The Paxillus involutus (Boletales, Paxillaceae) complex in Europe: Genetic diversity and morphological description of the new species Paxillus cuprinus, typification of P. involutus s.s., and synthesis of species boundaries

Paxillus involutus is a model species for ecological or physiological studies of ectomycorrhizal agaricomycetes. Three to six groups or species linked to it have been ecologically and morphologically distinguished. Phylogenetic studies have revealed the existence of four species in Europe: Paxillus ammoniavirescens, Paxillus obscurisporus, P. involutus, and a fourth as yet not described species. We studied 47 collections from 24 French and Italian locations, supplemented with GenBank data, in order to genetically and taxonomically delineate these species. Phylogenetic analyses of three nuclear DNA regions (rDNA internal transcribed spacer (ITS), tef1-α, and gpd) confirmed the four European species. Morphology, culture, and ecology features allowed us to delineate species boundaries and to describe the fourth species we named Paxillus cuprinus since it turns coppery with age. As there is no existing original herbarium specimen for P. involutus, one of our collections was chosen as the epitype. The low genetic diversity found in P. cuprinus correlates with stable morphological traits (basidiome colour, ovoid–amygdaliform spores with an apical constriction) and with ecological preferences (association with Betulaceae in open and temperate areas). In contrast, P. ammoniavirescens is characterized by a high genetic diversity and a high variation of its morphological and ecological features.     

Cortinarius sect. Brunnei (Basidiomycota, Agaricales) in North Europe

The section Brunnei was extensively studied based on material from North Europe. To stabilise the nomenclature we studied the relevant types of taxa included in this section. Phylogenetic relationships and species limits were investigated using rDNA ITS sequences and the results were compared with the morphological data. We recognised 11 species: Cortinarius brunneus, C. clarobrunneus comb. nov., C. coleoptera, C. ectypus, C. gentilis, C. glandicolor (neotypified), C. pseudorubricosus, and four species described as new C. caesiobrunneus, C. albogaudis, C. carabus, and C. cicindela. They are described here and their taxonomy, ecology, distribution, and relationships are discussed. In addition, a key to species of the section Brunnei is provided. A total of 77 new sequences of 11 species are published including nine type sequences. Also the taxonomic assignments of sequences in the public databases belonging to the section Brunnei are revised.     

Chemotaxonomy of Hypericum genus from Portugal: Geographical distribution and essential oils composition of Hypericum perfoliatum, Hypericum humifusum, Hypericum linarifolium and Hypericum pulchrum

The geographical distribution and analysis of the essential oils of species from three sections of Hypericum L. (Guttiferae/Clusiaceae/Hypericaceae) from Portugal are presented. Hypericum perfoliatum (section Drosocarpium) grows wild in the centre and south of Portugal; Hypericum humifusum and Hypericum linarifolium are both from section Oligostema, the former occurring throughout the country, while the second is distributed mainly in the north and centre; Hypericum pulchrum (section Taeniocarpium) is confined to the littoral north of Portugal. The essential oils were obtained by distillation–extraction, hydrodistillation and distillation in a modified Marcusson apparatus from the dried aerial parts of the different populations and were analysed by GC and GC–MS. Monoterpene hydrocarbons constituted the main fraction in all oils (43–69%, 53–85%, 28–45% and 48–65% for H. perfoliatum, H. humifusum, H. linarifolium and H. pulchrum, respectively). Sesquiterpene hydrocarbons (2–13%, 6–18%, 21–27% and 16–18%, respectively) and a third fraction of non-terpenic compounds (20–29%, 3–16%, 2–14% and 5–11%, respectively) from the four species attained relatively high amounts in all oils. Within each species, no major differences were detected in the essential oil composition, despite the fact that different locations, phenological phases and extraction methodologies were used. Notwithstanding the dominance of α-pinene in all four species' oils, cluster and principal components analysis on the identified components showed that the range of α-pinene, β-pinene and n-nonane supported a separation of the four species. The essential oil composition of the four species showed some qualitative resemblances, which correlate well with the taxonomical classification based on morphological characters.     

