Phylogenetic relationships amongTetrahymena species determined using the polymerase chain reaction

Summary The species of theTetrahymena pyriformis complex present a conundrum with regard to their highly conservative morphology and widely divergent molecular characteristics. We have investigated the phylogenetic relationships among these species using the nucleotide sequences from the histone H3II/H4II region of the genome. This region includes portions of the two histone coding sequences, as well as the intergenic region. The DNA sequences of these regions were amplified by the polymerase chain reaction (PCR) and the sequence of each was determined. Nucleotide substitutions and insertions/deletions within this set of sequences were compared to determine the phylogenetic relationships among the species of the complex. These data yield phylogenetic trees with identical topologies when different tree-building routines are used, indicating that the data are very robust.Glaucoma chattoni was used as an outgroup to root the trees for this analysis. The genome organization ofG. chattoni and the divergence of its histone H3II/H4II region sequence relative to those of the complex clearly indicate that this species has diverged considerably from the complex. These results show that PCR amplification analysis is feasible over considerable evolutionary distances. However, DNA-DNA hybridization may be more useful than sequence analysis in resolving the relationships among the closely related species in the complex.     

Phylogenetic relationships within the genus Tetrahymena inferred from the cytochrome c oxidase subunit 1 and the small subunit ribosomal RNA genes

Details of the phylogenetic relationships among tetrahymenine ciliates remain unresolved despite a rich history of investigation with nuclear gene sequences and other characters. We examined all available species of Tetrahymena and three other tetrahymenine ciliates, and inferred their phylogenetic relationships using nearly complete mitochondrial cytochrome c oxidase subunit 1 (cox1) and small subunit (SSU) rRNA gene sequences. The inferred phylogenies showed the genus Tetrahymena to be monophyletic. The three “classical” morphology-and-ecology-based groupings are paraphyletic. The SSUrRNA phylogeny confirmed the previously established australis and borealis groupings, and nine ribosets. However, these nine ribosets were not well supported. Using cox1 gene, the deduced phylogenies based on this gene revealed 12 well supported groupings, called coxisets, which mostly corresponded to the nine ribosets. This study demonstrated the utility of cox1 for resolving the recent phylogeny of Tetrahymena, whereas the SSU rRNA gene provided resolution of deeper phylogenetic relationships within the genus.     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.     

Evolutionary implications of a rRNA-based phylogeny of Harpellales

The Harpellales (Trichomycetes) are endosymbiotic microfungi, mostly unculturable and predominantly associated with larval aquatic insects worldwide. Molecular phylogenies including ‘gut fungi’ have included at most only four axenic isolates of the 38 genera of Harpellales. Cladistic analyses were used to infer the phylogeny of the Harpellales using partial 18S or 28S nu-rRNA sequences generated for 16 genera of Harpellales, with 64 of 72 sequences generated from unculturable samples. Both analyses placed Orphella outside an otherwise monophyletic group of Harpellales, more closely allied to the Kickxellales. The current classification recognizing two families is not corroborated and continued use of the family Legeriomycetaceae may not be supportable. The largest genera of Harpellales, Smittium and Stachylina, were polyphyletic and the 28S rRNA sequences separate Smittium culisetae from the remainder of its genus. The cladograms did not support the consistent mapping of important morphological taxonomic characters, including trichospore shape and zygospore type or appendage numbers for both. This study demonstrates the use of microscopic thalli from host guts for molecular phylogenies and suggests the need for more data from the remaining Harpellales, especially with the future inclusion of protein-coding genes.     

Multilocus sequence analysis of bradyrhizobia isolated from Aeschynomene species in Senegal

