Experimental Hybridization Reveals Biased Inheritance of the Internal Transcribed Spacer of the Nuclear Ribosomal DNA in Begonia ×taipeiensis

Begonia × taipeiensis C.-I Peng, a naturally occurring hybrid resulting from B. formosana × B. aptera, in Taiwan. To understand the inheritance of ribosomal DNA in unidirectional hybridization, experiments were conducted using B. formosana and B. aptera as ovule and pollen donors, respectively. The internal transcribed spacer region (ITS) of nuclear ribosomal DNA was amplified from the artificial hybrids, parental species, and natural hybrids. In contrast to the single type of ITS in the parental species, multiple sequences were cloned from both natural and artificial hybrids. A split decomposition network based on ITS nucleotide variation revealed that all but one (clone B14) of the hybrid sequences were “phylogenetically” closely related to B. formosana. Apparently, in such unidirectional hybridization, maternal DNA provided most of phylogenetic information. In the hybrid sequences, in addition to additive polymorphisms inherited from maternal (38.1%) and paternal (30.1%) plants, a novel nucleotide composition (31.8%) was also detected. The “new” characters are seen as noise in phylogenetic inference. They were probably obtained via intramolecular recombination, as gene conversion was not detected. The occurrence of genetic recombination appeared to be nonrandom, with a higher frequency in the ITS1 (3.14%) and ITS2 (3.42%) regions than in the 5.8S RNA gene (2.22%). Given the lack of sexual recombination in B. × taipeiensis and short time span, unequal crossing-over likely contributed to the heterogeneity of the ITS composition in the nuclear genome. Although the sterile hybrids have not attained their own lineage independent from the parental species, a high level of genetic diversity is transmitted asexually and maintained in these plants. Received 7 February 2001/ Accepted in revised form 31 May 2001     

Phylogenetic Analysis of a ‘Jewel Orchid’ Genus Goodyera (Orchidaceae) Based on DNA Sequence Data from Nuclear and Plastid Regions

A molecular phylogeny of Asiatic species of Goodyera (Orchidaceae, Cranichideae, Goodyerinae) based on the nuclear ribosomal internal transcribed spacer (ITS) region and two chloroplast loci (matK and trnL-F) was presented. Thirty-five species represented by 132 samples of Goodyera were analyzed, along with other 27 genera/48 species, using Pterostylis longifolia and Chloraea gaudichaudii as outgroups. Bayesian inference, maximum parsimony and maximum likelihood methods were used to reveal the intrageneric relationships of Goodyera and its intergeneric relationships to related genera. The results indicate that: 1) Goodyera is not monophyletic; 2) Goodyera could be divided into four sections, viz., Goodyera, Otosepalum, Reticulum and a new section; 3) sect. Reticulum can be further divided into two subsections, viz., Reticulum and Foliosum, whereas sect. Goodyera can in turn be divided into subsections Goodyera and a new subsection.     

Phylogenetic utility of protein (RPB2, β-tubulin) and ribosomal (LSU,SSU) gene sequences in the systematics of Sordariomycetes (Ascomycota,Fungi)

The Sordariomycetes is an important group of fungi whose taxonomic relationships and classification is obscure. There is presently no multi-gene molecular phylogeny that addresses evolutionary relationships among different classes and orders. In this study, phylogenetic analyses with a broad taxon sampling of the Sordariomycetes were conducted to evaluate the utility of four gene regions (LSU rDNA, SSU rDNA, beta-tubulin and RPB2) for inferring evolutionary relationships at different taxonomic ranks. Single and multi-gene genealogies inferred from Bayesian and Maximum Parsimony analyses were compared in individual and combined datasets. At the subclass level, SSU rDNA phylogenies demonstrate their utility as a marker to infer phylogenetic relationships at higher levels. All analyses with SSU rDNA alone, combined LSU rDNA and SSU rDNA, and the combined 28 S rDNA, SSU rDNA and RPB2 datasets resulted in three subclasses: Hypocreomycetidae, Sordariomycetidae and Xylariomycetidae, which correspond well to established morphological classification schemes. At the ordinal level, the best resolved phylogeny was obtained from the combined LSU rDNA and SSU rDNA datasets. Individually, the RPB2 gene dataset resulted in significantly higher number of parsimony informative characters. Our results supported the recent separation of Boliniaceae, Chaetosphaeriaceae and Coniochaetaceae from Sordariales and placement of Coronophorales in Hypocreomycetidae. Microascales was found to be paraphyletic and Ceratocystis is phylogenetically associated to Faurelina, while Microascus and Petriella formed another clade and basal to other members of Halosphaeriales. In addition, the order Lulworthiales does not appear to fit in any of the three subclasses. Congruence between morphological and molecular classification schemes is discussed.     

