Mitochondrial Small Subunit rRNA Gene Research Articles

Molecular phylogeny of the Pertusariaceae supports secondary chemistry as an important systematic character set in lichen-forming ascomycetes

The Pertusariaceae is a diverse, cosmopolitan group of crustose lichen-forming fungi. It is particularly rich in secondary metabolites, containing a variety of compounds from different substance classes, such as xanthones, depsides, depsidones, and depsones. Morphology and chemistry-based studies have provided conflicting views of relationships among taxa, and phylogenetic relationships among genera in the Pertusariaceae are still unsettled. To evaluate these relationships, we generated a phylogeny based on nucleotide sequences of the nuclear large subunit (nu LSU) and the mitochondrial small subunit (mt SSU) rRNA genes. For a subset of taxa, we additionally sequenced the mitochondrial large subunit (mt LSU) rDNA. We studied a total of 49 taxa from the family Pertusariaceae and the enigmatic genus Loxosporopsis including four Agyrialean taxa used as outgroups. The alignments were analyzed using a Bayesian approach with Markov Chain Monte Carlo tree sampling (B/MCMC), and maximum parsimony methods. In the resulting phylogenetic estimates the genus Pertusaria in its current circumscription is polyphyletic comprising three major clades: the Pertusaria s.str.-group, the Variolaria-group and the Varicellaria-group. Loxosporopsis is closely related to Pertusaria s.str. We re-analyzed morphological, anatomical, and chemical features in the light of the molecular study and found novel character combinations to describe monophyletic entities. The taxonomic significance of particular secondary chemical constituents in the Pertusariaceae is corroborated.

Phylogenetic analysis of Verticillium species based on nuclear and mitochondrial sequences.

Three different genes were sequenced from isolates of five plant-pathogenic Verticillium species, Verticillium albo-atrum, Verticillium dahliae, Verticillium longisporum, Verticillium nigrescens, and Verticillium tricorpus. The sequences covered parts of the mitochondrial cytochrome b gene ( cob), the mitochondrial small subunit rRNA gene ( rns) and the nuclear ITS2 region. When the sequences were combined, the five species clustered in five monophyletic groups, with V. nigrescens distantly related to the other species while V. tricorpus displayed a somewhat closer relationship to the three remaining species. V. albo-atrum, V. dahliae and V. longisporum were found to be very similar to each other, with V. albo-atrum and V. longisporum displaying the closest relationship. The species affiliation of V. longisporum is discussed.

Genetic Variations in the Internal Transcribed Spacer and Mitochondrial Small Subunit rRNA Gene of Naegleria spp.

Naegleria spp. are widely distributed free-living amebas, but one species in the genus, N. fowleri, causes acute fulminant primary amebic meningoencephalitis in humans and other animals. Thus, it is important to differentiate N. fowleri from the rest in the genus of Naegleria, and to develop tools for the detection of intra-specific genetic variations. In this study, one isolate each of N. australiensis, N. gruberi, N. jadini, and N. lovaniensis and 22 isolates of N. fowleri were characterized at the internal transcribed spacers (ITS) and mitochondrial small subunit rRNA (mtSSU rRNA) gene. The mtSSU rRNA primers designed amplified DNA of all isolates, with distinct sequences obtained from all species examined. In contrast, the ITS primers only amplified DNA from N. lovaniensis and N. fowleri, with minor sequence differences between the two. Three genotypes of N. fowleri were found among the isolates analyzed in both the mtSSU rRNA gene and ITS. The extent of sequence variation was greater in the mtSSU rRNA gene, but the ITS had the advantage of length polymorphism. These data should be useful in the development of molecular tools for rapid species differentiation and genotyping of Naegleria spp.

Advances in free-living amebae research 2003: workshop summary.

Advances in free-living amebae research 2003: workshop summary.

Nucleotide polymorphisms in three genes support host and geographic speciation in tree pathogens belonging toGremmeniellaspp.