This study reports the multilocus sequence analysis (MLSA) of nine house-keeping gene fragments (atpD, dnaK, glnA, glnB, gltA, gyrB, recA, rpoB and thrC) on a collection of 38 Bradyrhizobium isolated from Aeschynomene species in Senegal, which had previously been characterised by several phenotypic and genotypic techniques, allowing a comparative analysis of MLSA resolution power for species delineation in this genus. The nifH locus was also studied to compare house-keeping and symbiotic gene phylogenies and obtain insights into the unusual symbiotic properties of these Aeschynomene symbionts. Phylogenetic analyses (maximum likelihood, Bayesian) of concatenated nine loci produced a well-resolved phylogeny of the strain collection separating photosynthetic bradyrhizobial strains (PB) from non-photosynthetic bradyrhizobial (NPB) ones. The PB clade was interpreted as the remains an expanding ancient species that presently shows high diversification, giving rise to potential new species. B. denitrificans LMG8443 and BTAi1 strains formed a sub-clade that was identified as recently emerging new species. Congruence analyses (by Shimodaira–Hasegawa (S–H) tests) identified three gene-fragments (dnaK, glnB and recA) that should be preferred for MLSA analyses in Bradyrhizobium genus. The nine loci or nifH phylogenies were not correlated with the unusual symbiotic properties of PB (nod-dependent/nod-independent). Advantages and drawbacks of MLSA for species delineation in Bradyrhizobium are discussed.     

Phylogenetic relationships of Auriculoscypha based on ultrastructural and molecular studies

The phylogeny of Auriculoscypha anacardiicola, an associate of scale insects in India, is investigated using subcellular characters and MP and Bayesian analyses of combined nuLSU-rDNA, nuSSU-rDNA and 5.8S rDNA sequence data. It has simple septa with a pulley-wheel-shaped pore plug, which is diagnostic of phytoparasitic members of the Pucciniomycetes, and hyphal wall break on branching, a phenomenon unique to some simple septate heterobasidiomycetes. The septal ultrastructure of A. anacardiicola is similar to that of the genus Septobasidium. The close relationship to Septobasidium is also confirmed by rDNA sequence analyses. The polyphyletic nature of the order Platygloeales, noted in earlier studies, is evident from the present molecular analysis as well. The placement of Auriculoscypha in the Platygloeales can no longer be justified and both ultrastructural and molecular evidence strongly support the placement of Auriculoscypha in the Septobasidiales.     

A molecular phylogeny of Hebeloma species from Europe

In order to widen the scope of existing phylogenies of the ectomycorrhizal agaric genus Hebeloma a total of 53 new rDNA ITS sequences from that genus was generated, augmented by sequences retrieved from GenBank, and analysed using Bayesian, strict consensus and neighbour joining methods. The lignicolous Hebelomina neerlandica, Gymnopilus penetrans, and two species of Galerina served as outgroup taxa. Anamika indica, as well as representatives of the genera Hymenogaster and Naucoria, were included to test the monophyly of Hebeloma, which is confirmed by the results. Hebeloma, Naucoria, Hymenogaster and Anamika indica cluster in a strongly supported monophyletic hebelomatoid clade. All trees largely reflect the current infrageneric classification within Hebeloma, and divide the genus into mostly well-supported monophyletic groups surrounding H. crustuliniforme, H. velutipes, H. sacchariolens, H. sinapizans, and H. radicosum, with H. sarcophyllum being shown at an independent position; however this is not well supported. The section Indusiata divides with strong support into three groups, the position of the pleurocystidiate Hebeloma cistophilum suggests the possible existence of a third subsection within sect. Indusiata. Subsection Sacchariolentia is raised to the rank of section.     

Microcalorimetric Study on the Toxic Effect of Pb<Superscript>2+</Superscript> to <Emphasis Type="Italic">Tetrahymena</Emphasis>

The toxic effect of Pb²⁺ has been studied in eukaryotic cells by using Tetrahymena as a target. The maximum power (P _m) and the growth rate constant (k) were determined, which showed that values of P _m and k were linked to the concentration (C) of Pb²⁺. The addition of Pb²⁺ caused a decrease of the maximum heat production and growth rate constant, indicating that Tetrahymena growth was inhibited in the presence of Pb²⁺, and Pb²⁺ took part in the metabolism of cells. From micrographs, morphological changes of Tetrahymena were observed with addition of Pb²⁺, indicating that the toxic effect of Pb²⁺ derived from destroying the membrane of surface of Tetrahymena. According to the thermogenic curves and photos of Tetrahymena under different conditions, it is clear that metabolic mechanism of Halobacterium halobium R1 growth has been changed with the addition of Pb²⁺.     

