<Emphasis Type="Italic">Ophiocordyceps pulvinata</Emphasis> sp. nov., a pathogen of ants with a reduced stroma

Ophiocordyceps pulvinata, a pathogen of ants, is formally described as a new species. Genus level designation of this species is difficult due to several apparently conflicting morphological and ecological characters. Affinity with Ophiocordyceps is suggested by the dark color stroma and ascospore morphology. However, the species was included in a book of entomopathogenic fungi of Japan as Torrubiella sp. due to the production of perithecia on an astipitate stroma. Phylogenetic analyses of molecular data support a close relationship with O. unilateralis, a finding consistent with morphological characteristics of the color, asci and ascospores and ecological traits of host affiliation. Thus, O. pulvinata represents another example of the loss of stipe for the hypocrealean arthropod pathogenic fungi and highlights the utility of asci and ascospore morphology as taxonomically informative characters of closely related taxa.     

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.     

Polycephalomyces lianzhouensis sp. nov., a new species,co-occurs with Ophiocordyceps crinalis

A new species from Guangdong Province in south China, Polycephalomyces lianzhouensis, is described and illustrated here; this species was found to co-occur with Ophiocordyceps crinalis. The phylogenetic position of this species was investigated according to morphological features and phylogenetic inferences based on the nrSSU, nrLSU, tef1, rpb1, and ITS gene sequences. Results demonstrated that P. lianzhouensis was highly associated with P. ramosopulvinatus but could be distinguished from the latter using the sequences of rpb1, nrLSU, and ITS gene and the features of much smaller stromata and perithecia and longer part-spores.     

Ophiocordyceps halabalaensis: a new species of Ophiocordyceps pathogenic to Camponotus gigas in Hala Bala Wildlife Sanctuary, Southern Thailand

Several fungal pathogens of ants have been reported as members of the family Ophiocordycipitaceae in the order Hypocreales. Surveys in the south of Thailand have shown specimens showing characteristics that are morphologically similar to Ophiocordyceps unilateralis, a very common ant pathogen, by producing a lateral pad on one side of the stroma and producing whole ascospores. Phylogenetic analyses of the partial elongation factor tef1-α and the internal transcribed spacer regions ITS1-5.8S-ITS2 rDNA have shown that this is a distinct species from O. unilateralis. The morphological characters of Ophiocordyceps halabalaensis differs from O. unilateralis in the possession of bigger perithecia and ascospores, and molecular analyses have shown that this ant-specific fungus is sufficiently different from O. unilateralis, deserving the naming of a new species. Aspects of morphology, host association/host-specificity, and taxonomic position are discussed.     

Molecular phylogeny and morphology of <Emphasis Type="Italic">Ophiocordyceps unituberculata</Emphasis> sp. nov. (Ophiocordycipitaceae), a pathogen of caterpillars (Noctuidae,Lepidoptera) from Yunnan,China

Ophiocordyceps unituberculata, a new insect pathogenic fungus from southwestern China, is described using molecular phylogenetic and morphological data. This fungus differs from other Ophiocordyceps species by its enormously long monophialidic conidiogenous cells and a large periclinal protuberance often growing near the apex of conidiogenous cells, single conidia (lanceolate to fusiform) embedded in mucous sheath, and much larger conidial dimensions. Four-locus (nrSSU, nrLSU, tef-1α and rpb1) and ITS data phylogenetic analyses show that O. unituberculata belongs to the Hirsutella nodulosa clade within the genus Ophiocordyceps of Ophiocordycipitaceae and is a separate clade from other allied species. Molecular phylogeny and morphology both strongly support the distinctiveness of this taxon. The interspecific relationships in the H. nodulosa clade are discussed.     

Infection and behavior manipulation of social wasps (Vespidae: Polistinae) by Ophiocordyceps humbertii in Neotropical forests: new records of wasp-zombification by a fungus

ABSTRACT

Ophiocordyceps is a genus comprised by entomopathogenic fungi known to infect ten orders of insects, including Hymenoptera. Amongst the nearly 250 species described in the genus, few are known to manipulate their hosts, which are most notably ants. These species cause their hosts to die in an exposed position high above the ground while grabbing and/or biting the abaxial surface of leaves or branches, which in turn optimizes the fungus spore production and dispersal. Herein, we report on 14 social wasp species belonging to four genera (Agelaia, Mischocyttarus Polybia, and Pseudopolybia) infected by Ophiocordyceps humbertii, a common wasp pathogen. This study broadens the geographic and host range for O. humbertii and provides the first record of its ability to manipulate its host.     

