共存于厌氧真菌分离培养液中瘤胃甲烷菌的检测及其多样性分析

对分离自山羊瘤胃的真菌分离培养液中甲烷菌进行16SrDNA扩增、DGGE分析、RFLP及测序分析,研究共存于真菌分离培养液中甲烷菌的种类及其多样性。DGGE结果显示:从厌氧真菌分离至第45代,甲烷菌多样性指数由1·32降至0·99,相似性最低为34·7%;第45代至62代,多样性指数由0·99升至1·15,相似性最低为89·2%。RFLP多态性分析69个克隆共得到5个操作分类单元,选择其中6个具有代表性的序列进行测序。序列及系统进化分析表明,属于其中3个操作分类单元的克隆最相似菌都是UnculturedarchaealsymbiontPA202,相似性均为95%,没有与这些克隆相似性较高的已培养甲烷菌;属于另外2个操作分类单元的克隆最相似菌都是Unculturedrumenmethanogen956,相似性均为97%,最相似已知菌为Methanobrevibactersp.NT7,相似性为97%。结果表明,真菌培养液中存在目前尚未分离培养的瘤胃甲烷菌。     

A rapid screening test to distinguish between Candida albicans and Candida dubliniensis using NMR spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy combined with a statistical classification strategy (SCS) successfully distinguished between Candida albicans and Candida dubliniensis. 96% of the isolates from an independent test set were identified correctly. This proves that this rapid approach is a valuable method for the identification and chemotaxonomic characterisation of closely related taxa. Most discriminatory regions were correlated with metabolite profiles, indicating biochemical differences between the two species.     

Fungal secondary metabolism - from biochemistry to genomics

Much of natural product chemistry concerns a group of compounds known as secondary metabolites. These low-molecular-weight metabolites often have potent physiological activities. Digitalis, morphine and quinine are plant secondary metabolites, whereas penicillin, cephalosporin, ergotrate and the statins are equally well known fungal secondary metabolites. Although chemically diverse, all secondary metabolites are produced by a few common biosynthetic pathways, often in conjunction with morphological development. Recent advances in molecular biology, bioinformatics and comparative genomics have revealed that the genes encoding specific fungal secondary metabolites are clustered and often located near telomeres. In this review, we address some important questions, including which evolutionary pressures led to gene clustering, why closely related species produce different profiles of secondary metabolites, and whether fungal genomics will accelerate the discovery of new pharmacologically active natural products.     

The phylogenetic interpretation of biological surveys

The ecological attributes of two species may be similar through convergent evolution or common ancestry. The extent of similarity by descent can be evaluated by comparing them with their most closely‐related outgroup in a given phylogeny. I describe a method of nested sister‐group analysis for estimating ecological similarity based on landscape features or on co‐distribution. The phylogeny is dissected into triplets, each comprising two sister taxa and their outgroup. For a triplet at any phylogenetic level, the similarity of sister groups with respect to some given character can be compared with their joint similarity to the outgroup to give a single test of similarity by descent. Each comparison is independent, and the full set of triplets provides a complete accounting of phylogenetic variation at all levels. This procedure was applied to 188 moderately abundant species of dicots in two independent surveys from adjoining districts of midland England, supplemented by physical surveys of landscape attributes obtained from digitized maps of the same districts. The co‐distribution of sister species was consistently more positive than the co‐distribution of random species pairs, demonstrating the existence of a phylogenetic signal at some level. When sister species are compared with their most closely‐related outgroup, however, neither landscape attributes nor co‐distribution showed any overall similarity arising from common ancestry, in the sense that ecological attributes are not generally conserved after lineage splitting. Instead, the distribution of similarity is strikingly similar to random data. The lack of ecological similarity between closely‐related groups was attributed to rapid character change at or shortly after the splitting of lineages, coupled with a lack of correlation between successive lineage splits.     