Re-thinking the classification of corticioid fungi

Corticioid fungi are basidiomycetes with effused basidiomata, a smooth, merulioid or hydnoid hymenophore, and holobasidia. These fungi used to be classified as a single family, Corticiaceae, but molecular phylogenetic analyses have shown that corticioid fungi are distributed among all major clades within Agaricomycetes. There is a relative consensus concerning the higher order classification of basidiomycetes down to order. This paper presents a phylogenetic classification for corticioid fungi at the family level. Fifty putative families were identified from published phylogenies and preliminary analyses of unpublished sequence data. A dataset with 178 terminal taxa was compiled and subjected to phylogenetic analyses using MP and Bayesian inference. From the analyses, 41 strongly supported and three unsupported clades were identified. These clades are treated as families in a Linnean hierarchical classification and each family is briefly described. Three additional families not covered by the phylogenetic analyses are also included in the classification. All accepted corticioid genera are either referred to one of the families or listed as incertae sedis.     

Biosorption of Cu(II) ions from synthetic and actual wastewater using three algal species

The use of three freshwater microalgal cultures—Chlorella sorokiniana, Anabaena laxa, and Hapalosiphon welwitschii—for sorption of copper(II) from synthetic Cu(II) solutions and Marinduque, Philippines, wastewater was studied. The optimum amount of biomass for the three species was 0.025 g dry weight. The optimum contact time for both C. sorokiniana and A. laxa was 1 h, whereas that of H. welwitschii was 30 min. All three species exhibited maximum Cu(II) sorption at pH 4.0–6.0. The Langmuir adsorption isotherm was the best fit model for the three species. The three cultures were found to be effective biosorbents when used in synthetic wastewaters of low concentration (10–30 ppm). Maximum Cu(II) reductions obtained were 88.2, 88.6, and 91.7% for the C. sorokiniana, A. laxa, and H. welwitschii cultures, respectively. C. sorokiniana, A. laxa, and H. welwitschii removed 5.70, 11.16, and 7.15% of Cu(II), respectively, when applied to wastewater taken from Consolidated Mines Inc. (CMI) containing around 150 ppm Cu(II). C. sorokiniana and A. laxa, in combination, exhibited 14.05% Cu(II) removal from CMI wastewater. Desorption with 0.11 M HCl effected 73.20, 64.54, and 70.85% removal of Cu(II) from the surfaces of C. sorokiniana, A. laxa, and H. welwitschii, respectively. SEM-EDS spectra of the three species confirmed the presence of Cu(II) on their surfaces. Presented at the 6th Meeting of the Asia Pacific Society of Applied Phycology, Manila, Philippines.     

Characterization of the macronuclear DNA of different species ofTetrahymena

Summary The macronuclear DNAs from 20 different species ofTetrahymena were characterized using alternating Orthogonal Field (AOF) gel electrophoresis. Each species has approximately 300 different macronuclear DNA molecules that range in size from about 100–2000 kb pairs. Although the individual macronuclear DNA molecules are not well resolved on an AOF gel, most species have a unique profile of macronuclear DNA. The sequences that hybridize with histone H4 (Tetrahymena) and ubiquitin (yeast) genes were identified on the separated macronuclear DNA molecules of the different species. All species have 2 histone H4 genes located on macronuclear DNA molecules of different sizes. This is consistent with the duplication of the histone H4 gene prior to the speciation events leading to the various species ofTetrahymena. The number and sizes of the macronuclear DNA molecules that hybridize with the ubiquitin probe vary from species to species. A grouping of the different species ofTetrahymena based on this hybridization pattern paralels groupings of the species based on ribosomal RNA sequences and isoenzymes. Some intraspecific variation among different strains ofTetrahymena thermophila was detected using ubiquitin and 5S ribosomal RNA as probes.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

Social evolution in the genusHalictus: a phylogenetic approach

Summary Halictine bees exhibit an enormous diversity of solitary and social colony structures. To investigate social evolution in the genusHalictus, phylogenies of 15 species of the subgeneraH. (Halictus) andH. (Seladonia) were constructed based on protein electrophoretic data. Solitary, social, and socially polymorphic species were included.Halictus (Seladonia) apparently rendersH. (Halictus) paraphyletic. The common ancestor ofH. (Halictus) andH. (Seladonia) was probably social or socially polymorphic. This implies that some solitary and socially polymorphic species, such asH. confusus andH. tumulorum, represent evolutionary reversals from a completely eusocial condition to the solitary condition that is thought to be primitive for the subfamily as a whole.     

Taxonomy and ecology of some ciliates (Protozoa,Ciliophora) of the saprobic system. III. Revision of the genera Colpidium and Dexiostoma,and establishment of a new genus,Paracolpidium nov. gen.