Temporal and spatial gradients of humidity shape the occurrence and the behavioral manipulation of ants infected by entomopathogenic fungi in Central Amazon

Some species of the fungal genus Ophiocordyceps that parasitize ants have evolved some degree of behavioral manipulation to increase their own transmission. Carpenter ants (Camponotus spp.) infected by Ophiocordyceps unilateralis species climb vegetation and bite leaves or other plant parts before death, while other ants infected by other Ophiocordyceps species die buried within or on the leaf-litter, attached to stems and tree trunks. The microenvironment of the host's death location is important for fungal development and dispersal to new hosts, being an interesting example of an extended phenotype. This study investigated the effect of humidity variation in space (15 km²) and time (14 months) on the occurrence and expression of the extended Ophiocordyceps phenotype parasitizing ants in Central Amazonia. We found O. unilateralis s.l., O. kniphofioides s.l. and O. australis s.l. parasitizing 30 ant species, but O. unilateralis s.l. was by far the most abundant fungus. Parasitized Camponotus atriceps and C. bispinosus were more abundant and died in higher places in periods with greater air humidity. Otherwise, the abundance and height of C. senex cadavers were higher in drier plots. These results provide insights on how the spatial and temporal variation in air humidity may shape the occurrence and behavioral manipulation of ants infected by entomopathogenic fungi at larger scales.     

Systematic analyses of Ophiocordyceps lanpingensis sp. nov., a new species of Ophiocordyceps in China

A new species of Ophiocordyceps, Ophiocordyceps lanpingensis collected from Lanping County, Yunnan Province, southwestern China, was described based on morphological characteristic, ITS1-5.8S-ITS2 rDNA sequences analyses, 5-gene (nrSSU, nrLSU, tef-1α, rpb1 and rpb2) sequences analyses and MAT1-2-1 gene sequences analyses. This species was characterized by thinner stroma, smaller perithecium, thinner ascospore (multiseptate with short septation). The phylogenetic analyses based on the ITS, the 5-gene and MAT1-2-1 gene dataset showed O. lanpingensis had the closest evolution relationship with O. robertsii and O. sinensis, but still had obvious distances to them. Both morphological character and systematic analyses supported that O. lanpingensis was a new species of Ophiocordyceps.     

Relationship between intraspecific variations and host insects of Ophiocordyceps nutans collected in Japan

To investigate the host specificity of Ophiocordyceps nutans against hemipteran insects in the wild, we determined the relationship between host species and rDNA-internal transcribed spacer (ITS) variation in O. nutans. The analyzed fungal specimens infected 16 host species belonging to four families of Hemiptera. The molecular phylogenetic analysis revealed that O. nutans can be classified into two types corresponding to their host families. The genetic distance values between the two types were very remote (>0.084), and the strains of O. nutans that parasitized Halyomorpha halys and Plautia crossota stali, well-known insect pests, formed a subclade. The results suggest that O. nutans should have host specificity which can be valuable for developing biological control agents against specific hemipteran insects.     

A Polycephalomyces hyperparasite of Ophiocordyceps sinensis leads to shortened duration of production and reduced numbers of host ascospores

Ophiocordyceps sinensis, an entomopathogenic fungus of the Tibetan Plateau is one of the most valuable traditional Chinese medicines. A fungal pathogen of O. sinensis, Polycephalomyces sp. GIMCC 3.570 (isolate 2010A-0818) was characterized by morphology, related to sequences of six genes (SSU, ITS, LSU, TEF1-α, RPB1 and ATP6) found in a BLAST search and five genes (SSU, LSU, TEF1, RPB1 and RPB2) by phylogenetic analysis using maximum parsimony, maximum likelihood and Bayesian methods. The results strongly support placement of P. sp. GIMCC 3.570 in the genus Polycephalomyces. To determine the influence of P. sp. GIMCC 3.570 on the reproduction of its host, 30 artificially infected O. sinensis samples were compared with 30 uninfected controls. In the infected fungus, the life span of the stroma was significantly shorter and significantly fewer ascospores were produced compared with uninfected O. sinensis (p < 0.01).     

