Phylogenetic and chemical studies in the potential psychotropic species complex of Psilocybe atrobrunnea with taxonomic and nomenclatural notes.

Five Psilocybe species with unresolved systematic position (P. atrobrunnea, P. laetissima, P. medullosa, P. pelliculosa, and P. silvatica) were investigated using four molecular markers (EF1-α, ITS, LSU, and IGS). Phylogenetic analysis revealed that with the exception of P. laetissima, which is now rightfully classified in the genus Leratiomyces, all investigated species belong to Psilocybe sect. Psilocybe. For the first time, psychotropic compounds psilocin and psilocybin were detected in P. medullosa using gas chromatography-mass spectrometry. On the contrary, neither psilocin, nor psilocybin was detected in P. atrobrunnea and negative results were also obtained from mycelia grown in vitro on tryptamine/tryptophan-amended media. These results strongly suggest that biosynthesis of these alkaloids was lost in P. atrobrunnea. With the exception of minor differences detected in EF1-α marker, all sequences of American and European collections of P. atrobrunnea were identical. On the other hand, a thorough nomenclatural study revealed that the name P. atrobrunnea must be considered dubious; the oldest available candidate name, P. fuscofulva, was therefore adopted. The molecular data suggests that morphologically identical American P. silvatica and European P. medullosa likely represent distinct species; epitypes of both taxa were therefore designated.

Trypanosoma avium of raptors (Falconiformes): phylogeny and identification of vectors.

Avian trypanosomes are widespread parasites of birds, the transmission of which remains mostly unclear, with various blood-sucking insects mentioned as possible vectors. A search for vectors of trypanosomes of sparrowhawk (Accipiter nisus), buzzard (Buteo buteo), lesser-spotted eagle (Aquila pomarina) and kestrel (Falco tinnunculus) was performed in Czech and Slovak Republics. Black flies (Eusimulium spp.), hippoboscid flies (Ornithomyia avicularia), mosquitoes (Culex pipiens pipiens) and biting midges (Culicoides spp.), trapped while attempting to feed on raptor nestlings, were found to contain trypanosomatids in their intestine. Trypanosomes from the raptors and blood-sucking insects were isolated, and their 18S rRNA sequences were used for species identification and for the inference of intra- and interspecific relationships. Together with the trypanosome isolated from a black fly, the bird trypanosomes formed a well-supported Trypanosoma avium clade. The isolates derived from hippoboscid flies and mosquitoes are most likely also avian trypanosomes infecting birds other than the studied raptors. Analysis of the kinetoplast, that has features characteristic for the avian trypanosomes (minicircle size; dimensions of the kinetoplast disc), provided further evidence for the identification of vectors. It is suggested that all trypanosomes isolated from raptors included in this study belong to the T. avium complex and are transmitted by the ornithophilic simuliids such as Eusimulium securiforme.

Phylogeny of mitosporic entomopathogenic fungi: is the genus Paecilomyces polyphyletic?

We analyzed sequences of the divergent domain at the 5' end of the large subunit rRNA gene from the mitosporic entomopathogenic fungi Paecilomyces sp., Paecilomyces fumosoroseus, Paecilomyces farinosus, Paecilomyces lilacinus, Verticillium lecanii, Verticillium psalliotae, Beauveria bassiana, Aschersonia sp., Aschersonia placenta, ascomycetous Cordyceps sp., and Cordyceps militaris. Phylogenetic analysis showed P. fumosorseus as the best characterized out of the analyzed species with the B. bassiana clade as its sister group. Two of the P. farinosus isolates were invariably placed within the Verticillium cluster, which also contained C. militaris. The only analyzed P. lilacinus isolate appeared on the root of the hyphomycetous fungi and was characterized as the most distinct from all the hyphomycetous fungi tested. Polyphyly of the genus Paecilomyces was well supported by the Kishino-Hasegawa test. In all trees based on the small subunit rRNA gene sequences obtained from the GenBank, V. lecanii, V. psalliotae, P. fumosoroseus, P. tenuipes and B. bassiana form, together with that of C. militaris, the best supported cluster in the tree. The rest of Cordyceps spp. constitute a distinct clade. Phylogenetic relationships derived from both tested DNA regions show polyphyly of the genus Paecilomyces and close relationships among entomopathogenic species of the genera Verticillium, Paecilomyces, and Beauveria.