The genera Colpidium Stein, 1860 and Dexiostoma Jankowski, 1967 are revised. Our monographic treatment is based on a morphologic and biometric reinvestigation of the main species of these genera and on an evaluation of data from the literature. Colpidium comprises four species, namely C. colpoda (Losana, 1829), C. singulare Vuxanovici, 1961, C. acuminatum Vuxanovici, 1962, and C. kleini Foissner, 1969. The monotypic genus Dexiostoma contains D. campyla (Stokes, 1886) and is distinguished from Colpidium by a different oral infraciliature and the position and shape of the preoral suture. A new genus, Paracolpidium nov. gen., is suggested for Colpidium truncatum Stokes, 1885 because its oral infraciliature deviates from both Colpidium and Dexiostoma. Contrary to Colpidium and Dexiostoma, the silverline system of Paracolpidium truncatum lacks secondary meridians but shows short projections to the left of the primary meridians indicating a phylogenetic relationship to Tetrahymena.     

Implications ofrbcL sequence data for higher order relationships of theLoasaceae and the anomalous aquatic plantHydrostachys (Hydrostachyaceae)

Subclass and ordinal relationships ofLoasaceae, a small predominately New World family, are examined usingrbcL sequence data. Sequences were examined for eight of the fifteen genera of theLoasaceae and the morphologically anomalous aquatic genusHydrostachys (Hydrostachyaceae). Parsimony analyses of these sequences, combined with previously publishedrcbL data, indicate thatLoasaceae belong in theCornales, and are the sister group ofHydrangeaceae. This agrees with phylogenies based on chloroplast DNA inverted repeat restriction site, morphological and chemical data. TherbcL trees support the monophyly of theLoasaceae and most generic relationships correspond to current subfamily divisions. TherbcL phylogeny also provides the first suggestion thatHydrostachys is allied with theHydrangeaceae in theCornales.     

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.     

Phylogenetic analysis of <Emphasis Type="Italic">Orgyia pseudotsugata</Emphasis> single-nucleocapsid nucleopolyhedrovirus

The Douglas-fir tussock moth Orgyia pseudotsugata (Lepidoptera: Lymantriidae) is a frequent defoliator of Douglas-fir and true firs in western USA and Canada. A single nucleopolyhedrovirus (SNPV) isolated from O. pseudotsugata larvae in Canada (OpSNPV) was previously analyzed via its polyhedrin gene, but is phylogenetic status was ambiguous. Sequences of four conserved baculovirus genes, polyhedrin, lef-8, pif-2 and dpol, were amplified from OpSNPV DNA in polymerase chain reactions using degenerate primer sets and their sequences were analyzed phylogenetically. The analysis revealed that OpSNPV belongs to group II NPVs and is most closely related to SNPVs that infect O. ericae and O. anartoides, respectively. These results show the need for multiple, concatenated gene phylogenies to classify baculoviruses. Foundation item: Supported by a scholarship from the European Union (Functional Biodiversity and Crop Protection), contract n^o HPMT-CT-2000-00199.     

Morphological differences and molecular phylogeny of freshwater blooming species, Peridiniopsis spp. (Dinophyceae) from China

In 2008–2010, several freshwater dinoflagellate blooms caused by Peridiniopsis spp. were observed in China. P. penardii and P. niei sampled from various geographical localities were examined by means of light and scanning electron microscopy. After comparing morphological and molecular differences, the new freshwater variety Peridiniopsis penardii var. robusta var. nov. (Peridiniales, Dinophyceae) found in Manwan Reservoir, Yunnan Province was described. The new variety differed from P. penardii since it possessed numerous robust antapical spines and a conspicuous apical spine. Molecular phylogenetic analyses based on SSU, LSU and ITS indicated P. niei, P. penardii and P. penardii var. robusta were closely related with P. kevei, and clustered into a monophyletic clade. The new variety possessed an endosymbiotic diatom which was similar to P. penardii and P. kevei, whereas the endosymbiont was not present in cells of P. niei. The endosymbiont SSU and ITS phylogenies showed that the endosymbionts of these three dinoflagellates were closely related to members of Thalassiosirales. Furthermore it was concluded that the endosymbionts might originate from Discostella-like species.     