Characterization of <Emphasis Type="Italic">Penicillium</Emphasis> Species Isolated from Grape Berries by Their Internal Transcribed Spacer (ITS1) Sequences and by Gas Chromatography–Mass Spectrometry Analysis of Geosmin Production

Geosmin (trans-1,10-dimethyl-trans-9-decalol), an earthy-musty compound, has been identified in wines and in grape juice, in which its presence is highly detrimental to the aromatic quality. Geosmin has a biological origin, and the analysis of rotten grape microflora has been done on two grape varieties (Semillon, Cabernet Sauvignon) from six parcels of the Bordeaux region over 3 years (1999, 2000, 2001). Forty-three Penicillium-related species have been analyzed by gas chromatography–mass spectrometry (GC–MS) for their geosmin production. GC–MS analysis has demonstrated that the earthy odor was always correlated with the presence of geosmin. Phenotypic characterization of Penicillium spp. being ambiguous, a molecular characterization by rDNA internal transcribed spacer (ITS1) sequencing was performed for all strains. The results evidenced that all strains producing geosmin belonged to only one species, P. expansum, and that the other strains, not producing geosmin, belonged to three species: P. purpurogenum, P. thomii, and Talaromyces wortmanii.     

Nested in the Chlorellales or Independent Class? Phylogeny and Classification of the Pedinophyceae (Viridiplantae) Revealed by Molecular Phylogenetic Analyses of Complete Nuclear and Plastid-encoded rRNA Operons

The class Pedinophyceae was established for asymmetric uniflagellate green algae, and was originally considered as an ancestral lineage of viridiplants. However, analyses of 71 concatenated plastid proteins [Turmel et al. (2009): Mol. Biol. Evol. 26: 2317-2331] recovered Pedinomonas within the Chlorellales (Trebouxiophyceae), thereby questioning the Pedinophyceae as an independent class. For the present study, complete nuclear and plastid-encoded rRNA operon sequences have been determined for 37 taxa of green algae including 6 members of the Pedinophyceae, providing 9272 aligned nucleotide positions. Phylogenies using both rRNA operons consistently rejected any relationship between Pedinophyceae and the Chlorellales. Instead, the Pedinophyceae were significantly resolved as sister of all phycoplast-containing 'core' chlorophytes, i.e. Chlorodendrophyceae, Trebouxiophyceae, Ulvophyceae and Chlorophyceae. Reinvestigation of plastid proteins discovered biased phylogenetic signal among protein partitions, indicating the published Pedinomonas + Chlorellales association as likely artificial. Marine pedinophytes (Resultomonas and Marsupiomonas; Marsupiomonadales ord. nov.), formed a sister clade to the order Pedinomonadales, occurring in freshwater and soil habitats. Synapomorphies in rRNA secondary structures were integrated in taxonomic diagnoses of the Pedinophyceae and were also used for BLAST searches targeting environmental sequence databases. The latter approach revealed conserved habitat preferences for the Marsupiomonadales and Pedinomonadales, and identified several novel pedinophyte lineages.     

Evolution of ITS1 rDNA in the Digenea (Platyhelminthes: Trematoda): 3′ End Sequence Conservation and Its Phylogenetic Utility

A comparison of ribosomal internal transcribed spacer 1 (ITS1) elements of digenetic trematodes (Platyhelminthes) including unidentified digeneans isolated from Cyathura carinata (Crustacea: Isopoda) revealed DNA sequence similarities at more than half of the spacer at its 3′ end. Primary sequence similarity was shown to be associated with secondary structure conservation, which suggested that similarity is due to identity by descent and not chance. Using an analysis of apomorphies, the sequence data were shown to produce a distinct phylogenetic signal. This was confirmed by the consistency of results of different tree reconstruction methods such as distance approaches, maximum parsimony, and maximum likelihood. Morphological evidence additionally supported the phylogenetic tree based on ITS1 data and the inferred phylogenetic position of the unidentified digeneans of C. carinata met the expectations from known trematode life-cycle patterns. Although ribosomal ITS1 elements are generally believed to be too variable for phylogenetic analysis above the species or genus level, the overall consistency of the results of this study strongly suggests that this is not the case in digenetic trematodes. Here, 3′ end ITS1 sequence data seem to provide a valuable tool for elucidating phylogenetic relationships of a broad range of phylogenetically distinct taxa. Received: 20 October 1997 / Accepted: 24 March 1998     

