Morphology and multigene phylogeny reveal three new species of Samsoniella (Cordycipitaceae, Hypocreales) from spiders in China.

The genus Samsoniella was erected based on orange cylindrical to clavate stromata, superficial perithecia and conidiophores with Isaria-like phialides and to segregate them from the Akanthomyces group. In this study, based on morphological features and multigene (SSU, LSU, TEF, RPB1 and RPB2) phylogenetic analysis six Samsoniella species parasitizing spiders were collected in China. Three of them belong to known species S.alpina, S.erucae and S.hepiali. Three new species S.anhuiensissp. nov., S.araneasp. nov. and S.fusiformisporasp. nov. are illustrated and described. They are clearly distinct from other species in Samsoniella occurring in independent subclades. Furthermore, among the four insect-pathogenic fungi specimens collected from similar sites, three of them were identified as the new species described below. Our study significantly broadens the host range of Samsoniella from Insecta to Arachnida, marking a noteworthy expansion in understanding the ecological associations of these fungi. Additionally, the identification of both mononematous and synnematous conidiophores in our study not only expands the knowledge of Samsoniella species but also provides a basis for future research by comparing the ecological significance between these conidiophore types. In conclusion, our study enhances the understanding of Samsoniella diversity, presenting a refined phylogenetic framework and shedding light on the ecological roles of these fungi in spider parasitism.

Behavioral mechanisms and morphological symptoms of zombie ants dying from fungal infection.

BACKGROUND: Parasites that manipulate host behavior can provide prominent examples of extended phenotypes: parasite genomes controlling host behavior. Here we focus on one of the most dramatic examples of behavioral manipulation, the death grip of ants infected by Ophiocordyceps fungi. We studied the interaction between O. unilateralis s.l. and its host ant Camponotus leonardi in a Thai rainforest, where infected ants descend from their canopy nests down to understory vegetation to bite into abaxial leaf veins before dying. Host mortality is concentrated in patches (graveyards) where ants die on sapling leaves ca. 25 cm above the soil surface where conditions for parasite development are optimal. Here we address whether the sequence of ant behaviors leading to the final death grip can also be interpreted as parasite adaptations and describe some of the morphological changes inside the heads of infected workers that mediate the expression of the death grip phenotype. RESULTS: We found that infected ants behave as zombies and display predictable stereotypical behaviors of random rather than directional walking, and of repeated convulsions that make them fall down and thus precludes returning to the canopy. Transitions from erratic wandering to death grips on a leaf vein were abrupt and synchronized around solar noon. We show that the mandibles of ants penetrate deeply into vein tissue and that this is accompanied by extensive atrophy of the mandibular muscles. This lock-jaw means the ant will remain attached to the leaf after death. We further present histological data to show that a high density of single celled stages of the parasite within the head capsule of dying ants are likely to be responsible for this muscular atrophy. CONCLUSIONS: Extended phenotypes in ants induced by fungal infections are a complex example of behavioral manipulation requiring coordinated changes of host behavior and morphology. Future work should address the genetic basis of such extended phenotypes.

Samuelsia mundiveteris sp. nov. from Thailand.

Samuelsia mundiveteris is described as the first member of the genus from the Old World tropics.

Pyridone and tetramic acid alkaloids from the spider pathogenic fungus Torrubiella sp. BCC 2165.

Torrubiellones A-D (1-4), new pyridone and tetramic acid alkaloids, were isolated from the spider pathogenic fungus Torrubiella sp. BCC 2165. Torrubiellone A (1) exhibited antimalarial activity with an IC(50) value of 8.1 µM, while it showed very weak cytotoxic activity.

The life of a dead ant: the expression of an adaptive extended phenotype.