We detected nucleotide polymorphisms within the genus Gremmeniella in DNA sequences of β-tubulin, glyceraldehyde phosphate dehydrogenase, and mitochondrial small subunit rRNA (mtSSU rRNA) genes. A group-I intron was present in strains originating from fir (Abies spp.) in the mtSSU rRNA locus. This intron in the mtSSU rRNA locus of strains isolated from Abies sachalinensis (Fridr. Schmidt) M.T. Mast in Asia was also found in strains isolated from Abies balsamea (L.) Mill. in North America. Phylogenetic analyses yielded trees that grouped strains by host of origin with strong branch support. Asian strains of Gremmeniella abietina (Lagerberg) Morelet var. abietina isolated from fir (A. sachalinensis) were more closely related to G. abietina var. balsamea from North America, which is found on spruce (Picea spp.) and balsam fir, and European and North American races of G. abietina var. abietina from pines (Pinus spp.) were distantly related. Likewise, North American isolates of Gremmeniella laricina (Ettinger) O. Petrini, L.E. Petrini, G. Laflamme, & G.B. Ouellette, a pathogen of larch, was more closely related to G. laricina from Europe than to G. abietina var. abietina from North America. These data suggest that host specialization might have been the leading evolutionary force shaping Gremmeniella spp., with geographic separation acting as a secondary factor.Key words: Gremmeniella, geographic separation, host specialization, mitochondrial rRNA, nuclear genes.

Evidence for a slowed rate of molecular evolution in the order acipenseriformes.

A test of the hypothesis that the members of the order Acipenseriformes (sturgeons and paddlefishes) possess a slowed rate of molecular evolution was carried out by conducting relative-rate comparisons with representatives of four groups of teleost fishes (Cypriniformes, Elopomorpha, Salmonidae, and Percomorpha) using 21 nuclear or mitochondrial protein loci and the nuclear and mitochondrial small subunit rRNA genes, obtained from the literature or our own research. In 70 out of 81 comparisons between individual taxa (86%), acipenseriform sequences showed slower rates of change than the homologous teleost loci examined. When teleost sequences are considered together, 21 of the 23 loci show slower rates of substitution in the acipenseriform lineage. Teleost proteins show 1.85 times as many unique amino acid differences as acipenseriform proteins, when both are compared with outlier sequences. These results support a hypothesis of slowed molecular evolutionary rate in the Acipenseriformes.

First Report of Phomopsis longicolla from Velvetleaf Causing Stem Lesions on Inoculated Soybean and Velvetleaf Plants.

Reddish brown lesions were observed on the lower stem and upper root area of velvetleaf (Abutilon theophrasti Medik.) plants growing in an Illinois soybean field in June 2000. The lesions were similar in appearance to those caused by Rhizoctonia root rot of soybean. Stems and roots with lesions were cut into ≍5-mm pieces, surface-disinfested, and placed on 2% water agar at pH 4.5. The cultural morphology of the two isolates fit the description of Phomopsis longicolla Hobbs (1). Colonies on potato dextrose agar (PDA) were floccose, dense, and white. The undersides of the cultures were colorless. Stromata were large, black, and spreading. The pattern of stromata in one isolate was effuse, and most of the stromata were immersed or semiimmersed in the medium, whereas the stromata from the other isolate were massive and prominent. Neither isolate turned green on PDA. Alpha conidia were hyaline, ellipsoidal to fusiform, and guttulate. DAPI (4',6-diamidino-2-phenylindole)-stained alpha conidia were uninucleate. Beta conidia and perithecia did not occur on either PDA or oat flakes on water agar from 1 to 10 weeks at 25°C under a 12-h photoperiod. The DNA sequences of the mitochondrial small subunit rRNA genes of the two isolates were identical and shared 100% sequence identity with two P. longicolla soybean isolates that we had identified previously. Pathogenicity tests were conducted in a greenhouse by cutting the stems of 3-week-old soybean and velvetleaf plants at the second internode. Mycelial plugs (4 mm in diameter) from the margin of 1-week-old cultures of the two isolates from velvetleaf and one from soybean were individually placed mycelial side down directly on the top of cut stems of 10 to 15 plants per isolate. Controls included noninoculated plants with and without PDA plugs. Plants were kept in a mist chamber in the dark at 25°C for 4 days and were then transferred to a greenhouse with a 16-h photoperiod at 24 ± 3°C. Stem lesions were measured 7 days after inoculation. The experiment was repeated once. Mean stem lesion lengths caused by the velvetleaf and soybean isolates were 23 and 20 mm, respectively, on soybean stems, while negative controls produced no lesions. Mean stem lesion lengths caused by the velvetleaf and soybean isolates were 23.5 and 12 mm, respectively, on velvetleaf stems. P. longicolla was reisolated from the stem lesions of five randomly collected plants. This is the first report of P. longicolla being isolated from velvetleaf and causing stem lesions on inoculated soybean and velvetleaf plants. Reference: (1) T. W. Hobbs et al. Mycologia 77: 535, 1985.

