Molecular phylogeny of the entomopathogenic fungi of the genus Cordyceps (Ascomycota: Clavicipitaceae) and its evolutionary implications

Abstract Cordyceps is an endoparasite ascomycetous genus containing approximately 450 species with a diversity of insect hosts, traditionally included in the family Clavicipitaceae of Ascomycota. Establishing the relationships among species with a varied range of morphologies and hosts is of importance to our understanding of the phylogeny and co‐evolution of parasites and hosts in entomopathogenic ascomycetes. To this end, we used a combination of molecular index and morphological characters from 40 representative species to carry out comprehensive molecular phylogenetic analyses. Based on the phylogenetic tree, we used the program DISCRETE for inferring the rates of evolution and finding ancestral states of morphological character. The phylogenetic analyses revealed two important points. (i) Types of perithecia attached to stroma reflected an evolutionary trend in Cordyceps. The vertically immersed perithecia form was the ancestral state, superficial and obliquely immersed perithecia were derived characters, obliquely immersed was irreversible. Species with obliquely immersed perithecia were in a closely related group and were the derived group. (ii) A strong correlation between fungal relatedness and the microhabitat supported the hypothesis that the host jumps through commingling in soil microhabitats. Based on the results of these analyses, host switching explains the diversity of entomopathogenic fungi of the genus Cordyceps.     

A comparative study on the production of exopolysaccharides between two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis in submerged mycelial cultures

AIMS: The present study comparatively investigates the optimal culture conditions for the production of exopolysaccharides (EPS) and cordycepin during submerged mycelial culture of two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis. METHODS AND RESULTS: Fermentations were performed in flasks and in 5-l stirred-tank fermenters. In the case of C. militaris, the highest mycelial biomass (22.9 g l(-1)) and EPS production (5 g l(-1)) were achieved in a medium of 40 g l(-1) sucrose, 5 g l(-1) corn steep powder at 30 degrees C, and an initial pH 8.0. The optimum culture conditions for C. sinensis was shown to be (in g l(-1)) 20 sucrose, 25 corn steep powder, 0.78 CaCl2, 1.73 MgSO4.7H2O at 20 degrees C, and an initial pH 4.0, where the maximum mycelial biomass and EPS were 20.9 and 4.1 g l(-1) respectively. Cordycepin, another bioactive metabolite, was excreted at low levels during the early fermentation period (maximum 38.8 mg l(-1) in C. militaris; 18.2 mg l(-1) in C. sinensis). CONCLUSIONS: The two fungi showed different nutritional and environmental requirements in their submerged cultures. Overall, the concentrations of mycelial biomass, EPS and cordycepin achieved in submerged culture of C. militaris were higher than those of C. sinensis. SIGNIFICANCE AND IMPACT OF THE STUDY: C. militaris and C. sinensis are representative insect-born fungi which have been longstanding and widely used as traditional medicines in eastern Asia. Comparative studies between two fungi are currently not available and this is the first report on the optimum medium composition for submerged culture of C. sinensis.     

Molecular phylogeny revealed complex evolutionary process in Elymus species

Recent molecular phylogenetic studies on Elymus have added to our understanding of the origination of Elymus species. However, evolutionary dynamics and speciation of most species in Elymus are unclear. Molecular phylogeny has demonstrated that reticulate evolution has occurred extensively in the genus, as an example, the largest subunit of RNA polymerase II (rpb2) and phosphoenolpyruvate carboxylase (pepC) data revealed two versions of the St genome, St1 and St2contributing to speciation of E. caninus. Phylogenetic analyses of E. pendulinus uncovered additional genome-level complexity. Our data indicated that both chloroplast and nuclear gene introgression have occurred in the evolutionary process of E. pendulinus. Non-donor species genomes have been detected in severalElymus species, such as in allohexaploid E. repens (StStStStHH), a Taeniatherum-like (Ta genome in Triticeae) GBSSI sequence, Bromus- (Bromeae) and Panicum-like (Paniceae) ITS sequences have been detected. The chloroplast DNA data indicated that Pseudoroegneria is the maternal genome donor to Elymus species, but whether different Elymus species originated from different St donors remains an open question. The origin of the Y genome in Elymus is puzzling. It is clear that the Ygenome is distinct from the St genome, but unclear on the relationships of Y to other genomes in Triticeae. Introgressive hybridization may be an important factor complicating the evolutionary history of the species in Elymus. The extent of introgression and its role in creating diversity in Elymus species should be the objective of further investigations.     