Specialized parasites are expected to express complex adaptations to their hosts. Manipulation of host behavior is such an adaptation. We studied the fungus Ophiocordyceps unilateralis, a locally specialized parasite of arboreal Camponotus leonardi ants. Ant-infecting Ophiocordyceps are known to make hosts bite onto vegetation before killing them. We show that this represents a fine-tuned fungal adaptation: an extended phenotype. Dead ants were found under leaves, attached by their mandibles, on the northern side of saplings approximately 25 cm above the soil, where temperature and humidity conditions were optimal for fungal growth. Experimental relocation confirmed that parasite fitness was lower outside this manipulative zone. Host resources were rapidly colonized and further secured by extensive internal structuring. Nutritional composition analysis indicated that such structuring allows the parasite to produce a large fruiting body for spore production. Our findings suggest that the osmotrophic lifestyle of fungi may have facilitated novel exploitation strategies.

Isariotins E and F, spirocyclic and bicyclic hemiacetals from the entomopathogenic fungus Isaria tenuipes BCC 12625.

New spirocyclic and bicyclic hemiacetals, isariotins E (1) and F (2), together with TK-57-164A (3) were isolated from the entomopathogenic fungus Isaria tenuipes BCC 12625. The absolute configuration of 3 was addressed by application of the modified Mosher's method. Isariotin F (2) exhibited activity against the malaria parasite Plasmodium falciparum K1 with an IC(50) value of 5.1 microM and cytotoxic activities against cancer cell lines (KB, BC, and NCI-H187) and nonmalignant (Vero) cells with respective IC(50) values of 15.8, 2.4, 1.6, and 2.9 microM.

A beauvericin hot spot in the genus Isaria.

Beauvericin is a naturally occurring cyclohexadepsipeptide originally described from Beauveria bassiana but also reported from several Fusarium species as well as members of the genus Isaria. Twenty-six isolates of Isaria species and its Cordyceps teleomorph, and ten taxonomically close strains including Beauveria, Nomuraea and Paecilomyces species were sequenced and tested for beauvericin production. Trees using ITS rDNA and beta-tubulin sequence data were constructed and used to infer the phylogenetic distribution of beauvericin production. A group comprising Isaria tenuipes and its known teleomorph Cordyceps takaomontana, Isaria cicadae and its Cordyceps teleomorph, Isaria japonica and Isaria fumosorosea, showed positive beauvericin production which correlated well with combined ITS rDNA and beta-tubulin phylogenies. The results suggested that beauvericin can serve as a chemotaxonomic marker for these limited species of the I. tenuipes complex.

Hopane triterpenes as chemotaxonomic markers for the scale insect pathogens Hypocrella s. lat. and Aschersonia.

The scale insect pathogens Hypocrella s. lat. and their Aschersonia anamorphs, collected at various locations in Thailand, were surveyed for their productivity of three hopane triterpenes, zeorin (6alpha,22-dihydroxyhopane), dustanin (15alpha,22-dihydroxyhopane), and 3beta-acetoxy-15alpha,22-dihydroxyhopane, when cultured in a liquid medium (potato-dextrose broth) under static conditions. Among 53 strains of Aschersonia species, 48 strains (91 %) produced at least one of these compounds. Hypocrella and Moelleriella species (43 strains) also frequently produced these triterpenoids; only two strains lacked all of these triterpenes. The results demonstrate that hopane triterpenes may be suitable for use as chemotaxonomic markers for Hypocrella and Moelleriella species and their Aschersonia anamorphs.

A combined ITS rDNA and beta-tubulin phylogeny of Thai species of Hypocrella with non-fragmenting ascospores.

A combined ITS and beta-tubulin gene phylogeny has revealed new species of Hypocrella and Aschersonia related to the type species Hypocrella discoidea from natural forest in Thailand. As a result, Hypocrella calendulina and Hypocrella luteola are named as new species with Aschersonia sensu stricto anamorphs for specimens previously identified as Hypocrella discoidea sensu Petch. Hypocrella siamensis and Aschersonia minutispora are described as new species, both exhibiting brown stromata, with the former producing whole ascospores.

Comparison of the bioactive secondary metabolites from the scale insect pathogens, Anamorph Paecilomyces cinnamomeus, and Teleomorph Torrubiella luteorostrata.