Molecular Differentiation of Fusarium solani f. sp. glycines from Other F. solani Based on Mitochondrial Small Subunit rDNA Sequences

Fusarium solani is a soilborne plant pathogen that infects many different hosts. Within the species, there is some specialization, and a number of forma specialis have been described based on host affiliation. One of these, F. solani f. sp. glycines, infects soybean and causes sudden death syndrome. To differentiate between F. solani f. sp. glycines and other F. solani isolates, a partial sequence of the mitochondrial small subunit (mtSSU) rRNA gene was amplified by polymerase chain reaction and sequenced from 14 F. solani f. sp. glycines and 24 F. solani isolates from various plant hosts. All F. solani f. sp. glycines isolates had identical sequences. A single, unique insertion of cytosine occurred in all F. solaniisolates but not in any of the F. solani f. sp. glycines isolates. Two major lineages, distinguished by sequence divergence and the presence or absence of multiple insertions, occurred in F. solani isolates. Cladistic analysis produced a single most-parsimonious tree with three major clades. The first clade contained all F. solani f. sp. glycines isolates. A second clade grouped together all of the F. solani isolates that had only a single nucleotide insertion difference from the first clade. Genetic distance between these two clades was 0.016. A third clade was formed by five F. solaniisolates that had multiple insertions. Isolates in the third clade had a genetic distance of 0.040 from the first and second clades. Based on the sequence data, it is likely that F. solani f. sp. glycineshas a shorter evolutionary history than other F. solaniisolates that have either single or multiple nucleotide insertions. The differences in nucleotide insertions in part of the mtSSU rRNA gene between F. solani f. sp. glycinesand other F. solani isolates provide a direct and reliable way to distinguish isolates of F. solani.

Phylogenetic relationships among Acanthamoeba spp. based on PCR-RFLP analyses of mitochondrial small subunit rRNA gene.

We investigated the value of mitochondrial small subunit rRNA gene (mt SSU rDNA) PCR-RFLP as a taxonomic tool for Acanthamoeba isolates with close inter-relationships. Twenty-five isolates representing 20 species were included in the analysis. As in nuclear 18S rDNA analysis, two type strains (A. astronyxis and A. tubiashi) of morphological group 1 diverged earliest from the other strains, but the divergence between them was less than in 18S riboprinting. Acanthamoeba griffini of morphological group 2 branched between pathogenic (A. culbertsoni A-1 and A. healyi OC-3A) and nonpathogenic (A. palestinensis Reich, A. pustulosa GE-3a, A. royreba Oak Ridge, and A lenticulata PD2S) strains of morphological group 3. Among the remaining isolates of morphological group 2, the Chang strain had the identical mitochondrial riboprints as the type strain of A. hatchetti. AA2 and AA1, the type strains of A. divionensis and A. paradivionensis, respectively, had the identical riboprints as A. quina Vil3 and A. castellanii Ma. Although the branching orders of A. castellanii Neff, A. polyphaga P23, A. triangularis SH621, and A. lugdunensis L3a were different from those in 18S riboprinting analysis, the results obtained from this study generally coincided well with those from 18S riboprinting. Mitochondrial riboprinting may have an advantage over nuclear 18S rDNA riboprinting because the mt SSU rDNAs do not seem to have introns that are found in the 18S genes of Acanthamoeba and that distort phylogenetic analyses.

Rate and mode differences between nuclear and mitochondrial small-subunit rRNA genes in mushrooms.

Sequences from homologous regions of the nuclear and mitochondrial small-subunit rRNA genes from 10 members of the mushroom order Boletales were used to construct evolutionary trees and to compare the rates and modes of evolution. Trees constructed independently for each gene by parsimony and tested by bootstrap analysis have identical topologies in all statistically significant branches. Examination of base substitutions revealed that the nuclear gene is biased toward C-T transitions and that the distribution of transversions in the mitochondrial gene is strongly effected by an A-T bias. When only homologous regions of the two genes were compared, base substitutions per nucleotide were roughly 16-fold greater in the mitochondrial gene. The difference in the frequency of length mutations was at least as great but was impossible to estimate accurately because of their absence in the nuclear gene. Maximum likelihood was used to show that base-substitution rates vary dramatically among the branches. A significant part of the rate inconstancy was caused by an accelerated nuclear rate in one branch and a retarded mitochondrial rate in a different branch. A second part of the rate variability involved a consistent inconstancy: short branches exhibit ratios of mitochondrial to nuclear divergences of less than 1, while longer branches had ratios of approximately 4:1-8:1. This pattern suggests a systematic error in the branch length calculation. The error may be related to the simplicity of the divergence estimates, which assumes that all base positions have an equal probability of change.