Nitrogen-containing non-steroidal secondary metabolites of Solanum, Cyphomandra, Lycianthes and Margaranthus

Of the 49 species of Solanum studied, cuscohygrine has been detected in 25, solamine and related amines in 17 and solamine-derived amides in 16. Five species of Cyphomandra examined all contained both amines and amides. From roots of Margaranthus solanaceus cuscohygrine has been isolated which probably occurs, too, in roots of Lycianthes rantonnettii. The distribution of these compounds throughout the taxa could be of chemotaxonomic value.     

The genus Platychara from the Western Hemisphere

The systematics of four species belonging to the genus Platychara (Charophyta) from the Western Hemisphere is discussed. Three of the species, as defined herein, occur in Cretaceous and Paleocene rocks from Mexico through South America. The type species, P. compressa (Peck and Reker) Grambast, also of Cretaceous and Paleocene age, is herein restricted to deposits north of Mexico. These latter restrictions geographically separate P. compressa and P. perlata as presently defined but the relationship between these two species is still uncertain. A new species, P. grambastii, is proposed for specimens from Maestrichtian sediments in Jamaica.     

Systematics and evolution of the genus Torrubiella (Hypocreales, Ascomycota)

Torrubiella is a genus of arthropod-pathogenic fungi that primarily attacks spiders and scale insects. Based on the morphology of the perithecia, asci, and ascospores, it is classified in Clavicipitaceae s. lat. (Hypocreales), and is considered a close relative of Cordyceps s. 1., which was recently reclassified into three families (Clavicipitaceae s. str., Cordycipitaceae, Ophiocordycipitaceae) and four genera (Cordyceps s. str, Elaphocordyceps, Metacordyceps, and Ophiocordyceps). Torrubiella is distinguished morphologically from Cordyceps s. lat. mainly by the production of superficial perithecia and the absence of a well-developed stipitate stroma. To test and refine evolutionary hypotheses regarding the placement of Torrubiella and its relationship to Cordyceps s. lat., a multi-gene phylogeny was constructed by conducting ML and Bayesian analyses. The monophyly of Torrubiella was rejected by these analyses with species of the genus present in Clavicipitaceae, Cordycipitaceae, and Ophiocordycipitaceae, and often intermixed among species of Cordyceps s. lat. The morphological characters traditionally used to define the genus are, therefore, not phylogenetically informative, with the stipitate stromata being gained and/or lost several times among clavicipitaceous fungi. Two new genera (Conoideocrella, Orbiocrella) are proposed to accommodate two separate lineages of torrubielloid fungi in the Clavicipitaceae s. str. In addition, one species is reclassified in Cordyceps s. str. and three are reclassified in Ophiocordyceps. The phylogenetic importance of anamorphic genera, host affiliation, and stipitate stromata is discussed.     

Competition and co-existence of Zoophthora radicans and Pandora blunckii, two co-occurring fungal pathogens of the diamondback moth, Plutella xylostella

The entomopathogenic fungi Zoophthora radicans and Pandora blunckii co-occur in field populations of Plutella xylostella and, therefore, are likely to interact during the infection process. We have investigated the possible outcomes of these interactions in the laboratory. Using four isolates, two of each fungal species, inter-specific interaction experiments were done in Petri dishes and on intact plants. In Petri dish experiments, larvae were inoculated directly using sporulating mats of mycelium, both species had the same opportunity to infect and only the relative concentration of conidia of each pathogen species applied was manipulated. In the intact plant experiments, larvae were placed onto fungus-contaminated plants, inoculation was passive and the probability of infection by either or both species of fungi depended on larval activity and proximity to inoculum. In the Petri dish experiment, the species with the largest concentration of conidia out-competed the other regardless of virulence, and results were similar in the intact plant experiment. The ecological implications for competition or co-existence of these two pathogens in the field are discussed.     