Effect of temperature on the larval stage of Tetanocera elata (Diptera: Sciomyzidae) – Potential biological control agent of pestiferous slugs

Due to the considerable losses caused by slugs in terms of agricultural production and revenue, there is an urgent need for a cost effective biological control agent. The malacophagous nature of the sciomyzid fly, Tetanocera elata (Fab.) makes it a possible contender to meet this demand. This study examined the effect of constant temperatures (14, 17, 20, 23, and 26 °C), in addition to ambient outdoor and laboratory temperatures on T. elata larval duration and predation. In general, the mean and median larval stage duration decreased as temperature increased with percentage survival for the overall larval stage (62%) greatest at 20 °C with a median duration of 44 days. There was no significant difference between temperatures with regard to the number of slugs killed per larva and while predation rate increased with increasing constant temperature, there was also no significant difference between the constant temperatures. Our results show that puparial weight can be used to predict the sex of adult flies prior to their emergence. The results are discussed in the context of the suitability of T. elata as a biological control agent of pestiferous slugs.     

Phylogenetic position of the genus Bryhnia Kaurin (Brachytheciaceae,Bryophyta) inferred from chloroplast (rpl16, trnG,trnL–F) and nuclear (ITS) sequence data

Abstract

The systematic position of the genus Bryhnia Kaurin has been problematic and remains unsettled. A phylogenetic analysis of Bryhnia and closely related genera, based on the nuclear marker ITS1–5·8S–ITS2 and the plastid markers rpl16, trnG, and trnL–F, was carried out to clarify their relationships and systematic positions. The phylogenetic trees generated from Maximum parsimony and Bayesian analyses show that Bryhnia s.s., represented by B. novae-angliae (Sull. & Lesq.) Grout, B. hultenii E.B.Bartram, and B. scabrida (Lindb.) Kaurin, forms a maximally supported clade nested within Brachythecium Schimp. ‘Bryhnia scabrida’ sensu is Bryhnia brachycladula Cardot, and together they form a monophyletic group with Eurhynchiadelphus eustegia (Besch.) Ignatov & Huttunen and Myuroclada maximowiczii (G.G.Borshch.) Steere & W.B.Schofield. We propose the inclusion of the species currently recognized in Bryhnia in Brachythecium, with the transfer of Bryhnia brachycladula to the genus Myuroclada Besch. Bryhnia brachycladula is further treated as a synonym of the new combination, Myuroclada longiramea (Müll.Hal.) M.Li, Y.F.Wang, M.S.Ignatov & S.Huttunen.     

Phylogenetic relationships within the ‘core’ Laureae (Litsea complex, Lauraceae) inferred from sequences of the chloroplast gene matK and nuclear ribosomal DNA ITS regions

A phylogenetic analysis of the core Laureae (Litsea complex) was conducted using the chloroplast gene matK and nuclear ribosomal DNA ITS sequences to investigate generic relationships and boundaries within the complex. Despite low genetic divergence for matK, rooting of the tree with Sassafras resulted in Iteadaphne as the basal member of the complex and five resolved clades: a Neolitsea clade and then Laurus, Parasassafras, Litsea and Lindera clades in a large polytomy with unresolved Lindera sections plus Umbellularia. A combined analysis of the data (identical to the ITS results) provided a more resolved phylogeny of the Laureae, with four major lineages: the Laurus, Litsea, Lindera and Actinodaphne II clades. These clades also appear to reflect the importance of inflorescence structure and ontogeny within the Laureae, as well as data from cuticular micromorphology, but there was no support for traditional generic characters such as 2- versus 4- celled anthers. As a result, genera such as Actinodaphne, Litsea, Neolitsea and Lindera were polyphyletic in all analyses. Parasassafras was related to Sinosassafras by the matK data, but distant from it in the ITS and combined analyses.     