Molecular phylogeny of the Laboulbeniomycetes (Ascomycota)

A first molecular-based phylogeny is presented for the Laboulbeniomycetes, a group of ascomycete fungi that utilize arthropods for nutrition and/or dispersal. Morphological diversification and life-history evolution has made it difficult to resolve relationships within the group, and to identify close relatives. Here, we infer a preliminary phylogeny based on acquisition of 51 new SSU rDNA sequences, representing a total of 65 taxa. The results of this study demonstrate that Laboulbeniomycetes is monophyletic, and related to Sordariomycetes. The class could be divided into at least 4 or 5 orders, though we refrain from formally giving names to these at this stage. Further evidence for the occurrence of asexuality within the class is provided by the inclusion of the genera Chantransiopsis and Tetrameronycha, both known only as asexual taxa with thalli consisting of linearly superposed cells. The precise placement of the genus Herpomyces (Herpomycetaceae), on cockroaches, remains unresolved in our analysis, but lies outside of the main clade of sexually reproducing Laboulbeniales. There is good support for this latter grouping, comprising taxa that are found on both aquatic and terrestrial hosts. Within this large assemblage, we recognize 5 distinct clades (clades E, F, G, H, I). Relationships among the so-called “aquatic genera” (≡ Ceratomycetaceae + some Euceratomycetaceae and Zodiomyces) are poorly resolved in our analyses, accounting for 3 of these clades (E, F, G), with the remainder of the taxa (largely equivalent to Laboulbeniaceae) split into two major groupings (clades H, I). Across all taxa, antheridial characteristics, features on which the earliest classifications were based, are shown to be homoplastic. On the other hand, features of perithecial development show an overall trend towards reduction, and appear to be phylogenetically informative. Morphological characters are identified that support the dichotomy in the Laboulbeniaceae and subclades within the two major groupings are discussed further in light of information on thallus morphology, development, and host relationships.     

Isolation and pathogenicity of some South African entomopathogenic fungi (Ascomycota) against eggs and larvae of Cydia pomonella (Lepidoptera: Tortricidae)

Virulent entomopathogenic fungal strains against Cydia pomonella (Lepidoptera: Tortricidae) were isolated and identified in the Western Cape Province of South Africa. Thirty-nine isolates belonging to six species were obtained using the insect bait method. Generally, Metarhizium robertsii (Ascomycota: Hypocreales) was the most frequently encountered species representing 51% of the total number of isolates collected from the soil samples. This is the first report of M. robertsii from southern Africa. Mortality data from an immersion bioassay indicated that the 39 fungal isolates were pathogenic against fifth instar larvae of C. pomonella inducing 47–85% insect mortalities. Two M. robertsii strains, MTL151 and GW461, induced 85% larval mortality and were selected for further evaluations. The exposure of freshly laid eggs to wax papers that were pre-treated with fungal spores ranging from 10³ to 10⁸ spores ml^?1 of MTL151 and GW461 resulted in a significant reduction of egg hatchabilities, from 95 to 66% and 93 to 71%, respectively as spore concentration increased. First instar larval neonates were exposed to apples that were pre-sprayed topically with varied conidia suspensions (10³?10⁸ spores ml^?1). The mean percentage of participating apples with larvae in their cores/flesh significantly reduced from 53 to 10% (MTL151) and 76 to 10% (GW461) of 10 apples, and a concurrent decrease in the incidence of apple fruit rot was observed as conidia concentration increased. Up to 90% of apples treated with 1 × 10⁸ spores ml^?1 had no larvae present in their cores and this result compared favourably with the commercial pesticide Fruitfly®, containing the active ingredient cypermethrin (20 g/l) used at a recommended dose of 0.25 ml/250 ml of water.     