Mycoplasma feriruminatoris sp. nov., a fast growing Mycoplasma species isolated from wild Caprinae

Five Mycoplasma strains from wild Caprinae were analyzed: four from Alpine ibex (Capra ibex) which died at the Berlin Zoo between 1993 and 1994, one from a Rocky Mountain goat collected in the USA prior to 1987. These five strains represented a population different from the populations belonging to the ‘Mycoplasma mycoides cluster’ as tested using multi locus sequence typing, Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis and DNA–DNA hybridization. Analysis of the 16S rRNA gene (rrs), genomic sequence based in silico as well as laboratory DNA–DNA hybridization, and the analysis of phenotypic traits in particular their exceptionally rapid growth all confirmed that they do not belong to any Mycoplasma species described to date. We therefore suggest these strains represent a novel species, for which we propose the name Mycoplasma feriruminatoris sp. nov. The type strain is G5847^T (= DSM 26019^T = NCTC 1362^T).     

Chemical markers in Veronica sect. Hebe. II

In a continued chemosystematic investigation of the water-soluble compounds in Veronica sect. Hebe, four additional species were investigated. In comparison to other, Northern Hemisphere (NH) species of Veronica, those belonging to the New Zealand species in sect. Hebe are apparently more variable in chemical content. In addition to the compounds characteristic for NH Veronica, namely mannitol, aucubin, catalpol and 6-O-esters of catalpol as well as some caffeoyl phenylethanoid glucosides (CGPs), Veronica topiaria (syn. Hebe topiaria) also gave an unusual 6-O-ester of aucubin named topiarioside. The former Hebe species Veronica cupressoides and Veronica stenophylla each provided one of the two previously undescribed disaccharide esters named hebitol I and II, respectively, and the former plant also provided a CPG named cuproside, a 6-O-β-glucopyranosyl derivative of the known hebeoside. The last species, namely Veronica hulkeana (syn. Heliohebe hulkeana) only contained compounds common to other species of Veronica. The taxonomic results are discussed and it is concluded that carbohydrate esters are common in sect. Hebe. The data so far obtained indicate that the occurrences of esters of 6-O-rhamnopyranosylcatalpol are confined to the most derived species in the section.     

Molecular confirmation of natural hybridization between Lumnitzera racemosa and Lumnitzera littorea

The hypothesis of natural hybridization between Lumnitzera racemosa and Lumnitzera littorea, two mangrove species distributed in the Indo-West Pacific region, was proposed in 1970s based on morphological traits; however, no molecular evidence has been reported to support it. In this study, we sequenced two low-copy nuclear genes and one chloroplast intergenic spacer (trnS-trnG) in the two Lumnitzera species and their putative hybrid to test this hypothesis. Our results revealed that there were 9 and 27 nucleotide substitutions at the two nuclear loci, respectively, between one haplotype of L. racemosa and L. littorea, and that the putative hybrid showed additivity in chromatograms at these sites. Sequencing the chloroplast intergenic region trnS-trnG showed that the two Lumnitzera species differed by seven fixed nucleotide substitutions and four fixed insertions/deletions in this region, while the putative hybrid had identical sequences to L. racemosa. Molecular data clearly demonstrated that there indeed existed natural hybridization between L. racemosa and L. littorea and that L. racemosa was the maternal parent in this hybridization event. The uncommon direction of hybridization and F₁ nature of hybrids in this case, and in mangroves in general, is discussed.     

Genetic relationship among Paspalum species of the subgenus Anachyris: Taxonomic and evolutionary implications

