Phylogenetic affinities of an enigmatic nannoplankton,Braarudosphaera bigelowii based on the SSU rDNA sequences

The phylogenetic position of an enigmatic calcareous nannoplankton Braarudosphaera bigelowii, whose taxonomic position has long been questioned, was investigated using molecular genetic methods. The SSU rDNA sequences of B. bigelowii were obtained using a single cell PCR technique independently from two single cells isolated from field samples. A BLAST search revealed that the SSU rDNA of B. bigelowii was most similar to those of the division Haptophyta. Subsequent phylogenetic analyses showed that B. bigelowii was included in a clade comprising members of the orders Isochrysidales and Coccolithales, the genus Chrysoculter and two unidentified haptophytes. Morphometric measurements of extant B. bigelowii populations were made to obtain precise information on their intraspecific size variation. The results showed that these populations could be subdivided into a small form (< 2.4 μm), an intermediate form A (4.0–5.3 μm), and an intermediate form B (5.3–7.2 μm), although the borders of the latter two forms overlapped. The SSU rDNA sequences showed the presence of 10-bp substitutions plus six insertions/deletions between specimens of intermediate forms A and B. The levels of DNA variation between them suggested that these two forms are genetically distinct from each other.     

Discovery of an Endosymbiotic Nitrogen-Fixing Cyanobacterium UCYN-A in Braarudosphaera bigelowii (Prymnesiophyceae)

Braarudosphaera bigelowii (Prymnesiophyceae) is a coastal coccolithophore with a long fossil record, extending back to the late Cretaceous (ca. 100 Ma). A recent study revealed close phylogenetic relationships between B. bigelowii, Chrysochromulina parkeae (Prymnesiophyceae), and a prymnesiophyte that forms a symbiotic association with the nitrogen-fixing cyanobacterium UCYN-A. In order to further examine these relationships, we conducted transmission electron microscopic and molecular phylogenetic studies of B. bigelowii. TEM studies showed that, in addition to organelles, such as the nucleus, chloroplasts and mitochondria, B. bigelowii contains one or two spheroid bodies with internal lamellae. In the 18S rDNA tree of the Prymnesiophyceae, C. parkeae fell within the B. bigelowii clade, and was close to B. bigelowii Genotype III (99.89% similarity). Plastid 16S rDNA sequences obtained from B. bigelowii were close to the unidentified sequences from the oligotrophic SE Pacific Ocean (e.g. {"type":"entrez-nucleotide","attrs":{"text":"HM133411","term_id":"311035799","term_text":"HM133411"}}HM133411) (99.86% similarity). Bacterial16S rDNA sequences obtained from B. bigelowii were identical to the UCYN-A sequence {"type":"entrez-nucleotide","attrs":{"text":"AY621693","term_id":"54125940","term_text":"AY621693"}}AY621693 from Arabian Sea, and fell in the UCYN-A clade. From these results, we suggest that; 1) C. parkeae is the alternate life cycle stage of B. bigelowii sensu stricto or that of a sibling species of B. bigelowii, and 2) the spheroid body of B. bigelowii originated from endosymbiosis of the nitrogen-fixing cyanobacterium UCYN-A.     

Morphological, Ecological and Molecular Studies of Vannella simplex Wohlfarth-Bottermann 1960 (Lobosea, Gymnamoebia), with a new Diagnosis of this Species

Vannella simplex (Gymnamoebia, Vannellidae) is one of the most common amoebae species, recorded from a variety of regions. It was originally described as a freshwater species, but has also been reported from shallow-water regions of the Baltic Sea. In the present work, we investigated the morphology and biology of three V. simplex isolates, originating from geographically distant regions. Among them is one brackish water strain, isolated from artificial cyanobacterial mats, which were originally sampled in Nivå Bay (Baltic Sea, The Sound). The strain is cyst-forming and can thrive at salinity ranges from 0–50 ppt. Phylogenetic relationships were investigated by sequencing partial SSU rDNA of the cultured V. simplex isolates. Additional sequences were obtained from four environmental DNA extractions of sediment samples collected from different localities in Switzerland. Analysis of all obtained sequences revealed a monophyletic group. Based on the analysis and comparison of morphological, ecological and molecular data sets we compiled a distribution map of V. simplex and propose an emendation of this species.     