Development of twelve novel microsatellite markers in the medicinal mushroom Cordyceps militaris (Ascomycota: Clavicipitaceae) for its strain identification and genetic analysis

Cordyceps militaris (a caterpillar fungus), which belongs to the class Ascomycetes, has extensively been used for medicinal purposes in East Asia. Here, we isolated and characterized 12 microsatellite loci from the medicinal mushroom, C. militaris. Twenty‐nine individual samples were taken from a single locality in southwestern Korea and used to characterize the developed markers. The number of alleles of these loci ranged from two to 13 (mean allelic richness = 6.44). Observed heterozygosity and expected heterozygosity in the population ranged from 0.035 to 0.880 and from 0.035 to 0.886, respectively. Five of 12 loci significantly deviated from Hardy–Weinberg Equilibrium, most likely due to heterozygote deficiency caused by inbreeding. Tests of genotypic linkage disequilibrium between the 12 loci showed no significant association of alleles. These microsatellite markers will provide valuable tools for genetic analyses for the strain identification of C. militaris as well as for its resource conservation.     

Screening of entomopathogenic fungi against the European spruce bark beetle,Dendroctonus micans (Coleoptera: Scolytidae)

The European spruce bark beetle, Dendroctonus micans Kugelann (Coleoptera: Scolytidae), is one of the most serious destructive pests of oriental spruce (Picea orientalis L.). In order to find an effective biocontrol agent against this pest, we determined the effectiveness of entomopathogenic fungi against D. micans. Virulence of nine highly pathogenic strains including Beauveria bassiana (2), Beauveria cf. bassiana (2), Metarhizium anisopliae (2), Metarhizium sp. (1), Isaria fumosorosea (1) and Evlachovaea sp. (1) was evaluated on D. micans larvae and adults under laboratory conditions. Mortality values for both larvae and adults ranged from 83 to 100% and from 23 to 100%, respectively. Larvae were more susceptible to fungi than adults (P<0.05). Based on screening tests, B. cf. bassiana isolate KTU-53 was found the most effective isolate. LC₅₀ values were calculated as 1.77×10⁴ and 2.65×10⁴ conidia mL^?1 for isolate KTU-53 against larvae and adults, respectively. Consequently, B. cf. bassiana isolate KTU-53 appears to be the most promising microbial control agent for biocontrol of D. micans.     

台湾虫草及其无性型关系的分子确证

采用PCR技术,以rDNA-ITS区为分子指标,对台湾虫草Cordyceps formosana及其无性型黄山被毛孢Hirsutel lahuangshanensis进行测序和比较分析,以进一步确定两者的对应关系。结果表明:两者的序列完全相同,从而在分子水平上证明了黄山被毛孢是台湾虫草的无性阶段。系统进化树显示台湾虫草的无性型归为被毛孢属较为合理。     

Management of entomopathogenic fungi in cultures of Tenebrio molitor (Coleoptera: Tenebrionidae)