Paspalum is one of the most important genera of the Poaceae family due to its large number of species and diversity. The subgenus Anachyris comprises six species mainly from South America grouped together by sharing rare spikelet characteristics. A genetic analysis using ISSR markers, compared with the morphological and phenotypic variation observed in each one species, was used to establish genetic relationships among 40 accessions with several ploidy levels, belonging to 5 species of the subgenus Anachyris. Fourteen accessions of Paspalum malacophyllum (2x and 4x), 12 of P. simplex (2x, 3x, 4x and 6x), 4 of P. procurrens (2x and 4x), 4 of P. usterii (4x) and 6 of P. volcanensis (4x) were analysed. A total of 227 ISSR loci (98.7% polymorphic) were detected among all accessions, with variable loci number and percentages of polymorphism according to species delimitations. Six main groups were identified by cluster analysis based on Jaccard's genetic distance and UPGMA, four of which matched all the respective accessions of P. simplex, P. procurrens, P. usterii and P. volcanensis, while the other two were consistent with two different groups of accessions of P. malacophyllum, one involving most tetraploid accessions, and the other one grouping together a tetraploid and two diploid accessions. The distinctive morphological characteristics and the separate clustering of these tetraploid and diploid cytotypes suggest to consider a new multiploid species complex inside the subgenus Anachyris. Both cytotypes of P. procurrens, and the four co-specific cytotypes of P. simplex consistently clustered together forming two specific groups for the two multiploid taxons. This is in agreement with the existence of high phenotypic similarities between diploid and tetraploid cytotypes of P. procurrens, and among diploid, triploid, tetraploid and hexaploid cytotypes of P. simplex. Since the polyploid cytotypes of these species are reproduced by apomixis, the specific genetic clustering by ISSR markers and morphological and cytological results support the hypothesis that the two multiploid species were originated by autopolyploidy. Our results confirm previous studies suggesting a monophyletic origin for the subgenus Anachyris and are concordant with previous data regarding genomic homologies and phylogenetic analyses in the genus.     

The species of Coleosporium (Pucciniales) on Solidago in North America

Species of Coleosporium (Pucciniales) are rust fungi that typically alternate between pines and angiosperms. In North America, species of Coleosporium often infect Solidago (goldenrods), although their taxonomy on these hosts is unresolved. Joseph. C. Arthur and George B. Cummins regarded these as a single species, Coleosporium solidaginis (fide Arthur) or C. asterum (fide Cummins), but later inoculation studies demonstrated the presence of more than one species, distinguishable by their aecial hosts. A more recent taxonomic study of Coleosporium found that specimens on Solidago identified as C. asterum in North America were not conspecific with the type, which is from Japan, prompting the present study. Herein, we conducted a systematic study on ca. 60 collections of Coleosporium infecting species of Asteraceae from North America using regions of ribosomal DNA and morphology of teliospores and basidia. Our data indicate at least three species of Coleosporium occur on Solidago in North America, C. solidaginis, C. montanum comb. nov., which is proposed for the taxon that has commonly been identified as C. asterum in North America, and C. delicatulum, all of which can be differentiated by morphology of their basidia. In addition, the challenges of marker selection for molecular barcoding of rust fungi is discussed.     

Phylogenetic diversity of Bradyrhizobium strains nodulating Calicotome spinosa in the Northeast of Algeria

Fifty-two slow-growing strains were isolated from root nodules of Calicotome spinosa grown in the Northeast of Algeria and grouped in 24 rep-PCR clusters. One representative strain for each profile was further phylogenetically characterized. The nearly complete 16S rRNA gene sequence indicated that all strains were affiliated to Bradyrhizobium. Multi-Locus Sequence Analysis (MLSA) of the atpD, glnII and recA genes and of the 16S-23S rRNA internal transcribed spacer (ITS) showed that these strains formed four divergent clusters: one close to Bradyrhizobium canariense and Bradyrhizobium lupini and three others separate from all the described species, representing three putative new Bradyrhizobium species. A phylogenetic analysis based on the nodC gene sequence affiliated the strains to either of the two symbiovars, genistearum or retamae.     

Phosphoglucose isomerase and esterase phenotypes in Crassostrea angulata and C. gigas

The phenotype distributions and the allele frequencies of the phosphoglucose isomerase and esterase loci examined in the samples of Crassostrea angulata (Essex, England) and C. gigas (Brittany) do not differ significantly and the two populations as such are indistinguishable. The validity of the species C. angulata is questioned and it is postulated that the two samples may be geographic isolates of the same species, i.e. C. gigas. The hatchery reared population of C. gigas from Conway is distinguishable from the other samples of Crassostrea examined. The lack of phenotype diversity is attributed to founder effects of the small parental stock imported in 1965. The distributions of all phenotypes are in agreement with Hardy-Weinberg expectations. Phosphoglucose isomerase (E.C. 5.3.1.9.) is a dimer governed by at least four alleles in C. angulata and five alleles in C. gigas. The slower (Es-S) zone of the esterase electrophoretogram would appear, in both species to be governed by four alleles at a single locus. There was no esterase banding which was specific to either species of Crassostrea.     