On the reliability of DNA sequences of Ophiocordyceps sinensis in public databases

Some DNA sequences in the International Nucleotide Sequence Databases (INSD) are erroneously annotated, which has lead to misleading conclusions in publications. Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a fungus endemic to the Tibetan Plateau, and more than 100 populations covering almost its distribution area have been examined by us over recent years. In this study, using the data from authentic materials, we have evaluated the reliability of nucleotide sequences annotated as O. sinensis in the INSD. As of October 15, 2012, the INSD contained 874 records annotated as O. sinensis, including 555 records representing nuclear ribosomal DNA (63.5 %), 197 representing protein-coding genes (22.5 %), 92 representing random markers with unknown functions (10.5 %), and 30 representing microsatellite loci (3.5 %). Our analysis indicated that 39 of the 397 internal transcribed spacer entries, 27 of the 105 small subunit entries, and five of the 53 large subunit entries were incorrectly annotated as belonging to O. sinensis. For protein-coding sequences, all records of serine protease genes, the mating-type gene MAT1-2-1, the DNA lyase gene, the two largest subunits of RNA polymerase II, and elongation factor-1α gene were correct, while 14 of the 73 β-tubulin entries were indeterminate. Genetic diversity analyses using those sequences correctly identified as O. sinensis revealed significant genetic differentiation in the fungus although the extent of genetic differentiation varied with the gene. The relationship between O. sinensis and some other related fungal taxa is also discussed.     

Comparative phylogenetic relationships and genetic structure of the caterpillar fungus Ophiocordyceps sinensis and its host insects inferred from multiple gene sequences

Ophiocordyceps sinensis (Ascomycota: Ophiocordycipitaceae) is a native fungal parasite of Hepialidae caterpillars and one of the most economically important medicinal caterpillar fungi in China. However, little is known about the phylogenetic and evolutionary relationships between O. sinensis and its host insects. In this study, nuclear ITS and β-tubulin sequences from O. sinensis and mitochondrial COI, COII, and Cytb sequences from its hosts were analyzed across 33 populations sampled from five regions in China. Phylogenetically, both O. sinensis and its hosts were divided into three geographically correlated clades, and their phylogenies were congruent. Analysis of molecular variance and calculated coefficients of genetic differentiation revealed significant genetic divergence among the clades within both O. sinensis (F_ST= 0.878, N_ST=0.842) and its hosts (F_ST=0.861, N_ST=0.816). Estimated gene flow was very low for O. sinensis (Nm=0.04) and the host insects (Nm=0.04) among these three clades. Mantel tests demonstrated a significant correlation (P<0.01) between the genetic distances for O. sinensis and its hosts, as well as a significant association (P<0.05) between geographic and genetic distances in both. The similar phylogenetic relationships, geographic distributions, and genetic structure and differentiation between O. sinensis and its hosts imply that they have coevolved.     

The phylogenetic placement of hypocrealean insect pathogens in the genus Polycephalomyces: An application of One Fungus One Name

Understanding the systematics and evolution of clavicipitoid fungi has been greatly aided by the application of molecular phylogenetics. They are now classified in three families, largely driven by reevaluation of the morphologically and ecologically diverse genus Cordyceps. Although reevaluation of morphological features of both sexual and asexual states were often found to reflect the structure of phylogenies based on molecular data, many species remain of uncertain placement due to a lack of reliable data or conflicting morphological characters. A rigid, darkly pigmented stipe and the production of a Hirsutella-like anamorph in culture were taken as evidence for the transfer of the species Cordyceps cuboidea, Cordyceps prolifica, and Cordyceps ryogamiensis to the genus Ophiocordyceps. Data from ribosomal DNA supported these species as a single group, but were unable to infer deeper relationships in Hypocreales. Here, molecular data for ribosomal and protein coding DNA from specimens of Ophiocordyceps cuboidea, Ophiocordyceps ryogamiensis, Ophiocordyceps paracuboidea, Ophiocordyceps prolifica, Cordyceps ramosopulvinata, Cordyceps nipponica, and isolates of Polycephalomyces were combined with a broadly sampled dataset of Hypocreales. Phylogenetic analyses of these data revealed that these species represent a clade distinct from the other clavicipitoid genera. Applying the recently adopted single system of nomenclature, new taxonomic combinations are proposed for these species in the genus Polycephalomyces, which has been historically reserved for asexual or anamorphic taxa.     

High diversity of the fungal community structure in naturally-occurring Ophiocordyceps sinensis

Background

Ophiocordyceps sinensis (syn. Cordyceps sinensis), which is a parasite of caterpillars and is endemic to alpine regions on the Tibetan Plateau, is one of the most valuable medicinal fungi in the world. “Natural O. sinensis specimens” harbor various other fungi. Several of these other fungi that have been isolated from natural O. sinensis specimens have similar chemical components and/or pharmaceutical effects as O. sinensis. Nevertheless, the mycobiota of natural O. sinensis specimens has not been investigated in detail.

