Molecular characterization of Embellisia and Nimbya species and their relationship to Alternaria, Ulocladium and Stemphylium

DNA sequences from rDNA and protein-coding regions were determined for six Embellisia and two Nimbya spp. and were compared to those from Alternaria, Ulocladium and Stemphylium spp. Sequences determined included rDNA from the nuclear internal transcribed-spacer region (ITS1/5.8S/ITS2) and the mitochondrial small-subunit (mt SSU) and a portion of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene. Phylogenetic analyses were performed on each dataset separately and then combined for total evidence analysis using methods of maximum parsimony and maximum likelihood. Results revealed that Embellisia and Nimbya clustered within a large monophyletic Alternaria-Nimbya-Embellisia-Ulocladium clade with Stemphylium as the sister taxon. Members of the infectoria species-group were the most basal group in this large polygeneric clade. Embellisia and Nimbya were sister taxa of the remaining Alternaria and Ulocladium spp. and were related more closely to Alternaria than was Stemphylium. Four Embellisia spp. formed a monophyletic clade. However, E. allii clustered with the two Nimbya spp. and E. indefessa clustered with Alternaria and Ulocladium spp., revealing that Embellisia, as currently circumscribed, is polyphyletic. Potential revisions of taxonomy for all genera are discussed.     

Sinomyces: a new genus of anamorphic Pleosporaceae

Sinomyces gen. nov. is described for Ulocladium alternariae and two new species from China. These three species differ from Ulocladium in producing simple conidiophores with a single, cicatrized apical pore or 1 or 2 short, uniperforate, geniculate sympodial proliferations. Phylogenetic analysis of partial DNA sequences of the glyceraldehyde-3-phosphate dehydrogenase?gene (gpd) gene and a fragment of the Alternaria alternata major allergen (Alt a 1) gene, using Maximum-Parsimony (MP), Maximum-Likelihood (ML) and Bayesian approaches, indicates that Sinomyces is distinct within the Alternaria-Stemphylium complex, although its closest relatives could not be determined. Sinomyces alternariae comb. nov. and the new species Sinomyces obovoideus and Sinomyces fusoideus are proposed.     

Sensitivity of the Alternaria infectoria species-group 1 to cycloheximide

Aims:  A possibility of using cycloheximide tolerance and/or sensitivity as an additional diagnostic tool for distinguishing morphologically related species within common small-spored Alternaria has been tested during this study.
Methods and Results:  A total of 33 strains from four Alternaria species-groups, namely Alternaria alternata , Alternaria arborescens , Alternaria infectoria and Alternaria tenuissima were tested for their growth response to 100 μg m⁻¹ cycloheximide in potato carrot agar. All A. infectoria strains were completely inhibited, showing no growth at all even after prolonged incubation. In contrast, all other strains representing the remnant three species exhibited a high resistance to this antibiotic.
Conclusions:  Cycloheximide sensitivity represents a further important physiological character for distinguishing A. infectoria from the three similar species.
Significance and Impact of the Study:  The relevance of these findings corresponds with the potential ability of the Alternaria species produce mycotoxins. Cycloheximide may be in future used in the design of selective media for the isolation of some potentially toxigenic food-borne Alternaria species such as A. alternata , A. tenuissima and/or A. arborescens , for example in screening cereals for toxigenic Alternaria spp. and for their direct separation from nontoxigenic representatives of A. infectoria species-group.     

A re-examination of the phylogenetic relationship between the causal agents of carrot black rot, Alternaria radicina and A. carotiincultae