First Molecular Data and the Phylogenetic Position of the Millipede-Like Centipede Edentistoma octosulcatum T?m?sváry, 1882 (Chilopoda: Scolopendromorpha: Scolopendridae)

Edentistoma octosulcatum Tömösváry, 1882, is a rare, superficially millipede-like centipede known only from Borneo and the Philippines. It is unique within the order Scolopendromorpha for its slow gait, robust tergites, and highly modified gizzard and mandible morphology. Not much is known about the biology of the species but it has been speculated to be arboreal with a possibly vegetarian diet. Until now its phylogenetic position within the subfamily Otostigminae has been based only on morphological characters, being variably ranked as a monotypic tribe (Arrhabdotini) or classified with the Southeast Asian genus Sterropristes Attems, 1934. The first molecular data for E. octosulcatum sourced from a newly collected specimen from Sarawak were analysed with and without morphology. Parsimony analysis of 122 morphological characters together with two nuclear and two mitochondrial loci resolves Edentistoma as sister group to three Indo-Australian species of Rhysida, this clade in turn grouping with Ethmostigmus, whereas maximum likelihood and parsimony analyses of the molecular data on their own ally Edentistoma with species of Otostigmus. A position of Edentistoma within Otostigmini (rather than being its sister group as predicted by the Arrhabdotini hypothesis) is consistently retrieved under different analytical conditions, but support values within the subfamily remain low for most nodes. The species exhibits strong pushing behaviour, suggestive of burrowing habits. Evidence against a suggested vegetarian diet is provided by observation of E. octosulcatum feeding on millipedes in the genus Trachelomegalus.     

Nuclear Overhauser Data and Stereochemical Considerations Suggest that Netropsin Binds Symmetrically Within the Minor Groove of Poly(dA)·Poly(dT), Forming Hydrogen Bonds with Both Strands of the Double Helix

Abstract

Sarma et al. (J. Biomol. Str. and Dynam. 2, 1085 (1985) have proposed, on the basis of nuclear magnetic resonance experiments on the complex of netropsin with poly(dA)·poly(dT), that the drug molecule lies asymmetrically along the dA side of the minor groove and makes hydrogen bonds only with the dA strand. If the crystal structure analyses of B-DNA (Fratini et al., J. Biol. Chem. 257, 14686 (1982)) and of its complex with netropsin (Kopka et al., J. Mol. Biol. 183, 553 (1985)) are any guide, this off-center, wide-groove model is stereochemically unlikely. More to the point, the off-center model is unnecessary to explain the observed nmr data. All of the nuclear Overhauser and other observations are fully explained by the structure seen in the x-ray crystal analysis, in which netropsin sits squarely centered within the minor groove, making bifurcated hydrogen bonds with both strands.     

Escherichia coli Virulence Protein NleH1 Interaction with the v-Crk Sarcoma Virus CT10 Oncogene-like Protein (CRKL) Governs NleH1 Inhibition of the Ribosomal Protein S3 (RPS3)/Nuclear Factor κB (NF-κB) Pathway

Enterohemorrhagic Escherichia coli and other attaching/effacing bacterial pathogens cause diarrhea in humans. These pathogens use a type III secretion system to inject virulence proteins (effectors) into host cells, some of which inhibit the innate immune system. The enterohemorrhagic E. coli NleH1 effector prevents the nuclear translocation of RPS3 (ribosomal protein S3) to inhibit its participation as a nuclear “specifier” of NF-κB binding to target gene promoters. NleH1 binds to RPS3 and inhibits its phosphorylation on Ser-209 by IκB kinase-β (IKKβ). However, the precise mechanism of this inhibition is unclear. NleH1 possesses a Ser/Thr protein kinase activity that is essential both for its ability to inhibit the RPS3/NF-κB pathway and for full virulence of the attaching/effacing mouse pathogen Citrobacter rodentium. However, neither RPS3 nor IKKβ is a substrate of NleH1 kinase activity. We therefore screened ∼9,000 human proteins to identify NleH1 kinase substrates and identified CRKL (v-Crk sarcoma virus CT10 oncogene-like protein), a substrate of the BCR/ABL kinase. Knockdown of CRKL abundance prevented NleH1 from inhibiting RPS3 nuclear translocation and NF-κB activity. CRKL residues Tyr-198 and Tyr-207 were required for interaction with NleH1. Lys-159, the kinase-active site of NleH1, was necessary for its interaction with CRKL. We also identified CRKL as an IKKβ interaction partner, mediated by CRKL Tyr-198. We propose that the CRKL interaction with IKKβ recruits NleH1 to the IKKβ complex, where NleH1 then inhibits the RPS3/NF-κB pathway.