Speciation ofBotryobasidium subcoronatum (Basidiomycota) collected in Taiwan: Morphology, mating tests, and molecular data

Taiwan is a phytogeographically isolated area, with a rate of ca. 40% endemism in higher plants. In this study, the strong tendency towards genetic isolation and speciation ofBotryobasidium subcoronatum (Basidiomycota) from Taiwan is evaluated in comparison with European specimens. Mating tests amongB. subcoronatum specimens collected in Taiwan and Europe were performed. Mon-mon and di-mon matings confirm different intersterility groups or biological species. The biological species concept was compared with results from morphological comparison and molecular data from mtSSU rDNA and the nuclear ITS2 region. Three intersterility groups have been detected comprising European and Taiwanese strains from temperate and subtropical areas. MtSSU rDNA and the ITS2 rDNA sequences confirm consepecifity ofB. subcoronatum.     

Relationships betweenNor-loci from differentTriticeae species

TheNor-loci of polyploid wheats and their putative diploid progenitor species were assayed by probing isolated nuclear DNA with ribosomal DNA spacer sequences (spacer rDNA sequences, isolated by cloning), from theNor-loci of genomes B (Triticum aestivum), G (T. timopheevi), B (syn. S,T. speltoides), A (T. monococcum) and V (Dasypyrum villosum). DNA samples for analysis were digested with the restriction endonuclease Taq 1 and assayed by DNA-DNA hybridization under standard (37°C) and high stringency (64°C) conditions. The assay procedure emphasized differences between the divergent spacer sequences of the polyploid species and allowed relative homologies to the respective sequences in diploid species to be established. — The studies indicated thatT. timopheevi andT. speltoides contain different sets of spacer rDNA sequences which were readily distinguishable and, in the case ofT. timopheevi, assigned toNor-loci on different chromosomes. This contrast with the spacer rDNA sequences of the majorNor-loci on chromosomes 1 B and 6 B inT. aestivum, which were difficult to distinguish and were deduced to contain very similar sequences. Among the diploid progenitor species only the spacer rDNA fromT. speltoides shared close homology with polyploid wheat species. OneNor-locus inT. timopheevi (on chromosome 6 G) did not show close homology with any of the rDNA spacer probes available. — The data suggestsT. speltoides was the origin of someNor-loci for both theT. timopheevi andT. turgidum lines of tetraploid wheats. The possibility that the 6GNor-locus inT. timopheevi may have derived from an unknown diploid species by introgressive hybridization is discussed. The spacer rDNA sequence probe fromT. monococcum shared good homology with some accessions ofD. villosum and a line ofT. dicoccoides; the implications of this finding for evolution of present-day wheats are discussed.     

Molecular features of a defined genetic marker for the determination of the Porphyra tenera lineage

Nucleotide sequences of the nuclear SSU rDNA and ITS1 are presented as a defined genetic marker for Porphyra tenera as a species. Exon nucleotide sequences were identical within all the P. tenera specimens. Intron nucleotide sequences varied between populations. The introns and ITS1 variations are presented as defined genetic markers to establish the Porphyra tenera strains. Wild-collected thalli identified by morphological systematics, from five populations of Porphyra tenera throughout Japan, were discriminated by comparing sequences of the various regions utilizing the results of this and previous studies.     

The karyotype and 5S rRNA genes from Spanish individuals of the bat species <Emphasis Type="Italic">Rhinolophus</Emphasis><Emphasis Type="Italic">hipposideros</Emphasis> (Rhinolophidae; Chiroptera)

The karyotype of individuals of the species Rhinolophus hipposideros from Spain present a chromosome number of 2n = 54 (NFa = 62). The described karyotype for these specimens is very similar to another previously described in individual from Bulgaria. However, the presence of one additional pair of autosomal acrocentric chromosomes in the Bulgarian karyotype and the differences in X chromosome morphology indicated that we have described a new karyotype variant in this species. In addition, we have analyzed several clones of 1.4 and 1 kb of a PstI repeated DNA sequence from the genome of R. hipposideros. The repeated sequence included a region with high identity with the 5S rDNA genes and flanking regions, with no homology with GenBank sequences. Search for polymerase III regulatory elements demonstrated the presence of type I promoter elements (A-box, Intermediate Element and C-box) in the 5S rDNA region. In addition, upstream regulatory elements, as a D-box and Sp1 binding sequences, were present in flanking regions. All data indicated that the cloned repeated sequences are the functional rDNA genes from this species. Finally, FISH demonstrated the presence of rDNA in nine chromosome pairs, which is surprising as most mammals have only one carrier chromosome pair.     