Segregation of secondary metabolite biosynthesis in hybrids of Fusarium fujikuroi and Fusarium proliferatum

Fusarium fujikuroi and Fusarium proliferatum are two phylogenetically closely related species of the Gibberella fujikuroi species complex (GFC). In some cases, strains of these species can cross and produce a few ascospores. In this study, we analyzed 26 single ascospore isolates of an interspecific cross between F. fujikuroi C1995 and F. proliferatum D4854 for their ability to produce four secondary metabolites: gibberellins (GAs), the mycotoxins fusarin C and fumonisin B(1), and a family of red polyketides, the fusarubins. Both parental strains contain the biosynthetic genes for all four metabolites, but differ in their ability to produce these metabolites under certain conditions. F. fujikuroi C1995 produces GAs and fusarins, while F. proliferatum D4854 produces fumonisins and fusarubins. The segregation amongst the progeny of these traits is not the expected 1:1 Mendelian ratio. Only eight, six, three and three progeny, respectively, produce GAs, fusarins, fumonisin B(1) and fusarubins in amounts similar to those synthesized by the producing parental strain. Beside the eight highly GA(3)-producing progeny, some of the progeny produce small amounts of GAs, predominantly GA(1), although these strains contain the GA gene cluster of the non-GA-producing F. proliferatum parental strain. Some progeny had recombinant secondary metabolite profiles under the conditions examined indicating that interspecific crosses can yield secondary metabolite production profiles that are atypical of the parent species.     

Headspace sorptive extraction and GC-TOFMS for the identification of volatile fungal metabolites

Volatiles from fungi cultivated in Petri dishes were collected by a simple headspace polydimethylsiloxane (PDMS) sorptive extraction technique (HSSE), thermally desorbed into a gas chromatographic capillary column and detected and identified by gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). The method was used to compare metabolite profiles of seven species of fungi grown on two types of sterile agars - potato dextrose and Sabouraud dextrose. Three species from the genus Penicillium (P. italicum, P. camemberti, and P. roqueforti) and four outgroups, each from a different phylum (Saprolegnia sp.; Sordaria fimicola, wild-type; Coprinus cinereus; and Rhizopus stolonifer) were grown on the two types of agars and analyzed. Multivariate analysis (PCA) was used to determine whether separate classes of fungi can be distinguished from one another based on their metabolite profiles. PCA showed clear class separation between the three Penicillium samples and the outgroups. Slight differences were observed in metabolite profiles as a function of growth medium. HSSE/GC-TOFMS appears to be a relatively simple and accurate technique for classification of fungi based on their volatile metabolite profiles. The volatiles sampling technique reported here is non-destructive, so it can be applied with traditional methods for studying fungal growth and metabolism.     

Neutral lipid fatty acid composition as trait and constraint in Collembola evolution

Functional traits determine the occurrence of species along environmental gradients and their coexistence with other species. Understanding how traits evolved among coexisting species helps to infer community assembly processes. We propose fatty acid composition in consumer tissue as a functional trait related to both food resources and physiological functions of species. We measured phylogenetic signal in fatty acid profiles of 13 field‐sampled Collembola (springtail) species and then combined the data with published fatty acid profiles of another 24 species. Collembola fatty acid profiles generally showed phylogenetic signal, with related species resembling each other. Long‐chain polyunsaturated fatty acids, related to physiological functions, demonstrated phylogenetic signal. In contrast, most food resource biomarker fatty acids and the ratios between bacterial, fungal, and plant biomarker fatty acids exhibited no phylogenetic signal. Presumably, fatty acids related to physiological functions have been constrained during Collembola evolutionary history: Species with close phylogenetic affinity experienced similar environments during divergence, while niche partitioning in food resources among closely related species favored species coexistence. Measuring phylogenetic signal in ecologically relevant traits of coexisting species provides an evolutionary perspective to contemporary assembly processes of ecological communities. Integrating phylogenetic comparative methods with community phylogenetic and trait‐based approaches may compensate for the limitations of each method when used alone and improve understanding of processes driving and maintaining assembly patterns.     