Buddleja davidii and Buddleja yunnanensis: Exploring features associated with commonness and rarity in Buddleja

Buddleja davidii is a widespread shrub in Asia while B. yunnanensis is a narrowly endemic species limited to Yunnan Province, China. To explore whether floral volatiles, morphological characters of flower and seed and breeding system are correlated with their distributions, we measured length and width of corolla, trichome density at corolla throat, level of stigma/anthers relationship, seed size and weight. The results indicated that these characteristics were significantly different between the two species (P < 0.01). Bagging experiments revealed that B. davidii is a self-incompatible plant while B. yunnanensis is self-compatible. Thick trichome density at the corolla throat may reduce out-crossing in B. yunnanensis. Autogamy plays an important role in fruit production of this species while B. davidii requires pollinators for fruiting. Scents were collected using dynamic headspace adsorption method and identified with coupled gas chromatography and mass spectrometry. In total, 27 floral scent compounds were identified. The volatile composition in the two species was very different. We attempted to determine if these features, associated with commonness and with rarity found in these two taxa, could also help to explain the distribution pattern of other species of the genus Buddleja.     

A multiplex real-time PCR method using hybridization probes for the detection and the quantification of Fusarium proliferatum, F. subglutinans, F. temperatum, and F. verticillioides

Maize contamination with Fusarium species is one of the major sources of mycotoxins in food and feed derivates. In the present study, a LightCycler^® real-time PCR method using hybridization probes was developed for the specific identification, detection, and quantification of Fusarium proliferatum, Fusarium subglutinans, Fusarium temperatum, and Fusarium verticillioides, four mycotoxin-producing pathogens of maize. Primers and hybridization probes were designed to target the translation elongation factor 1α (EF-1α) gene of F. subglutinans and F. temperatum or the calmodulin (Cal) gene of F. proliferatum and F. verticillioides. The specificity of the real-time PCR assays was confirmed for the four Fusarium species, giving no amplification with DNA from other fungal species commonly recovered from maize. The assays were found to be sensitive, detecting down to 5 pg and 50 pg of Fusarium DNA in simplex and multiplex conditions respectively, and were able to quantify pg-amounts of Fusarium DNA in artificially Fusarium-contaminated maize samples. The real-time PCR method developed provides a useful tool for routine identification, detection, and quantification of toxigenic Fusarium species in maize.     

Phylogenetic analyses of Bradyrhizobium symbionts associated with invasive Crotalaria zanzibarica and its coexisting legumes in Taiwan

In this study, the genetic diversity and identification of Bradyrhizobium symbionts of Crotalaria zanzibarica, the most widely-distributed invasive legume in Taiwan, and other sympatric legume species growing along riverbanks of Taiwan were evaluated for the first time. In total, 59 and 54 Bradyrhizobium isolates were obtained from C. zanzibarica and its coexisting legume species, respectively. Based on the multilocus sequence analysis (MLSA) of concatenated four housekeeping genes (dnaK-glnII-recA-rpoB gene sequences, 1901 bp), the 113 isolates displayed 53 unique haplotypes and grouped into 21 clades. Of these clades, 11 were found to be congruent to already defined Bradyrhizobium species, while the other 10 clades were found to not be congruent to any defined species. In particular, the C. zanzibarica isolates belong to 14 MLSA clades, six of which overlapped with the isolates of coexisting legumes. According to the nodA gene sequences (555 bp) obtained from the 105 isolates, these isolates were classified into three known nodA clades, III.2, III.3 and VII and were further clustered into 10 groups. Furthermore, the C. zanzibarica isolates were clustered into 8 nodA groups, five of which overlapped with the isolates from coexisting legumes. Additionally, the nodA genes of the isolates from native species were dominated by Asian origin, while those from C. zanzibarica were dominated by American origin. In conclusion, C. zanzibarica is a promiscuous host capable of recruiting diverse Bradyrhizobium symbionts, some of which are phylogenetically similar to the symbionts of coexisting legumes in Taiwan.     