The phylogenetic relationship between Alternaria radicina and A. carotiincultae was reexamined based on morphology, sequence analysis of rDNA (ITS and mitochondrial small subunit [mtSSU]), protein coding genes (actin [ACT], beta-tubulin, chitin synthase [CHS], translation elongation factor [EF-1a], Alternaria allergen a1 [Alt a1], and glyceraldehyde-3-phosphate dehydrogenase [gpd]), and RAPD and ISSR analysis of total genomic DNA. Although some morphological characters overlapped to a degree, with A. radicina isolates expressing moderate variation and A. carotiincultae isolates being highly uniform, A. carotiincultae could be differentiated from A. radicina based on significantly greater growth rate on potato dextrose agar (PDA) or acidified PDA (APDA) and average number of transverse septa per conidium. Sequence of rDNA and two protein coding genes, ACT and CHS, were invariant between species. However polymorphism with the EF-1a, beta-tubulin, and Alt a1 gene strictly separated the population of A. radicina and A. carotiincultae as distinct lineages, as did RAPD and ISSR analysis. The polymorphic gpd gene did not strictly separate the two species. However isolates of A. radicina encompassed several haplotypes, one of which was the exclusive haplotype possessed by A. carotiincultae isolates, suggesting evidence of incomplete lineage sorting. The results suggest that A. carotiincultae is closely related to A. radicina but is a recently divergent and distinct lineage, which supports its status as a separate species.     

A new Alternaria species from grapevine in China

Twelve Alternaria strains were isolated from pedicels and rachis from different parts of grapevines in China. In a phylogenetic analysis, nine showed a close relationship with Alternaria longipes, and are described as a new species, A. viniferae sp. nov., but the other four clustered with A. alternata and A. arborescens within the alternata species-group. The new species can clearly be separated from other related small-spored Alternaria species based on sequences of portions of the glyceraldehyde-3-phosphate dehydrogenase (gpd) and Alternaria major allergen (Alt a 1) genes as well as by distinctive morphology.     

Morphometric and molecular analysis of the Encarsia inaron species-group (Hymenoptera: Aphelinidae), parasitoids of whiteflies (Hemiptera: Aleyrodidae)

Several series of host-reared specimens of an Encarsia species, initially thought to be the cosmopolitan Encarsia inaron (Walker), were collected in the Azores Islands (Portugal). Subsequent morphometric analysis supported the presence of two species: E. inaron and a new species, described herein as Encarsia estrellae Manzari & Polaszek sp. n. Encarsia estrellae was reared from Aleyrodes singularis Danzig, A. ?singularis, and Bemisia sp. afer-group on several host plants. In addition, the D2 region of the 28S rDNA gene was sequenced in eight individuals belonging to these species, as well as single representatives of two closely related and one distantly related species. Phylogenetic analysis of these DNA sequences, together with 23 additional Encarsia sequences retrieved from the European Molecular Biology Laboratory (EMBL) and GenBank databases, further supported the specific status of E. estrellae, and the placement of E. dichroa (Mercet) in the E. inaron species-group. Additionally, E. inaron is redescribed and some taxonomic problems in the E. inaron species-group are discussed.     

小孢子链格孢endoPG基因核苷酸序列分析及系统发育研究

对供试小孢子链格孢菌株的内聚半乳糖醛酸酶(endoPG)基因进行扩增,大部分菌株都可获得PCR产物。核苷酸和氨基酸序列比较表明:不同种小孢子链格孢endoPG基因核苷酸序列存在明显差异,甚至表现在氨基酸水平,这些差异可以作为一些种如梨黑斑链格孢、长柄链格孢区分的分子性状。利用邻近结合法构建系统发育树,所有菌株被分为8个聚类组。在系统发育树上,链格孢的一些不同分离物被聚在不同组中,而细极链格孢、链格孢的部分菌株、苹果链格孢、柑橘链格孢、粗柠檬褐斑链格孢、橘树链格孢被聚为一组,显示根据形态学特征划分的这些种与分子性状的不一致性。endoPG基因核苷酸序列富于变化,为小孢子链格孢系统发育研究提供了一种有用的手段。     

Common names for Australian ants (Hymenoptera: Formicidae)

Abstract Most insects do not have common names, and this is a significant barrier to public interest in them, and to their study by non-specialists. This holds for even highly familiar insect groups such as ants. Here, I propose common names for all major native Australian ant genera and species-groups, as well as for many of the most abundant and distinctive species. Sixty-two genera, 142 species-groups and 50 species are given names. The naming system closely follows taxonomic structure; typically a genus is given a general common name, under which species-group and species names are nested.     