A scale insect pathogen Paecilomyces cinnamomeus BCC 9616 and its teleomorph Torrubiella luteorostrata BCC 9617, collected on the same host specimen, were fermented and chemically explored. Both fungi produced paecilodepsipeptide A (1) and zeorin (4) as major constituents of mycelia extracts. The culture broth extract of BCC 9616 provided a known diketopiperazine, terezine D (5), and a new xanthone glycoside, norlichexanthone-6-O-beta-(4-O-methylglucopyranoside) (6). On the other hand, the broth extract of BCC 9617 contained small amounts of a new naphthopyrone glycoside, rubrofusarin-6-O-beta-(4-O-methylglucopyranoside) (7) along with 5. Structures of the new compounds, 6 and 7, were elucidated by interpretation of NMR and mass spectroscopic data. The overall results demonstrated that the metabolite profiles of the cultured anamorph (BCC 9616) and teleomorph (BCC 9617) originating from the same host specimen resemble each other closely. The (1)H-NMR spectroscopic analysis of the culture extracts from other strains of P. cinnamomeus and T. luteorostrata revealed that zeorin is the most commonly occurring fermentation product of these fungi, whereas paecilodepsipeptide A was the metabolite specific to the particular isolate BCC 9616/BCC 9617.

Phylogenetic classification of Cordyceps and the clavicipitaceous fungi.

Cordyceps, comprising over 400 species, was historically classified in the Clavicipitaceae, based on cylindrical asci, thickened ascus apices and filiform ascospores, which often disarticulate into part-spores. Cordyceps was characterized by the production of well-developed often stipitate stromata and an ecology as a pathogen of arthropods and Elaphomyces with infrageneric classifications emphasizing arrangement of perithecia, ascospore morphology and host affiliation. To refine the classification of Cordyceps and the Clavicipitaceae, the phylogenetic relationships of 162 taxa were estimated based on analyses consisting of five to seven loci, including the nuclear ribosomal small and large subunits (nrSSU and nrLSU), the elongation factor 1alpha (tef1), the largest and the second largest subunits of RNA polymerase II (rpb1 and rpb2), beta-tubulin (tub), and mitochondrial ATP6 (atp6). Our results strongly support the existence of three clavicipitaceous clades and reject the monophyly of both Cordyceps and Clavicipitaceae. Most diagnostic characters used in current classifications of Cordyceps (e.g., arrangement of perithecia, ascospore fragmentation, etc.) were not supported as being phylogenetically informative; the characters that were most consistent with the phylogeny were texture, pigmentation and morphology of stromata. Therefore, we revise the taxonomy of Cordyceps and the Clavicipitaceae to be consistent with the multi-gene phylogeny. The family Cordycipitaceae is validated based on the type of Cordyceps, C. militaris, and includes most Cordyceps species that possess brightly coloured, fleshy stromata. The new family Ophiocordycipitaceae is proposed based on Ophiocordyceps Petch, which we emend. The majority of species in this family produce darkly pigmented, tough to pliant stromata that often possess aperithecial apices. The new genus Elaphocordyceps is proposed for a subclade of the Ophiocordycipitaceae, which includes all species of Cordyceps that parasitize the fungal genus Elaphomyces and some closely related species that parasitize arthropods. The family Clavicipitaceaes. s. is emended and includes the core clade of grass symbionts (e.g., Balansia, Claviceps, Epichloë, etc.), and the entomopathogenic genus Hypocrella and relatives. In addition, the new genus Metacordyceps is proposed for Cordyceps species that are closely related to the grass symbionts in the Clavicipitaceaes. s.Metacordyceps includes teleomorphs linked to Metarhizium and other closely related anamorphs. Two new species are described, and lists of accepted names for species in Cordyceps, Elaphocordyceps, Metacordyceps and Ophiocordyceps are provided.

A phylogenetically-based nomenclature for Cordycipitaceae (Hypocreales).