Phylogenetic investigations of Sordariaceae based on multiple gene sequences and morphology

The family Sordariaceae incorporates a number of fungi that are excellent model organisms for various biological, biochemical, ecological, genetic and evolutionary studies. To determine the evolutionary relationships within this group and their respective phylogenetic placements, multiple-gene sequences (partial nuclear 28S ribosomal DNA, nuclear ITS ribosomal DNA and partial nuclear β-tubulin) were analysed using maximum parsimony and Bayesian analyses. Analyses of different gene datasets were performed individually and then combined to generate phylogenies. We report that Sordariaceae, with the exclusion Apodus and Diplogelasinospora, is a monophyletic group. Apodus and Diplogelasinospora are related to Lasiosphaeriaceae. Multiple gene analyses suggest that the spore sheath is not a phylogenetically significant character to segregate Asordaria from Sordaria. Smooth-spored Sordaria species (including so-called Asordaria species) constitute a natural group. Asordaria is therefore congeneric with Sordaria. Anixiella species nested among Gelasinospora species, providing further evidence that non-ostiolate ascomata have evolved from ostiolate ascomata on several independent occasions. This study agrees with previous studies that show heterothallic Neurospora species to be monophyletic, but that homothallic ones may have a multiple origins. Although Gelasinospora and Neurospora are closely related and not resolved as monophyletic groups, there is insufficient evidence to place currently accepted Gelasinospora and Neurospora species into the same genus.     

Molecular Systematics of Zopfiella and allied genera: evidence from multi-gene sequence analyses

This study aims to reveal the phylogenetic relationships of Zopfiella and allied genera in the Sordariales. Multiple gene sequences (partial 28 S rDNA, ITS/5.8 S rDNA and partial β-tubulin) were analysed using MP and Bayesian analyses. Analyses of different gene datasets were performed individually and then combined to infer phylogenies. Phylogenetic analyses show that currently recognised Zopfiella species are polyphyletic. Based on sequence analyses and morphology, it appears that Zopfiella should be restricted to species having ascospores with a septum in the dark cell. Our molecular analysis also shows that Zopfiella should be placed in Lasiosphaeriaceae rather than Chaetomiaceae. Cercophora and Podospora are also polyphyletic, which is in agreement with previous studies. Our analyses show that species possessing a Cladorrhinum anamorph are phylogenetically closely related. In addition, there are several strongly supported clades, characterised by species possessing divergent morphological characters. It is difficult to predict which characters are phylogenetically informative for delimiting these clades.     

H9, H10, and H11 compose a cluster of Hessian fly-resistance genes in the distal gene-rich region of wheat chromosome 1AS

H9, H10, and H11 are major dominant resistance genes in wheat, expressing antibiosis against Hessian fly [(Hf) Mayetiola destructor (Say)] larvae. Previously, H9 and H10 were assigned to chromosome 5A and H11 to 1A. The objectives of this study were to identify simple-sequence-repeat (SSR) markers for fine mapping of these genes and for marker-assisted selection in wheat breeding. Contrary to previous results, H9 and H10 did not show linkage with SSR markers on chromosome 5A. Instead, H9, H10, and H11 are linked with SSR markers on the short arm of chromosome 1A. Both H9 and H10 are tightly linked to flanking markers Xbarc263 and Xcfa2153 within a genetic distance of 0.3–0.5 cM. H11 is tightly linked to flanking markers Xcfa2153 and Xbarc263 at genetic distances of 0.3 cM and 1.7 cM. Deletion bin mapping assigned these markers and genes to the distal 14% of chromosome arm 1AS, where another Hf-resistance gene, Hdic (derived from emmer wheat), was also mapped previously. Marker polymorphism results indicated that a small terminal segment of chromosome 1AS containing H9 or H10 was transferred from the donor parent to the wheat lines Iris or Joy, and a small intercalary fragment carrying H11 was transferred from the resistant donor to the wheat line Karen. Our results suggest that H9, H10, H11, Hdic, and the previously identified H9- or H11-linked genes (H3, H5, H6, H12, H14, H15, H16, H17, H19, H28, and H29) may compose a cluster (or family) of Hf-resistance genes in the distal gene-rich region of wheat chromosome 1AS; and H10 most likely is the same gene as H9.Mention of commercial or proprietary product does not constitute an endorsement by the USDA.