyo; Aaf1). Each probe was species specific when applied using fluorescence in situ hybridization (FISH). None of the probes reacted with three other Alexandrium spp., A. lusitanicum Balech, A. ostenfeldii (Paulsen) Balech & Tangen, and A. insuetum Balech, or with eight other microalgae, including Gymnodinium mikimotoi Miyake et Kominami ex Oda and Heterosigma akashiwo (Hada) Hara et Chihara, suggesting that the species specificity of each probe was very high. Cells labeled with fluorescein 5-isothiocyanate–conjugated probes showed strong green fluorescence throughout the whole cell except for the nucleus. FISH could be completed within 1 h and largely eliminated the need for identifying species based on key morphological criteria. More than 80% of targeted cells of both species could be identified by microscopy and quantified during growth up to the early stationary phase; more than 70% of cells could be detected in the late stationary phase. The established FISH protocol was found to be a specific, rapid, precise, and quantitative method that might prove to be a useful tool to distinguish and quantify Alexandrium cells collected from Japanese coastal waters. [ABSTRACT FROM AUTHOR]

Phylogenetic relationships within the green algal class Cladophorophyceae were investigated. For 37 species, representing 18 genera, the sequences of the 5′-end of the large subunit rRNA were aligned and analysed. Ulva fasciata and Acrosiphonia spinescens (Ulvophyceae) were used as outgroup taxa. The final alignment consisted of 644 positions containing 208 parsimony-informative sites. The analysis showed three lineages within the Cladophorophyceae: Cladophora horii diverged first, followed by two main lineages. The first lineage includes some Cladophora species and genera with a reduced thallus architecture. The second lineage comprises siphonocladalean taxa (excluding part of Cladophoropsis and including some Cladophora species). From this perspective the Siphonocladales forms a monophyletic group, the Cladophorales remaining paraphyletic. [ABSTRACT FROM AUTHOR]

Some species within the genus Pseudo-nitzschia H. Peragallo are associated with production of domoic acid, the agent responsible for amnesic shellfish poisoning (ASP). Identification and enumeration of particular Pseudo-nitzschia in natural populations is often difficult and time consuming because of the need for detailed morphological observations, which often require scanning or transmission electron microscopy. In earlier publications we described the development of large subunit ribosomal RNA (LSU rRNA)-targeted fluorescent DNA probes for discriminating among a variety of Pseudo-nitzschia species collected from Monterey Bay, California. Probes are applied using whole cell hybridization and a custom filtration manifold, enabling rapid identification and quantification of target species in cultured as well as field samples. In this work we compared a variety of preservation techniques and assessed the stability of stored samples with respect to their reactivity towards the probes. Of the preservatives tested, a saline ethanol-based treatment gave the best results in terms of probes yielding a bright and uniform cell label. Culture samples treated with this fixative continued to react well with the probes for at least 6 weeks post-fixation whether stored in the preservative or dried post-preservation, with samples being kept at either room temperature or -20° C. Likewise, field samples containing a variety of diatoms and dinoflagellate species stored in the saline ethanol solution at room temperature were also stable for at least 4–6 weeks, reacting brilliantly towards a positive control probe. After prolonged storage, however, cell reactivity towards the probes diminished dramatically. Post-hybridization, samples stored at 4° C were found to retain their fluorescence for at least 1 week. These results indicate a wider window of opportunity for Pseudo-nitzschia analysis using whole cell hybridization than previously reported. Sample collection, preservation, and probing protocols optimized for Pseudo-nitzschia are also applicable to a wide range of phytoplankton species. The time required to execute the whole cell hybridization protocol was reduced by premixing probe with hybridization buffer. The premixed probe solutions as well as fixative and wash solutions are all stable at room temperature for at least 6 weeks. Application of two different species-specific probes, each labeled with a different fluorochrome, allowed detection of two species on a single filter. The latter could be adopted in the future to increase the rate of sample processing and decrease the cost of sample analysis. [ABSTRACT FROM AUTHOR]