New genes for phylogenetic studies of lichenized fungi: glyceraldehyde-3-phosphate dehydrogenase and beta-tubulin genes

Abstract:Primers for amplification and sequencing of partial glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were designed for lichenized fungi. The 5′ gpd primer is most probably fungal specific, since a BLAST search in GenBank found identical sequences only from ascomycetous taxa, whereas the 3′ gpd primer was more universal. Utility of the gpd primers and previously designed beta-tubulin primers was tested in nine lichen taxa. Both the gpd and beta-tubulin primer pairs amplified in most of the taxa examined: the gpd primers generated a c. 1100 nucleotide fragment, whereas the PCR product obtained from the beta-tubulin primers was c. 900 nucleotides long. The gpd amplification products of Cladonia arbuscula and C. rangiferina were sequenced and both were found to contain three introns, the length of which varied between 49 to 83 nucleotides. To examine the applicability of gpd sequences in resolving relationships within Ascomycota, trees were calculated from 22 fungal gpd sequences obtained from GenBank together with the twoCladonia sequences using parsimony jackknifing. The gpd tree was compared with the SSU rDNA tree of the respective species (or genera). A similar analysis of the beta-tubulin gene was not performed, because only a few beta-tublin sequences from the same taxa were available in GenBank. The gpd tree was well resolved but in conflict with the SSU rDNA tree. In contrast to the SSU rDNA tree, the gpd tree did not support the monophyly of the Ascomycota. Analysis of the combined data set produced a tree very similar to that of the SSU rDNA data. However, the relationship of Lecanorales to the other orders remained unresolved. Even though gpd and beta-tubulin are highly conserved proteins, the third codon positions and introns are variable and both genes have the potential for inferring phylogenetic relationships at the lower taxonomic levels in the lichenized fungi. The two genes may be useful even below species level, depending on the species investigated.     

Molecular characterization, morphology, and pathogenicity of Alternaria panax from araliaceous plants in Korea

Alternaria blight on araliaceous plants is a common disease caused by Alternaria panax Whetzel and occurs worldwide. Genetic diversity among 58 isolates of A. panax from different araliaceous hosts in Korea was determined by amplified fragment length polymorphism (AFLP) analysis. Selected isolates from genetic groups (determined by AFLP analysis) were examined based on the results of phylogenetic analyses of eight genes (Alt a1, BT1, BT2, EF-1α, gpd, H3, ITS, and RPB2), morphological characteristics, and pathogenicity tests. Isolates were divided into two distinct genetic groups (A and B) by AFLP analysis and based on the results of sequence analyses of the BT1, BT2, gpd, and RPB2 genes. Isolates from ginseng plants (Panax ginseng and P. quinquefolius) fell into group B, whereas isolates from the other araliaceous plants clustered in group A. Morphologically, although some overlap was observed among isolates in the two groups, isolates in group B had longer and narrower conidia, distinct sporulation patterns at 15 °C, and did not secrete pigment into PDA media. Pathogenicity tests revealed that isolates in groups A and B only induced severe disease symptoms on leaves of their hosts, such as isolates in group A on Aralia elata, Aralia continentalis, and in group B on Panax ginseng. The results indicate that the two genetic groups of Alternaria from araliaceous plants should be considered as two different species, and we propose that group A is a candidate of new Alternaria species distinguished from A. panax.     

Molecular and morphological description of a new species of Ulocladium from Southern China

A new species of Ulocladium was discovered from diseased leaves of Lycopersicon esculentum and Duchesnea indica from Hunan Province of China. Morphologically, this species is very close to U. consortiale, U. cucurbitae, and U. subcucurbitae in producing narrow ellipsoid conidia at 1–3 days, but the conidial size range of this species at this stage could distinguish it from three well-known species. It also exhibits the multiplex conidium morphology at the different growth-stages (1–3 days and 4–7 days). The results of maximum parsimony (MP) and neighbor-joining (NJ) phylogenetic analyses of combined glyceraldehyde-3-phosphate dehydrogenase (gpd) gene and Alternaria alternata major allergen (Alt a 1) genes show that U. solani and U. subcucurbitae cluster in a unique and separate subclade with no clear affinities to a specific sistergroup, and demonstrate that the Ulocladium species group is monphyletic, but two clades of this section are recognized. Morphological features of this new species, the sequences of the Alt a 1 and gpd gene regions, and its comparison with related species in this genus are discussed.     