Methodology/Principal Findings

Based on the technique of PCR-single-strand conformation polymorphism (PCR-SSCP), the mycobiota of three different sections (stromata, sclerotia, and mycelial cortices) from natural O. sinensis specimens were investigated using both culture-dependent and -independent methods. For the culture-dependent method, 572 fungal strains were isolated, and 92 putative operational taxonomic units (OTUs) were identified from 226 sequenced strains with the threshold of 97%. For the culture-independent method, 490 fungal clones were identified from about 3000 clones of ITS fragments from the whole-community DNA; based on PCR-SSCP analyses, 266 of these clones were selected to be sequenced, and 118 putative OTUs were detected. The overwhelming majority of isolates/clones and OTUs were detected from mycelial cortices; only a few were detected from stromata and sclerotia. The most common OTUs detected with both methods belonged to Ascomycota; however, only 13 OTUs were detected simultaneously by both methods. Potential novel lineages were detected by each of the two methods.

Conclusions/Significance

A great number of fungal species present in the mycobiota of naturally-occurring O. sinensis specimens were detected, and many of them may represent undescribed lineages. That only a few of the same OTUs were detected by both methods indicated that different methods should be used. This study increased our understanding about the fungal community structure of this valuable medicinal herb.     

A genome scan of diversifying selection in Ophiocordyceps zombie‐ant fungi suggests a role for enterotoxins in co‐evolution and host specificity

Identification of the genes underlying adaptation sheds light on the biological functions targeted by natural selection. Searches for footprints of positive selection, in the form of rapid amino acid substitutions, and the identification of species‐specific genes have proved to be powerful approaches to identifying the genes involved in host specialization in plant‐pathogenic fungi. We used an evolutionary comparative genomic approach to identify genes underlying host adaptation in the ant‐infecting genus Ophiocordyceps, which manipulates ant behaviour. A comparison of the predicted genes in the genomes of species from three species complexes—O. unilateralis, O. australis and O. subramanianii—revealed an enrichment in pathogenesis‐associated functions, including heat‐labile enterotoxins, among species‐specific genes. Furthermore, these genes were overrepresented among those displaying significant footprints of positive selection. Other categories of genes suspected to be important for virulence and pathogenicity in entomopathogenic fungi (e.g., chitinases, lipases, proteases, core secondary metabolism genes) were much less represented, although a few candidate genes were found to evolve under positive selection. An analysis including orthologs from other entomopathogenic fungi in a broader context showed that positive selection on enterotoxins was specific to the ant‐infecting genus Ophiocordyceps. Together with previous studies reporting the overexpression of an enterotoxin during behavioural manipulation in diseased ants, our findings suggest that heat‐labile enterotoxins are important effectors in host adaptation and co‐evolution in the Ophiocordyceps entomopathogenic fungi.     

Phylogeny and morphology of Hirsutella tunicata sp. nov. (Ophiocordycipitaceae), a novel mite parasite from Peru

A new species of Hirsutella was isolated from unidentified mites on Petri plates inoculated with soil and root fragments collected from asparagus rhizosphere at Virú, Northern Peru. The fungus differs from other Hirsutella species by an envelope surrounding the conidium, conidia dimension and DNA sequences. In PDA cultures, the mycelium produced aerial hyphae with conidiogenous cells mainly at right angles, occasionally showing a secondary conidiophore. The solitary conidia are cymbiform, slightly apiculate, 5.0–6.0 × 3.0–4.0 μm. Phylogenetic analyses with partial rRNA and β-tubulin gene sequences confirmed the fungus as an Hirsutella (Ophiocordycipitaceae). Closest species shown by maximum likelihood and neighbor-joining trees were H. nodulosa and H. aphidis, from which the new species differs for conidium or conidiogenous cells dimensions, lack of synnemata and host type. A recombination event was also detected in the rRNA of the holotype strain, involving Ophiocordyceps sinensis as major parent and O. cochlidiicola as minor parent. A complement, inverted insertion was also found in its rRNA, involving part of the ITS2 and 5.8S regions, flanked by two short nucleotide arrays. Due to conidia dimension and phylogenetic position, the fungus is described as Hirsutella tunicata sp. nov. A review of mononematous Hirsutella species is provided.     