Identity of Naegleria strains isolated from organs of freshwater fishes.

Eighteen Naegleria strains were isolated from organs of freshwater fishes belonging to 5 species. Morphometric study allowed the separation of the Naegleria strains from the non-vahlkampfiid amoeboflagellates, but was inadequate for species determination. Six strains, representatives of groups that had a slightly different cyst size, were selected and corresponding derived clones were subjected to sequence analysis and riboprinting restriction fragment length polymorphism (RFLP)-PCR analysis of the small subunit (SSU) rRNA genes. One strain isolated from the brain of a fish with systemic infection was characterised by an intronless 2 kb long SSU rRNA gene and was identified as N. australiensis. Another 5 strains had a 1.3 kb long group I intron in their SSU rRNA gene and, based on the SSU rRNA sequences and riboprints, RFLP-PCR patterns appeared in phylogenetic trees to be closely related to Naegleria clarki.

Phylogenetic position of a renal coccidium of the European green frogs, 'Isospora' lieberkuehni Labbé, 1894 (Apicomplexa: Sarcocystidae) and its taxonomic implications.

'Isospora' lieberkuehni, an unusual isosporoid renal coccidium that parasitizes the European water frog was isolated from the edible frog, Rana kl. esculenta, in the Czech Republic. Sequencing of the small-subunit (SSU) rRNA gene showed that it belongs to the family Sarcocystidae, being closely related to a clade comprising members of the subfamily Toxoplasmatinae. The position within Sarcocystidae correlates with the mode of excystation via collapsible plates as postulated by previous authors. Phylogenetic, morphological and biological differences between 'Isospora' lieberkuehni and the other Stiedabody-lacking members of the genus Isospora justify separation of this coccidium on a generic level. Hyaloklossia Labbé, 1896 is the oldest available synonym and is herein re-erected. The original definition of the genus Hyaloklossia is emended based on recent observations.

Eimeria telekii n.sp. (Apicomplexa: Coccidia) from Lemniscomys striatus (Rodentia: Muridae): morphology, pathology and phylogeny.

Using a combination of morphological, life-cycle and molecular data, we describe a new apicomplexan parasite Eimeria telekii n.sp. from a striped grass mouse Lemniscomys striatus captured in Kenya. Oocysts are oval to spherical or ellipsoidal, 20.4 x 15.7 (15.5-25.0 x 12.0-20.0) microm with a colourless, smooth and bilayered wall. Sporocysts are ellipsoidal, 11.2 x 7.8 (10.0-12.0 x 7.0-9.0) microm with a small Stieda body and granular sporocyst residuum and contain 2 elongated, banana-shaped sporozoites with a single refractile body. Life-cycle, pathogenicity and host specificity of this parasite were studied in laboratory-bred Lemniscomys barbarus and BALB/c mice. Two asexual stages and the sexual phase took place within the enterocytes of the caecum and colon of L. barbarus but not in inoculated BALB/c mice. An infectious dose of 5000 oocysts caused severe clinical illness and mortality in 2/2 (100%) L. barbarus. Phylogenetic analysis of the small subunit rRNA gene of E. telekii and members of the genera Eimeria, Cyclospora and Isospora placed E. telekii within the eimerian rodent clade.

Phylogenetic analysis of Sarcocystis spp. of mammals and reptiles supports the coevolution of Sarcocystis spp. with their final hosts.

Sequences of the small subunit rRNA genes were obtained for two coccidians, Sarcocystis dispersa and an unnamed Sarcocystis sp. which parasitise the European barn owl and an African viperid snake as their final host, respectively, and share mouse as their intermediate host. Phylogenetic analysis of the sequence data showed that Sarcocystis sp. from the viperid snake is most closely related to another Sarcocystis sp. isolated from an American crotalid snake, while S. dispersa grouped with other bird-transmitted species. The available dataset failed to resolve the evolutionary relationships among four major branches into which all Sarcocystidae and Isospora spp. were split. However, within these branches, the phylogenetic relationships of the majority of analysed members of the genus Sarcocystis reflected coevolution with their final, rather than intermediate hosts.