Free from mitochondrial DNA: Nuclear genes and the inference of species trees among closely related darter lineages (Teleostei: Percidae: Etheostomatinae)

Investigations into the phylogenetics of closely related animal species are dominated by the use of mitochondrial DNA (mtDNA) sequence data. However, the near-ubiquitous use of mtDNA to infer phylogeny among closely related animal lineages is tempered by an increasing number of studies that document high rates of transfer of mtDNA genomes among closely related species through hybridization, leading to substantial discordance between phylogenies inferred from mtDNA and nuclear gene sequences. In addition, the recent development of methods that simultaneously infer a species phylogeny and estimate divergence times, while accounting for incongruence among individual gene trees, has ushered in a new era in the investigation of phylogeny among closely related species. In this study we assess if DNA sequence data sampled from a modest number of nuclear genes can resolve relationships of a species-rich clade of North American freshwater teleost fishes, the darters. We articulate and expand on a recently introduced method to infer a time-calibrated multi-species coalescent phylogeny using the computer program ^*BEAST. Our analyses result in well-resolved and strongly supported time-calibrated darter species tree. Contrary to the expectation that mtDNA will provide greater phylogenetic resolution than nuclear gene data; the darter species tree inferred exclusively from nuclear genes exhibits a higher frequency of strongly supported nodes than the mtDNA time-calibrated gene tree.     

A polyphasic approach for the characterization of endophytic Alternaria strains isolated from grapevines

A polyphasic approach was set up and applied to characterize 20 fungal endophytes belonging to the genus Alternaria, recovered from grapevine in different Italian regions.Morphological, microscopical, molecular and chemical investigations were performed and the obtained results were combined in a pooled cluster analysis. Following morphological analyses, all strains were grouped according to their three-dimensional sporulation pattern on PCA and to the colony characteristics on different substrates. After DNA extraction, all strains were analyzed by RAPD-PCR and the resulting profiles were subjected to cluster analysis. The metabolites extracted from the 20 Alternaria endophytes were analyzed by a HPLC and the resulting metabolite profiles were subjected to multivariate statistic analyses. In comparison with reference ‘small-spored’ Alternaria species, the 20 strains were segregated into two morphological groups: one belonging to the A. arborescens species-group and a second to the A. tenuissima species-group. RAPD analysis also showed that grapevine endophytes belonged to either the A. arborescens or the A. tenuissima species-group and that they were molecularly distinct from strains belonging to A. alternata. Chemotaxonomy gave the same grouping: the grapevine endophytic strains belong to A. arborescens or A. tenuissima species-groups producing known metabolites typical of these species-groups. Interestingly, the 20 grapevine endophytes were able to produce also a number of unknown metabolites, whose characterization could be useful for a more precise segregation of the two species-groups.The results show how complementary morphological, molecular and chemical data can clarify relationships among endophyte species-groups of low morphological divergence.     

Out-of-Africa again: A phylogenetic hypothesis of the genus Charaxes (Lepidoptera: Nymphalidae) based on five gene regions

Despite the long popularity of Charaxes among collectors and researchers, their evolutionary history is largely unknown. The current and accepted species groupings and relationships within the genus are based exclusively on adult morphology and life histories. Here, we examine the monophyly and evolutionary affinities of the species-groups within the genus Charaxes and explore how they relate to members of their closest genera (Euxanthe, Polyura and Palla) using 4167 bp of sequence data from five (1 mitochondrial and 4 nuclear) gene regions. Within the proposed phylogenetic framework, we estimate ages of divergence within the genus and also reconstruct their historical biogeography. We included representatives of all known species-groups in Africa and Asia, all known species of Euxanthe and Palla and two exemplar species of Polyura. We found the genus Charaxes to be a paraphyletic group with regard to the genera Polyura and Euxanthe, contrary to the earlier assumption of monophyly. We found that 13 out of 16 morphologically defined species-groups with more than one species were strongly supported monophyletic clades. Charaxes nichetes is the sister group to all the other Charaxes. Polyura grouped with the Zoolina and Pleione species-groups as a well-supported clade, and Euxanthe grouped with the Lycurgus species-group. Our results indicated that the common ancestor of Charaxes diverged from the common ancestor of Palla in the mid Eocene (45 million years ago) in (Central) Africa and began diversifying to its extant members 15 million years later. Most of the major diversifications within the genus occurred between the late Oligocene and Miocene when the global climates were putatively undergoing drastic fluctuations. A considerable number of extant species diverged from sister species during the Pliocene. A dispersal–vicariance analysis suggests that many dispersal rather than vicariance events resulted in the distribution of the extant species. The genus Polyura and the Indo-Australian Charaxes are most likely the results of three independent colonizations of Asia by African Charaxes in the Miocene. We synonymize the genera Polyura (syn. nov.) and Euxanthe (syn. nov.) with Charaxes, with the currently circumscribed Charaxes subdivided into five subgenera to reflect its phylogeny.     