The ending of dual nomenclatural systems for pleomorphic fungi in 2011 requires the reconciliation of competing names, ideally linked through culture based or molecular methods. The phylogenetic systematics of Hypocreales and its many genera have received extensive study in the last two decades, however resolution of competing names in Cordycipitaceae has not yet been addressed. Here we present a molecular phylogenetic investigation of Cordycipitaceae that enables identification of competing names in this family, and provides the basis upon which these names can be maintained or suppressed. The taxonomy presented here seeks to harmonize competing names by principles of priority, recognition of monophyletic groups, and the practical usage of affected taxa. In total, we propose maintaining nine generic names, Akanthomyces, Ascopolyporus, Beauveria, Cordyceps, Engyodontium, Gibellula, Hyperdermium, Parengyodontium, and Simplicillium and the rejection of eight generic names, Evlachovaea, Granulomanus, Isaria, Lecanicillium, Microhilum, Phytocordyceps, Synsterigmatocystis, and Torrubiella. Two new generic names, Hevansia and Blackwellomyces, and a new species, Beauveria blattidicola, are described. New combinations are also proposed in the genera Akanthomyces, Beauveria, Blackwellomyces, and Hevansia.

Ascochlorin derivatives from the leafhopper pathogenic fungus Microcera sp. BCC 17074.

Two new ascochlorin derivatives, nectchlorins A (1) and B (2), together with eight known compounds (3-10), were isolated from cultures of the leafhopper pathogen Microcera sp. BCC 17074. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. The absolute configuration of 2 was determined by application of the modified Mosher's method. The absolute configuration of LL-Z 1272α epoxide (9), which is a plausible biosynthetic precursor of ascochlorins, was established by chemical correlations. Cytotoxic activities of these ascochlorin derivatives were evaluated.

New 1F1N Species Combinations in Ophiocordycipitaceae (Hypocreales).

Based on the taxonomic and nomenclatural recommendations of Quandt et al. (2014) new species combinations are made for Ophiocordycipitaceae. These new combinations are compliant with recent changes in the International Code of Nomenclature for algae, fungi, and plants (ICN) and the abolition of the dual system of nomenclature for fungi. These changes include 10 new combinations into Drechmeria, four new combinations into Harposporium, 23 new combinations and 15 synonymies in Ophiocordyceps, and one new combination into Purpureocillium.

The polyphyletic nature of Paecilomyces sensu lato based on 18S-generated rDNA phylogeny.

Nuclear-encoded small-subunit ribosomal DNA was used to examine phylogenetic relationships in Paecilomyces sensu lato. Phylogenetic analysis of the 18S nr DNA demonstrates that Paecilomyces is polyphyletic across two subclasses, Sordariomycetidae and Eurotiomycetidae. The type species, Paecilomyces variotii, and thermophilic relatives belong in the order Eurotiales (Trichocomaceae), while mesophilic species related to Paecilomyces farinosus are in the order Hypocreales (Clavicipitaceae and Hypocreaceae). One species, Paecilomyces inflatus, had affinities for the order Sordariales. Within the Eurotiales, Paecilomyces is monophyletic. Within the Hypocreales, species of Paecilomyces are polyphyletic, although the data failed to fully resolve these relationships.

Torrubiellone E, an antimalarial N-hydroxypyridone alkaloid from the spider pathogenic fungus Torrubiella longissima BCC 2022.

Torrubiellone E (1), a new N-hydroxypyridone alkaloid, was isolated from the spider pathogenic fungus Torrubiella longissima BCC 2022, together with the known compounds, torrubiellones A (2) and B (3), and JBIR-130 (4). Compound 1 exhibited antimalarial activity against Plasmodium falciparum K1 with an IC5 value of 3.2 microg/mL, while it also showed weak cytotoxic activities.

Purpureocillium, a new genus for the medically important Paecilomyces lilacinus.

Paecilomyces lilacinus was described more than a century ago and is a commonly occurring fungus in soil. However, in the last decade this fungus has been increasingly found as the causal agent of infections in man and other vertebrates. Most cases of disease are described from patients with compromised immune systems or intraocular lens implants. In this study, we compared clinical isolates with strains isolated from soil, insects and nematodes using 18S rRNA gene, internal transcribed spacer (ITS) and partial translation elongation factor 1-α (TEF) sequences. Our data show that P. lilacinus is not related to Paecilomyces, represented by the well-known thermophilic and often pathogenic Paecilomyces variotii. The new genus name Purpureocillium is proposed for P. lilacinus and the new combination Purpureocillium lilacinum is made here. Furthermore, the examined Purpureocillium lilacinum isolated grouped in two clades based on ITS and partial TEF sequences. The ITS and TEF sequences of the Purpureocillium lilacinum isolates used for biocontrol of nematode pests are identical to those causing infections in (immunocompromised) humans. The use of high concentrations of Purpureocillium lilacinum spores for biocontrol poses a health risk in immunocompromised humans and more research is needed to determine the pathogenicity factors of Purpureocillium lilacinum.