Verrucophora farcimen gen. et sp. nov. (Dictyochophyceae,Heterokonta)—a bloom‐forming ichthyotoxic flagellate from the Skagerrak,Norway1

Since 1998, a heterokont flagellate initially named Chattonella aff. verruculosa has formed recurrent extensive blooms in the North Sea and the Skagerrak, causing fish mortalities. Cells were isolated from the 2001 bloom off the south coast of Norway, and monoalgal cultures were established and compared with the Chattonella verruculosa Y. Hara et Chihara reference strain NIES 670 from Japan. The cells in Norwegian cultured isolates were very variable in size and form, being large oblong (up to 34 μm long) to small rounded (5–9 μm in diameter) with two unequal flagella, numerous chloroplasts, and mucocysts. The SSU and partial LSU rDNA sequences of strains from Norway and Japan were compared and differed by 0.4% (SSU) and 1.3% (LSU), respectively. Five strains from Norway were identical in the LSU rDNA region. Phylogenetic analyses based on heterokont SSU and concatenated SSU + LSU rDNA sequences placed C. aff. verruculosa and the Japanese C. verruculosa within the clade of Dictyochophyceae, with the picoflagellate Florenciella parvula Eikrem as the closest relative. Ultrastructure, morphology, and pigment composition supported this affinity. We propose the name Verrucophora farcimen sp. et gen. nov. for this flagellate and systematically place it within the class Dictyochophyceae. Our studies also show that C. verruculosa from Japan is genetically and morphologically different but closely related to V. farcimen. The species is transferred from the class Raphidophyceae to the class Dictyochophyceae and renamed Verrucophora verruculosa. We propose a new order, Florenciellales, to accommodate V. farcimen, V. verruculosa, and F. parvula.     

Corylomyces: a new genus of Sordariales from plant debris in France

The new genus Corylomyces, isolated from the surface of a hazelnut (Corylus avellana) in the French Pyrenees, is described, illustrated and compared with morphologically similar taxa. It is characterised by tomentose, ostiolate ascomata possessing long necks composed of erect to sinuose hairs, and one- or two-celled, opaque, lunate to reniform ascospores. Analyses of the SSU and LSU fragments rDNA gene sequences support its placement in the Lasiosphaeriaceae (Sordariales).     

Actin Phylogeny and Intron Distribution in Bangiophyte Red Algae(Rhodoplantae)

The molecular phylogeny of red algal actin genes, with emphasis on the paraphyletic “Bangiophyceae,” was examined and compared to the rhodophyte SSU rDNA phylogeny. Nineteen new genomic actin sequences and seven SSU rDNA sequences were obtained and subjected to diverse phylogenetic analyses (maximum likelihood, distance/neighbor-joining, maximum parsimony, Bayesian analyses, and, with respect to protein sequences, also quartet puzzling). The actin trees confirmed most of the major clades found in the SSU rDNA phylogenies, although with a lower resolution. An actin gene duplication in the florideophycean lineage is reported, presumably related to an increased complexity of sexual reproduction. In addition, the distribution and characteristics of spliceosomal introns found in some of the actin sequences were examined. Introns were found in almost all florideophycean actin genes, whereas only two bangiophyte sequences contained introns. One intron in the florideophycean actin genes was also found in metazoan, and, shifted by one or two nucleotides, in a glaucocystophyte, a cryptophyte, and two fungal actin genes, and thus may be an ancient intron.[Reviewing Editor: Dr. Yves Van de Peer]     

A New Heterotrophic Dinoflagellate from the North‐eastern Pacific,Protoperidinium fukuyoi: Cyst–Theca Relationship,Phylogeny, Distribution and Ecology

The cyst–theca relationship of Protoperidinium fukuyoi n. sp. (Dinoflagellata, Protoperidiniaceae) is established by incubating resting cysts from estuarine sediments off southern Vancouver Island, British Columbia, Canada, and San Pedro Harbor, California, USA. The cysts have a brown‐coloured wall, and are characterized by a saphopylic archeopyle comprising three apical plates, the apical pore plate and canal plate; and acuminate processes typically arranged in linear clusters. We elucidate the phylogenetic relationship of P. fukuyoi through large and small subunit (LSU and SSU) rDNA sequences, and also report the SSU of the cyst‐defined species Islandinium minutum (Harland & Reid) Head et al. 2001. Molecular phylogenetic analysis by SSU rDNA shows that both species are closely related to Protoperidinium americanum (Gran & Braarud 1935) Balech 1974. Large subunit rDNA phylogeny also supports a close relationship between P. fukuyoi and P. americanum. Three subgroups in total are further characterized within the Monovela group. The cyst of P. fukuyoi shows a wide geographical range along the coastal tropical to temperate areas of the North‐east Pacific, its distribution reflecting optimal summer sea‐surface temperatures of ~14–18 °C and salinities of 22–34 psu.     

