Background: The right sampling of homologous sequences for phylogenetic or molecular evolution analyses is a crucial step, the quality of which can have a significant impact on the final interpretation of the study. There is no single way for constructing datasets suitable for phylogenetic analysis, because this task intimately depends on the scientific question we want to address, Moreover, database mining softwares such as BLAST which are routinely used for searching homologous sequences are not specifically optimized for this task. Results: To fill this gap, we designed BLAST-Explorer, an original and friendly web-based application that combines a BLAST search with a suite of tools that allows interactive, phylogenetic-oriented exploration of the BLAST results and flexible selection of homologous sequences among the BLAST hits. Once the selection of the BLAST hits is done using BLAST-Explorer, the corresponding sequence can be imported locally for external analysis or passed to the phylogenetic tree reconstruction pipelines available on the Phylogeny.fr platform. Conclusions: BLAST-Explorer provides a simple, intuitive and interactive graphical representation of the BLAST results and allows selection and retrieving of the BLAST hit sequences based a wide range of criterions. Although BLAST-Explorer primarily aims at helping the construction of sequence datasets for further phylogenetic study, it can also be used as a standard BLAST server with enriched output. BLAST-Explorer is available at http://www.phylogeny.fr

In an effort to establish a suitable alternative to the widely used 18S rRNA system for molecular systematics of fungi, we examined the nuclear gene RPB2, encoding the second largest subunit of RNA polymerase II. Because RPB2 is a single-copy gene of large size with a modest rate of evolutionary change, it provides good phylogenetic resolution of Ascomycota. While the RPB2 and 18S rDNA phylogenies were highly congruent, the RPB2 phylogeny did result in much higher bootstrap support for all the deeper branches within the orders and for several branches between orders of the Ascomycota. There are several strongly supported phylogenetic conclusions. The Ascomycota is composed of three major lineages: Archiascomycetes, Saccharomycetales, and Euascomycetes. Within the Euascomycetes, plectomycetes, and pyrenomycetes are monophyletic groups, and the Pleosporales and Dothideales are distinct sister groups within the Loculoascomycetes. We confirm the placement of Neolecta within the Archiascomycetes, suggesting that fruiting body formation and forcible discharge of ascospores were characters gained early in the evolution of the Ascomycota. These findings show that a slowly evolving protein-coding gene such as RPB2 is useful for diagnosing phylogenetic relationships among fungi.

All plants in natural ecosystems appear to be symbiotic with fungal endophytes. This highly diverse group of fungi can have profound impacts on plant communities through increasing fitness by conferring abiotic and biotic stress tolerance, increasing biomass and decreasing water consumption, or decreasing fitness by altering resource allocation. Despite more than 100 yr of research resulting in thousands of journal articles, the ecological significance of these fungi remains poorly characterized. Historically, two endophytic groups (clavicipitaceous (C) and nonclavicipitaceous (NC)) have been discriminated based on phylogeny and life history traits. Here, we show that NC-endophytes represent three distinct functional groups based on host colonization and transmission, in planta biodiversity and fitness benefits conferred to hosts. Using this framework, we contrast the life histories, interactions with hosts and potential roles in plant ecophysiology of C- and NC-endophytes, and highlight several key questions for future work in endophyte biology.

A new pleosporalean family Tetraplosphaeriaceae is established to accommodate five new genera; 1) Tetraplosphaeria with small ascomata and anamorphs belonging to Tetraploa s. str., 2) Triplosphaeria characterised by hemispherical ascomata with rim-like side walls and anamorphs similar to Tetraploa but with three conidial setose appendages, 3) Polyplosphaeria with large ascomata surrounded by brown hyphae and anamorphs producing globose conidia with several setose appendages, 4) Pseudotetraploa, an anamorphic genus, having obpyriform conidia with pseudosepta and four to eight setose appendages, and 5) Quadricrura, an anamorphic genus, having globose conidia with one or two long setose appendages at the apex and four to five short setose appendages at the base. Fifteen new taxa in these genera mostly collected from bamboo are described and illustrated. They are linked by their Tetraploa s. l. anamorphs. To infer phylogenetic placement in the Pleosporales, analyses based on a combined dataset of small- and large-subunit nuclear ribosomal DNA (SSU+LSU nrDNA) was carried out. Tetraplosphaeriaceae, however, is basal to the main pleosporalean clade and therefore its relationship with other existing families was not completely resolved. To evaluate the validity of each taxon and to clarify the phylogenetic relationships within this family, further analyses using sequences from ITS-5.8S nrDNA (ITS), transcription elongation factor 1-alpha (TEF), and beta-tubulin (BT), were also conducted. Monophyly of the family and that of each genus were strongly supported by analyses based on a combined dataset of the three regions (ITS+TEF+BT). Our results also suggest that Tetraplosphaeria (anamorph: Tetraploa s. str.) is an ancestral lineage within this family. Taxonomic placement of the bambusicolous fungi in Astrosphaeriella, Kalmusia, Katumotoa, Massarina, Ophiosphaerella, Phaeosphaeria, Roussoella, Roussoellopsis, and Versicolorisporium, are also discussed based on the SSU+LSU phylogeny.

Detailed restriction analyses of many samples often require substantial amounts of time and effort for DNA extraction, restriction digests, Southern blotting, and hybridization. We describe a novel approach that uses the polymerase chain reaction (PCR) for rapid simplified restriction typing and mapping of DNA from many different isolates. DNA fragments up to 2 kilobase pairs in length were efficiently amplified from crude DNA samples of several pathogenic Cryptococcus species, including C. neoformans, C. albidus, C. laurentii, and C. uniguttulatus. Digestion and electrophoresis of the PCR products by using frequent-cutting restriction enzymes produced complex restriction phenotypes (fingerprints) that were often unique for each strain or species. We used the PCR to amplify and analyze restriction pattern variation within three major portions of the ribosomal DNA (rDNA) repeats from these fungi. Detailed mapping of many restriction sites within the rDNA locus was determined by fingerprint analysis of progressively larger PCR fragments sharing a common primer site at one end. As judged by PCR fingerprints, the rDNA of 19 C. neoformans isolates showed no variation for four restriction enzymes that we surveyed. Other Cryptococcus spp. showed varying levels of restriction pattern variation within their rDNAs and were shown to be genetically distinct from C. neoformans. The PCR primers used in this study have also been successfully applied for amplification of rDNAs from other pathogenic and nonpathogenic fungi, including Candida spp., and ought to have wide applicability for clinical detection and other studies.