Achromobacter animicus sp. nov., Achromobacter mucicolens sp. nov., Achromobacter pulmonis sp. nov. and Achromobacter spiritinus sp. nov., from human clinical samples

The phenotypic and genotypic characteristics of fourteen human clinical Achromobacter strains representing four genogroups which were delineated by sequence analysis of nusA, eno, rpoB, gltB, lepA, nuoL and nrdA loci, demonstrated that they represent four novel Achromobacter species. The present study also characterized and provided two additional reference strains for Achromobacter ruhlandii and Achromobacter marplatensis, species for which, thus far, only single strains are publicly available, and further validated the use of 2.1% concatenated nusA, eno, rpoB, gltB, lepA, nuoL and nrdA sequence divergence as a threshold value for species delineation in this genus. Finally, although most Achromobacter species can be distinguished by biochemical characteristics, the present study also highlighted considerable phenotypic intraspecies variability and demonstrated that the type strains may be phenotypically poor representatives of the species. We propose to classify the fourteen human clinical strains as Achromobacter mucicolens sp. nov. (with strain LMG 26685^T [=CCUG 61961^T] as the type strain), Achromobacter animicus sp. nov. (with strain LMG 26690^T [=CCUG 61966^T] as the type strain), Achromobacter spiritinus sp. nov. (with strain LMG 26692^T [=CCUG 61968^T] as the type strain), and Achromobacter pulmonis sp. nov. (with strain LMG 26696^T [=CCUG 61972^T] as the type strain).     

A new clade of Mesorhizobium nodulating Alhagi sparsifolia

We isolated 33 nodule bacteria from the legume Alhagi sparsifolia growing in the desert of northwest China. They fell into three groups by restriction analysis of their rrs (small subunit ribosomal RNA) genes, and these, together with dnaK and dnaJ genes, were sequenced from representative isolates to assess their taxonomic position by phylogenetic analysis. The bacteria in each group belonged to different lineages that might represent three different new Mesorhizobium species, two of which form a novel clade very distinct from other species in the genus. Most A. sparsifolia symbionts harboured closely related nodA and nodC genes forming new lineages. The presence of these closely related symbiosis genes in various genomic backgrounds and the incongruence observed between the different gene phylogenies indicate a history of horizontal gene transfer of symbiosis genes between the A. sparsifolia symbionts.     

Bradyrhizobium arachidis sp. nov., isolated from effective nodules of Arachis hypogaea grown in China

Twenty-three bacterial strains isolated from root nodules of Arachis hypogaea and Lablab purpureus grown in five provinces of China were classified as a novel group within the genus Bradyrhizobium by analyses of PCR-based RFLP of the 16S rRNA gene and 16S–23S IGS. To determine their taxonomic position, four representative strains were further characterized. The comparative sequence analyses of 16S rRNA and six housekeeping genes clustered the four strains into a distinctive group closely related to the defined species Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense, Bradyrhizobium huanghuaihaiense, Bradyrhizobium japonicum and Bradyrhizobium daqingense. The DNA–DNA relatedness between the reference strain of the novel group, CCBAU 051107^T, and the corresponding type strains of the five mentioned species varied between 46.05% and 13.64%. The nodC and nifH genes of CCBAU 051107^T were phylogenetically divergent from those of the reference strains for the related species. The four representative strains could nodulate with A. hypogaea and L. purpureus. In addition, some phenotypic features differentiated the novel group from the related species. Based on all the results, we propose a new species Bradyrhizobium arachidis sp. nov. and designate CCBAU 051107^T (=CGMCC 1.12100^T = HAMBI 3281^T = LMG 26795^T) as the type strain, which was isolated from a root nodule of A. hypogaea and had a DNA G + C mol% of 60.1 (Tm).