Characterization of Alternaria isolates from the infectoria species-group and a new taxon from Arrhenatherum,Pseudoalternaria arrhenatheria sp. nov.

The infectoria species-group within the genus Alternaria was originally conceived by Simmons in 1993 and was based upon common morphological characteristics that included the development of conidial chains with primary, secondary, and tertiary branching resulting in substantial three-dimensional complexity. These characters can overlap to varying degrees with numerous taxa in another Alternaria group, the alternata species-group, making species-group differentiation difficult. However, members of the infectoria species-group are also distinguished from other small-spored Alternaria species based upon colony characteristics that typically include white or nearly white floccose colonies on DRYES medium and clumps of sporulation islands on low sugar media such as V8 agar, PCA, and weak PDA. In addition, the infectoria species-group contains representatives that are known to produce teleomorphs (Lewia), whereas the members of the alternata species-group and other Alternaria species-groups are strictly asexual. In this study, an assemblage of isolates recovered from varied hosts from the west coast of the United States were examined based upon morphological characters and compared to previously described members of the infectoria species-group. These isolates and members of the infectoria species-group typically produce arachnoid vegetative hyphae with multiple primary conidiophores, whereas other small-spored Alternaria species produce primary conidiophores predominately directly from the agar surface. Additionally, molecular phylogenetic analyses resolved these isolates and members of the infectoria species-group as distinctly nested amongst other sexual taxa in Allewia (Embellisia anamorph) and Macrospora (Nimbya anamorph) and phylogenetically distant to asexual lineages of Alternaria. One taxon among these isolates was novel and clustered with the asexual A. rosae in a distinct clade basal to all other members of the infectoria species-group. A new genus is proposed, Pseudoalternaria gen. nov. and a new taxon is described, Pseudoalternaria arrhenatheria sp. nov.. Moreover, a second taxon is reclassified, Pseudoalternaria rosae comb. nov.     

Phylogenetic relationships of five Oriental Apanteles species-groups (Hymenoptera: Braconidae: Microgastrinae) by concatenating four molecular markers

Genus Apanteles is a cosmopolitan and largest genus in Microgastrinae that can be set as chief example of the subfamily to be studied for. However, highly similar morphological characters intraspecifically lead to difficulty in identifying this genus. In this study, 25 individuals of Apanteles were identified morphologically and molecularly by using error rates approach of barcoding analysis. The phylogenetic of the Apanteles that consisted of five species-groups were studied together with their molecular time divergences by using four molecular markers i.e., COI, 16S, 28S and Cyt B. The species groups are mycetophilus, ultor, merula, merope and ater. The phylogenetic analyses show that each species groups were monophyletic with formation of sister clad for merula and merope-group. The phylogenetic analyses of Apanteles species-group were well resolved and supported by their morphological and taxonomical status. This study is a first record on the phylogenetic of five Oriental Apanteles species-group.     

Alt a 1 from Alternaria interacts with PR5 thaumatin-like proteins

Alt a 1 is a protein found in Alternaria alternata spores related to virulence and pathogenicity and considered to be responsible for chronic asthma in children. We found that spores of Alternaria inoculated on the outer surface of kiwifruits did not develop hyphae. Nevertheless, the expression of Alt a 1 gene was upregulated, and the protein was detected in the pulp where it co-localized with kiwi PR5. Pull-down assays demonstrated experimentally that the two proteins interact in such a way that Alt a 1 inhibits the enzymatic activity of PR5. These results are relevant not only for plant defense, but also for human health as patients with chronic asthma could suffer from an allergic reaction when they eat fruit contaminated with Alternaria.