Larvae of mealworms Tenebrio molitor L. (Coleoptera: Tenebrionidae) have been used as animal feed, but fungal pathogens rapidly downsize the populations, resulting in economic losses. In this work, we established an effective management strategy for fungal pathogens. An entomopathogenic fungus, Beauveria bassiana, was isolated from mealworm cadavers. The bioassay of some isolates of this species at >90% relative humidity revealed that the ERL1575 isolate had the highest virulence. At 20–30% RH, ERL1575 conidia when ingested produced 80% mortality but when sprayed topically produced only <10% mortality. Mealworms that had ingested conidia were exposed to 20, 25, 30 and 35°C and high humidity (>95%) for 5 days. This experiment produced about 90% mortality except at 35°C where mortality was <20%. When 40 fungicides were assayed against ERL1575, fluazinam (1000‐fold) and mancozeb (667‐fold) significantly inhibited conidial germination and/or hyphal growth. When fluazinam and mancozeb were added to the mealworm diet of conidia‐inoculated wheat bran, most were alive 3 days post application. However, 100% mortality resulted 3 days post application in the conidia‐inoculated wheat bran without any fungicides. In conclusion, B. bassiana isolates are pathogenic at <30°C when they are ingested by mealworms but fluazinam and mancozeb can be used for management to control the pathogen in their cultures.     

台湾虫草及其无性型关系的分子确证

采用PCR技术,以rDNA-ITS区为分子指标,对台湾虫草Cordyceps formosana及其无性型黄山被毛孢Hirsutella huangshanensis进行测序和比较分析,以进一步确定两者的对应关系。结果表明：两者的序列完全相同,从而在分子水平上证明了黄山被毛孢是台湾虫草的无性阶段。系统进化树显示台湾虫草的无性型归为被毛孢属较为合理。     

The phylogeny of the Praomys complex (Rodentia: Muridae) and its phylogeographic implications

Among the African Murinae (Rodentia, Muridae), the Praomys complex, whose systematics has been studied by different approaches, has raised numerous taxonomic problems. Different taxa, namely Praomys, Mastomys , Myomys and Hylomyscus have been considered either as separate genera or subgenera of Praomys . In order to understand the relationships within the Praomys complex and to test the monophyly of the genus Praomys , a cladistic analysis was conducted, based on morpho-anatomical factors involving different species of Praomys , Mastomys , Myomys and Hylomyscus. The results indicate that the Praomys complex is monophyletic, as are the genera Hylomyscus , Mastomys and Myomys , whereas the genus Praomys appears paraphyletic. Indeed, a group of species including Praomys jacksoni was found to be more closely related to the genera Mastomys and Myomys than to a Praomys tullbergi -group. The biotopes and the distribution areas of the species were mapped on the phylogeny. It appears that the different clades each present a relative ecological cohesion and are arranged according to a gradient from closed to open habitats. From there, an evolutionary scenario is proposed for the emergence of the different clades and species of the genus Praomys sensu stricto.     

First record of Beauveria bassiana (Ascomycota: Clavicipitaceae) infecting Cerambyx welensii (Coleoptera: Cerambycidae) and pathogenicity tests using a new bioassay method

The pathogenicity of Beauveria bassiana on Cerambyx welensii is first reported. Three native fungal isolates from Badajoz (Spain) were bioassayed on neonates using cork oak disks and LC₅₀s were 1.16–1.76 × 10⁵ and 0.30–0.61 × 10⁵ spores/ml on the 7th and 14th day post-inoculation. The fungus is potentially a biocontrol agent of C. welensii.     

Biochemical properties of different entomopathogenic fungi and their virulence against Chilo suppressalis (Lepidoptera: Crambidae) larvae

We determined the virulence of Beauveria bassiana, Metarhizium anisopliae, Isaria fumosoroseus and Lecanicilium lecanii against larvae of Chilo suppressalis Walker by bioassay and evaluated several enzymatic and non-enzymatic components. LC₅₀ values of the entomopathogenic fungi revealed 90, 32, 45,000, 4600, 42,000 and 1,540,000 spores/larva for isolates BB1–BB3 of B. bassiana, I. fumosoroseus, M. anisopliae and L. lecanii, respectively. Isolate BB3 and I. fumosoroseus had the highest amounts of total protein and hydrophobin and isolates BB3 and M. anisopliae showed the highest activities of lipases and chitinases. In case of proteases, the highest activities were observed for Pr1 of BB1 and Pr2 of L. lecanii. The highest general esterase activities were obtained in I. fumosoroseus and BB1 when 1-naphtyl acetate and 2-naphtyl acetate were used as substrates. The highest activity of glutathione S-transferase (GST) was observed in I. fumosoroseus by using both reagents but BB1 demonstrated the highest activities of alkaline phosphatase and acid phosphatase. Clustering of the fungi using biochemical enzymes revealed BB2 and BB3 as a separate group of entomopathogenic fungi. In another group, I. fumosoroseus and L. lecanii had the most similarity and were separated from BB1 and M. anisopliae. The fungi exhibited different virulence on larvae of C. suppressalis by producing adhering protein and extracellular enzymes. Overall, results of the bioassays and clustering based on enzymatic activities revealed that isolate BB2 was the most effective fungus against larvae of C. suppressalis.     