Relationships of the Insect-Pathogenic Order Entomophthorales (Zygomycota, Fungi) Based on Phylogenetic Analyses of Nuclear Small Subunit Ribosomal DNA Sequences (SSU rDNA)

We sequenced the nuclear small subunit of ribosomal DNA (SSU rDNA) from seven species within the insect-pathogenic order Entomophthorales. These sequences were aligned with other published SSU rDNA sequences and phylogenies were inferred using phenetic and cladistic methods. Based on three different phylogenetic methods the Entomophthorales (excludingBasidiobolus ranarum) is monophyletic;B. ranarumwas more closely related to chytrids from Chytridiales and Neocallimasticales than to Entomophthorales, as was proposed by Nagahamaet al.(Mycologia87:203–209, 1995). Nuclear characters (large nuclei containing conspicuous condensed chromatin and lack of a prominent nucleolus) were of predictive value for the monophyly of the family Entomophthoraceae. Conidial characters separate the Entomophthoraceae, which only includes obligate pathogens, into at least two lineages: one lineage with uninucleate conidia and another with multinucleate conidia. The two species ofConidiobolusstudied were paraphyletic in our analyses and only distantly related to each other. This information may prove to be important in the use of these fungi as biocontrol agents.     

Lineage-specific variations of congruent evolution among DNA sequences from three genomes,and relaxed selective constraints on <Emphasis Type="Italic">rbc</Emphasis>L in <Emphasis Type="Italic">Cryptomonas</Emphasis> (Cryptophyceae)

Background  

Plastid-bearing cryptophytes like Cryptomonas contain four genomes in a cell, the nucleus, the nucleomorph, the plastid genome and the mitochondrial genome. Comparative phylogenetic analyses encompassing DNA sequences from three different genomes were performed on nineteen photosynthetic and four colorless Cryptomonas strains. Twenty-three rbc L genes and fourteen nuclear SSU rDNA sequences were newly sequenced to examine the impact of photosynthesis loss on codon usage in the rbc L genes, and to compare the rbc L gene phylogeny in terms of tree topology and evolutionary rates with phylogenies inferred from nuclear ribosomal DNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA), and nucleomorph SSU rDNA.     

Microarray-based survey of repetitive genomic sequences in Vicia spp.

A modified DNA microarray-based technique was devised for preliminary screening of short fragment genomic DNA libraries from three Vicia species (V. melanops, V. narbonensis, and V. sativa) to isolate representative highly abundant DNA sequences that show different distribution patterns among related legume species. The microarrays were sequentially hybridized with labeled genomic DNAs of thirteen Vicia and seven other Fabaceae species and scored for hybridization signals of individual clones. The clones were then assigned to one of the following groups characterized by hybridization to: (1) all tested species, (2) most of the Vicia and Pisum species, (3) only a few Vicia species, and (4) preferentially a single Vicia species. Several clones from each group, 65 in total, were sequenced. All Group I clones were identified as rDNA genes or fragments of chloroplast genome, whereas the majority of Group II clones showed significant homologies to retroelement sequences. Clones in Groups III and IV contained novel dispersed repeats with copy numbers 10²–10⁶/1C and two genus-specific tandem repeats. One of these belongs to the VicTR-B repeat family, and the other clone (S12) contains an amplified portion of the rDNA intergenic spacer. In situ hybridization using V. sativa metaphase chromosomes revealed the presence of the S12 sequences not only within rDNA genes, but also at several additional loci. The newly identified repeats, as well as the retroelement-like sequences, were characterized with respect to their abundance within individual genomes. Correlations between the repeat distributions and the current taxonomic classification of these species are discussed.     

Pfiesteria piscicida and Pfiesteria shumwayae in coastal waters of Long Island, New York, USA

Water and sediment samples were collected during summer and early fall 1999–2004 from coastal waters of New York State, USA, to test for the presence of Pfiesteria piscicida and Pfiesteria shumwayae. Physical and chemical conditions were characterized, and real-time polymerase chain reaction assays were conducted. Both species were relatively common and found at most sites at least once, and the frequency of positive assays was higher in sediments than in the water column. In a subset of the data from Suffolk County, Long Island, the presence of Pfiesteria was related to high chlorophyll a and relatively high nutrient concentrations. Partial SSU rDNA sequences of four PCR amplicons generated using P. shumwayae primers indicated two sequences: three were identical to GenBank P. shumwayae entries, but one showed enough sequence difference (15 positions in a 454 bp amplicon) to suggest a possible new species. Three isolates were tested for toxicity, and one was found to kill fish in bioassays. Despite the widespread presence of both Pfiesteria species and demonstration of potential to harm fish, no blooms of these dinoflagellates have been observed, nor has there been evidence of Pfiesteria-related fish or human health problems in these waters, likely related to colder temperatures than optimal for Pfiesteria species.     