Chemical taxonomy of Torrubiella s. lat.: zeorin as a marker of Conoideocrella.

The insect pathogens in the genus Torrubiella s. lat. were recently divided into new genera based on molecular phylogenetic characters. Isolates collected at various locations in Thailand, were tested for their productivity of a hopane-type triterpene, zeorin (6α,22-dihydroxyhopane), when cultured in potato dextrose broth under static conditions. Among the 49 strains of Torrubiella s. lat. species, Conoideocrella luteorostrata (ten strains) and C. tenuis (seven strains), all collected on scale insects (Hemiptera), produced zeorin, whereas another six strains of Orbiocrella petchii (which was recently removed from Torrubiella) failed in the detection of this secondary metabolite. All other Torrubiella s. lat. (26 strains), collected on other insect hosts including leafhoppers (eight strains), Lepidoptera (one strain), and spiders (17 strains), produced no detectable zeorin. Paecilomyces cinnamomeus (nine strains), the anamorph of C. luteorostrata, also produced zeorin. These results correspond with the recent taxonomic reclassification based on multigene phylogeny.

Ophiocordyceps barnesii and its relationship to other melolonthid pathogens with dark stromata.

A hypocrealean Coleoptera pathogen with characteristic part-spores, collected from Khao Yai National Park and Kaeng Krachan National Park in Thailand, is reported. The overall morphology was similar to Cordyceps barnesii, which is known from Sri Lanka, with ascospores disarticulating into four unusually long part-spores that were 30-40 µm long. This disarticulation and part-spore size is, so far, unique within Cordyceps sensu lato. The Thai material was identified with C. barnesii and its placement in the genus Ophiocordyceps was confirmed. Multigene analyses based on the ribosomal small subunit, RPB1 and RPB2 genes revealed the close relationship of the Thai material to Ophiocordyceps konnoana as well as O. ravenelii, O. superficialis, and O. nigrella (all of which have significantly smaller part-spores). However, Ophiocordyceps barnesii and these related species were all characterised by dark-brown to purplish stromata and an affinity for melolonthid larval hosts. No anamorph was seen in the field and was not produced in the slow-growing cultures.

Graveyards on the move: the spatio-temporal distribution of dead ophiocordyceps-infected ants.

Parasites are likely to play an important role in structuring host populations. Many adaptively manipulate host behaviour, so that the extended phenotypes of these parasites and their distributions in space and time are potentially important ecological variables. The fungus Ophiocordyceps unilateralis, which is pan-tropical in distribution, causes infected worker ants to leave their nest and die under leaves in the understory of tropical rainforests. Working in a forest dynamic plot in Southern Thailand we mapped the occurrence of these dead ants by examining every leaf in 1,360 m(2) of primary rainforest. We established that high density aggregations exist (up to 26 dead ants/m(2)), which we coined graveyards. We further established that graveyards are patchily distributed in a landscape with no or very few O. unilateralis-killed ants. At some, but not all, spatial scales of analysis the density of dead ants correlated with temperature, humidity and vegetation cover. Remarkably, having found 2243 dead ants inside graveyards we only found 2 live ants of the principal host, ant Camponotus leonardi, suggesting that foraging host ants actively avoid graveyards. We discovered that the principal host ant builds nests in high canopy and its trails only occasionally descend to the forest floor where infection occurs. We advance the hypothesis that rare descents may be a function of limited canopy access to tree crowns and that resource profitability of such trees is potentially traded off against the risk of losing workers due to infection when forest floor trails are the only access routes. Our work underscores the need for an integrative approach that recognises multiple facets of parasitism, such as their extended phenotypes.