Isolation and virulence of entomopathogenic fungi against the great spruce bark beetle,Dendroctonus micans (Kugelann) (Coleoptera: Scolytidae)

Twelve fungal strains including Lecanicillium muscarium (Petch.) Zare and Gams, Isaria farinosa (Holmsk.) Fr., Fusarium sp., Beauveria bassiana Sensu Lato and Beauveria sp. were isolated from larvae and adults of D. micans. In addition, virulence of these isolates against this pest was determined. Conidia suspensions of 1×10⁶ conidia mL^–1 were applied to larvae and adults. The highest mortality and mycosis for larvae were obtained from isolate ARSEF 9271 (Beauveria bassiana) with 90% mortality and mycosis within 10 days. ARSEF 9271 also produced 93% mortality and mycosis in adults. On the other hand, the highest mortality and mycosis for adults were obtained with isolate ARSEF 9272 (Beauveria sp.), with 100% mortality and 80% mycosis within 10 days. These results indicate that isolates ARSEF 9271 and ARSEF 9272 seem to be the most promising potential fungal biocontrol agents against D. micans.     

Molecular identification and phylogeny of arbuscular mycorrhizal fungi

The fossil record and molecular data show that the evolutionary history of arbuscular mycorrhizal fungi (Glomales) goes back at least to the Ordovician (460 million years ago), coinciding with the colonization of the terrestrial environment by the first land plants. At that time, the land flora only consisted of plants on the bryophytic level. Ribosomal DNA sequences indicate that the diversity within the Glomales on the family and genus level is much higher than previously expected from morphology-based taxonomy. Two deeply divergent lineages were found and described in two new genera, Archaeospora and Paraglomus, each in its own family. Based on a fast-growing number of available DNA sequences, several systems for molecular identification of the Glomales within roots have been designed and tested in the past few years. These detection methods have opened up entirely new perspectives for studying the ecology of arbuscular mycorrhiza.     

Susceptibility of the tobacco whitefly,Bemisia tabaci (Hemiptera: Aleyrodidae) biotype Q to entomopathogenic fungi

Recently, the Q biotype of tobacco whitefly has been recognized as the most hazardous strain of Bemisia tabaci worldwide because of increased resistance to some insecticide groups requiring alternative strategies for its control. We studied the susceptibility of this biotype of B. tabaci to 21 isolates of Beauveria bassiana, three isolates of Isaria fumosorosea, one isolate of I. cateni, three isolates of Lecanicillium lecanii, one isolate of L. attenuatum, and one isolate of Aschersonia aleyrodis. These isolates were evaluated on pruned eggplant seedlings, at a concentration of 10⁸ conidia/mL (deposited at 6000±586 conidia mm^?2). The mortality based on mycosis varied from 18 to 97% after 6 days. Isaria fumosorosea isolate Pf04, B. bassiana isolates Bb06, Bb12, and L. lecanii L14 were found the most effective. Furthermore, five isolates were chosen for concentration–mortality response assays and compared to B. bassiana GHA as a standard. The numbers of nymphs infected by fungi were correlated with the spore concentration. L. lecanii L14 and I. fumosorosea Pf04 had the shortest LT₅₀ at 3.5 and 3.3 days at 6000±586 conidia mm^?2. Mortality declined and LT₅₀ values were longer as the concentration of conidia was reduced. The LD₅₀ values were calculated as 87, 147, 191, 263, and 269 conidia mm^?2 for isolates L14, GHA, AS1260, Bb13, and Pf04, respectively. These results indicated that the Q biotype of sweetpotato whitefly was susceptible to the five isolates of entomopathogenic fungi and these isolates have potential to be developed as microbial pesticides for whitefly control.     