Molecular phylogeny of the lionfish genera Dendrochirus and Pterois (Scorpaenidae, Pteroinae) based on mitochondrial DNA sequences

This study investigates the molecular phylogeny of seven lionfishes of the genera Dendrochirus and Pterois. MP, ML, and NJ phylogenetic analysis based on 964 bp of partial mitochondrial DNA sequences (cytochrome b and 16S rDNA) revealed two main clades: (1) “Pterois” clade (Pterois miles and Pterois volitans), and (2) “Pteropterus–Dendrochirus” clade (remainder of the sampled species). The position of Dendrochirus brachypterus either basal to the main clades or in the “Pteropterus–Dendrochirus” clade cannot be resolved. However, the molecular phylogeny did not support the current separation of the genera Pterois and Dendrochirus. The siblings P. miles and P. volitans are clearly separated and our results support the proposed allopatric or parapatric distribution in the Indian and Pacific Ocean. However, the present analysis cannot reveal if P. miles and P. volitans are separate species or two populations of a single species, because the observed separation in different clades can be either explained by speciation or lineage sorting. Molecular clock estimates for the siblings P. miles and P. volitans suggest a divergence time of 2.4–8.3 mya, which coincide with geological events that created vicariance between populations of the Indian and Pacific Ocean.     

Morphology,ultrastructure and molecular phylogeny of cyst-producing Caladoa arcachonensis gen. et sp. nov. (Peridiniales,Dinophyceae) from France and Indonesia

The dinoflagellate order Peridiniales encompasses several well circumscribed families. However, the family level of some genera, such as Bysmatrum and Vulcanodinium, has remained elusive for many years. Four Peridinium-like strains were established from the Atlantic coast of France and North Sulawesi, Indonesia through cyst germination or isolation of single cells. The cyst-theca relationship was established on specimens from the French Atlantic. Their morphologies were examined using light, scanning and transmission electron microscopy. The cells were characterized by a much larger epitheca relative to the hypotheca, a large anterior sulcal (Sa) plate deeply intruding the epitheca and a small first anterior intercalary plate. The plate formula was identified as Po, cp, X, 4′, 3a, 7′′, 6C, 5S, 5′′′, 2′′′′, shared by Apocalathium, Chimonodinium, Fusiperidinium and Scrippsiella of the family Thoracosphaeraceae but the configuration of Sa plate and anterior intercalary plates is different. Transmission electron microscopy showed that the eyespot was located within a chloroplast comprising two rows of lipid globules and thus belongs to type A. All four strains were classified within a new genus Caladoa as C. arcachonensis gen. et sp. nov. Small subunit ribosomal DNA (SSU rDNA), partial large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer ribosomal DNA (ITS rDNA) sequences were obtained from all strains. Genetic distance based on ITS rDNA sequences between French and Indonesian strains reached 0.17, suggesting cryptic speciation in C. arcachonensis. The maximum likelihood and Bayesian inference analysis based on concatenated data from SSU and LSU rDNA sequences revealed that Caladoa is monophyletic and closest to Bysmatrum. Our results supported that Caladoa and Bysmatrum are members of the order Peridiniales but their family level remains to be determined. Our results also support that Vulcanodinium is closest to the family Peridiniaceae.     

Molecular phylogeny of the genus Stereocaulon (Stereocaulaceae, lichenized ascomycetes)

In the present study phylogenetic relationships of the genus Stereocaulon (lichenized ascomycetes) were examined using DNA sequences from the ITS1–5.8 S–ITS2 rDNA gene cluster and from the protein-coding β-tubulin gene. In addition to the fruticose species traditionally classified in Stereocaulon, representatives of the crustose species that have recently been transferred to the genus were included. Muhria, a monotypic genus that is morphologically similar to Stereocaulon, differing only in apothecia ontogeny, was also incorporated. The analyses included 101 specimens from the ingroup representing 49 taxa. Sequences from both DNA regions were analysed simultaneously using direct optimization under the parsimony optimality criterion. The results support the inclusion of the crustose species and Muhria in Stereocaulon, while the current infrageneric classification is not supported. As Muhria is securely nested within Stereocaulon the new combination Stereocaulon urceolatum comb. nov. (syn. Muhria urceolata) is made. Further, species concepts need to be re-examined, as some species do not appear as monophyletic entities in the phylogeny.