The Invasive Wetland Plant Alternanthera philoxeroides Shows a Higher Tolerance to Waterlogging than Its Native Congener Alternanthera sessilis

Plant invasion is one of the major threats to natural ecosystems. Phenotypic plasticity is considered to be important for promoting plant invasiveness. High tolerance of stress can also increase survival of invasive plants in adverse habitats. Limited growth and conservation of carbohydrate are considered to increase tolerance of flooding in plants. However, few studies have examined whether invasive species shows a higher phenotypic plasticity in response to waterlogging or a higher tolerance of waterlogging (lower plasticity) than native species. We conducted a greenhouse experiment to compare the growth and morphological and physiological responses to waterlogging of the invasive, clonal, wetland species Alternanthera philoxeroides with those of its co-occurring, native, congeneric, clonal species Alternanthera sessilis. Plants of A. philoxeroides and A. sessilis were subjected to three treatments (control, 0 and 60 cm waterlogging). Both A. philoxeroides and A. sessilis survived all treatments. Overall growth was lower in A. philoxeroides than in A. sessilis, but waterlogging negatively affected the growth of A. philoxeroides less strongly than that of A. sessilis. Alternanthera philoxeroides thus showed less sensitivity of growth traits (lower plasticity) and higher waterlogging tolerance. Moreover, the photosynthetic capacity of A. philoxeroides was higher than that of A. sessilis during waterlogging. Alternanthera philoxeroides also had higher total non-structural and non-soluble carbohydrate concentrations than A. sessilis at the end of treatments. Our results suggest that higher tolerance to waterlogging and higher photosynthetic capacity may partly explain the invasion success of A. philoxeroides in wetlands.     

Foliar nutrient allocation patterns in Banksia attenuata and Banksia sessilis differing in growth rate and adaptation to low-phosphorus habitats

Background and AimsPhosphorus (P) and nitrogen (N) are essential nutrients that frequently limit primary productivity in terrestrial ecosystems. Efficient use of these nutrients is important for plants growing in nutrient-poor environments. Plants generally reduce foliar P concentration in response to low soil P availability. We aimed to assess ecophysiological mechanisms and adaptive strategies for efficient use of P in Banksia attenuata (Proteaceae), naturally occurring on deep sand, and B. sessilis, occurring on shallow sand over laterite or limestone, by comparing the allocation of P among foliar P fractions.MethodsWe carried out pot experiments with slow-growing B. attenuata, which resprouts after fire, and faster growing opportunistic B. sessilis, which is killed by fire, on substrates with different P availability using a randomized complete block design. We measured leaf P and N concentrations, photosynthesis, leaf mass per area, relative growth rate and P allocated to major biochemical fractions in B. attenuata and B. sessilis.Key ResultsThe two species had similarly low foliar total P concentrations, but distinct patterns of P allocation to P-containing fractions. The foliar total N concentration of B. sessilis was greater than that of B. attenuata on all substrates. The foliar total P and N concentrations in both species decreased with decreasing P availability. The relative growth rate of both species was positively correlated with concentrations of both foliar nucleic acid P and total N, but there was no correlation with other P fractions. Faster growing B. sessilis allocated more P to nucleic acids than B. attenuata did, but other fractions were similar.ConclusionsThe nutrient allocation patterns in faster growing opportunistic B. sessilis and slower growing B. attenuata revealed different strategies in response to soil P availability which matched their contrasting growth strategy.     

A real-time qPCR assay to quantify Ophiocordyceps sinensis biomass in Thitarodes larvae

Ophiocordyceps sinensis, an entomogenous fungus parasitic in the larvae of moths (Lepidoptera), is one of the most valuable medicinal fungi, and it only distributed naturally on the Tibetan Plateau. The parasitical amount of O. sinensis in various tissues of the host Thitarodes larvae has an important role in study the occurrence and developmental mechanisms of O. sinensis, but there no an effective method to detect the fungal anamorph. A real-time quantitative PCR (qPCR) system, including a pair of species-specific ITS primers and its related program, was developed for O. sinensis assay with high reliability and efficiency. A calibration curve was established and exhibited a very good linear correlation between the fungal biomass and the C _T values (R ²=0.999419) by the qPCR system. Based on this method, O. sinensis was detected rapidly in four tissues of its host caterpillars, and the results were shown as following: the maximum content of O. sinensis parasitized in the fat-body, and next came body-wall; both of them were much larger than that observed in the haemolymph and intestinal-wall. Taken together, these results show that qPCR assays may become useful tools for study on developmental mechanism of O. sinensis.