The phylogeny of the annelid genus Ophryotrocha (Dorvilleidae)

Ophyotrocha is easy to keep in the laboratory and has therefore been used in several studies of evolution and speciation. The phylogenetic relationships within the group are, however, still not clear and morphological and molecular data are contradictory. Here we attempt to shed light on the phylogeny by adding an additional gene (cytochrome c oxidase subunit I) to the previous analyses of the group. However, the results are still incongruent with the results from the morphological data. We also include a species of the genus Iphitime, and conclude that this species falls within the Ophryotrocha clade. The implications are discussed.     

Molecular diversification of salamanders of the tropical American genus Bolitoglossa (Caudata: Plethodontidae) and its evolutionary and biogeographical implications

The largest genus of salamanders, Bolitoglossa (Plethodontidae), is widespread in tropical America, where it occurs in diverse habitats and elevations, from high elevation grasslands to lowland rain forest . It has the most extensive geographical range of any salamander genus. While most species occur in Middle America, it ranges throughout most of tropical South America as well. Phylogenetic analysis of 1196 bp of two mitochondrial genes (cytochrome b , 16S RNA) from 55 species offers strong support for the monophyly of the genus and sorts the species into a number of clades. Taking into account morphology, distribution, general ecology, and prior systematic and taxonomic studies, we recognize seven subgenera, four of them new: Bolitoglossa Duméril, Bibron et Duméril, 1854, Eladinea Miranda Ribeiro, 1937, Magnadigita Taylor, 1944, Mayamandra , Nanotriton , Oaxakia and Pachymandra . All South American and some lower Middle American species are included in a single well -supported clade, Eladinea . At the species level our analyses uncover the existence of large genetic diversity within morphologically homogeneous taxa. We propose the new combination: B. (Eladinea) paraensis (Unterstein, 1930) stat. nov. , for Brazilian salamanders previously included under B. altamazonica . We evaluate evidence for the multiple colonization of the tropical lowlands by morphologically derived species groups. South America was invaded by members of one clade, Eladinea , which we infer to have dispersed to South America prior to closure of the Panamanian Portal. Despite the relatively long history of salamanders in South America, that continent now accounts for a relatively small proportion of the lineages and species of neotropical salamanders.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 81 , 325–346.     

Molecular phylogeny and the historical biogeography of the warblers of the genus Sylvia (Aves)

A molecular phylogeny based on DNA/DNA hybridization revealed that the Sylvia-Parisoma complex is monophyletic and includes three main groups of species, the “mid-European” warblers, the genus Parisoma, and the “eu-Mediterranean” Sylvia species sensu stricto. The latter can be assigned to three main clusters, a “West-Mediterranean” group, a “Central-Mediterranean group”, and an “East-Mediterranean” group. The radiation of the whole complex is much more ancient than formerly believed. It started ca 12–13 Ma ago and the ancestors of the main extant groups differentiated during the Pliocene. Only speciation events within the “eu-Mediterranean” lineages occurred during the Pleistocene. The paleoclimatical and paleoecological history of the Mediterranean region is too complicated to provide any evidence for direct relationships between past events and evolutionary steps of these taxa which did not leave any reliable fossil record. However, some major speciation events may be related to well documented climatical crises as well as paleobotanical data. The largely man-induced extension of matorrals over several millenia presumably extended the range of several species that were formerly much more restricted, which complicates reconstruction of the spatio-temporal course of speciation.