Phytochemical analysis and genetic characterization of six Hypericum species from Serbia

The secondary metabolite contents and genetic profiles of six Hypericum species (H. barbatum Jacq., H. hirsutum L., H. linarioides Bosse, H. maculatum Crantz, H. rumeliacum Boiss. and H. tetrapterum Fries), collected from different locations in Serbia, have been analyzed. Methanol extracts of the aerial parts of the plants were obtained by accelerated solvent extraction (ASE) at 40 degrees C and 100 bar, and analyzed for five pharmacologically important standard constituents (hyperoside, quercitrin, pseudohypericin, hyperforin and hypericin) by LC-MS/MS. The highest content of hypericin and pseudohypericin was observed in the H. barbatum extract, while the highest content of hyperforin and quercitrin was found in the H. tetrapterum extract and the highest content of hyperoside in the H. maculatum extract. A literature survey shows that the above six Hypericum species, with the exception of H. maculatum, have not been previously genetically profiled. In order to correlate the chemical constituents of the species under investigation with their genetic factors, genetic profiling of these species was undertaken using the random amplification of polymorphic DNA (RAPD) and single sequence repeat (SSR) profiles of the above selected plants. Among the 52 random primers used for the initial screening, only 10 yielded polymorphic RAPD profiles. A total of 111 polymorphic markers were generated using these primers. The SSR analysis shows that 8 out of the 10 primers used were polymorphic. The correlation among the species under investigation using the two genetic markers was performed using Jaccuard's coefficients of similarity and a high correlation (r=0.99) was obtained. The main conclusion from the above data is that there exists a stronger correlation for secondary metabolite contents with RAPD data than with SSR data among the six Hypericum species from Serbia.     

The Influence of Genotype and Environment on the Physiological and Metabolic Diversity ofFusarium compactum

Talbot, N. J., Vincent, P., and Wildman, H. G. 1996. The influence of genotype and environment on the physiological and metabolic diversity ofFusarium compactum. Fungal Genetics and Biology20,254–267. Fungal species produce a large variety of secondary metabolites which are of considerable interest to the pharmaceutical industry. It is clear that the secondary metabolite production of a species varies significantly in strains from different geographic locations and from different habitats. The influence of genotype and environment on metabolite production is, however, poorly understood. In this study we examined the influence of genotypic variability, physiological variability, environmental location, and habitat on metabolite production byFusarium compactum.Isolates of the fungus from two geographic locations and two distinct habitat types were examined for growth on 95 different carbon sources, and genotypic variability was determined using RAPDs and rDNA–RFLP analysis. In a blind test secondary metabolite production was assessed using HPLC profiles of methanolic cell extracts. A number of correlations were observed between genotypic groupings, as determined using parsimony, and specific metabolite production. Similar correlations were also observed with physiological groups although genotypic analysis proved to be a more sensitive predictor of metabolite variability. The data suggest a complex relationship between environment, genotype, and metabolite production but highlight the use of genetic screening as a means of optimizing the chances of identifying a wide range of metabolites from a given species.     

Differentiation in the seed protein profiles of two closely related species of Narcissus

In the present paper we have analysed the relationship between two closely related species of Narcissus L. [N. cantabricus D.C. and N. hedraeanthus (Webb & Heldr.) Colmeiro] by means of seed protein electrophoresis. We have calculated two different coefficients of similarity between the seed protein profiles of both species. With the aim of knowing the meaning of such coefficients we have compared them with the ones obtained in several other different groups.     

Ecological constraints limit the fitness of fungal hybrids in the Heterobasidion annosum species complex

The ability of two closely related species to maintain species boundaries in spite of retained interfertility between them is a documented driving force of speciation. Experimental evidence to support possible interspecific postzygotic isolation mechanisms for organisms belonging to the kingdom Fungi is still missing. Here we report on the outcome of a series of controlled comparative inoculation experiments of parental wild genotypes and F(1) hybrid genotypes between closely related and interfertile taxa within the Heterobasidion annosum fungal species complex. Results indicated that these fungal hybrids are not genetically unfit but can fare as well as parental genotypes when inoculated on substrates favorable to both parents. However, when placed in substrates favoring one of the parents, hybrids are less competitive than the parental genotypes specialized on that substrate. Furthermore, in some but not all fungus x plant combinations, a clear asymmetry in fitness was observed between hybrids carrying identical nuclear genomes but different cytoplasms. This work provides some of the first experimental evidence of ecologically driven postzygotic reinforcement of isolation between closely related fungal species characterized by marked host specificity. Host specialization is one of the most striking traits of a large number of symbiotic and parasitic fungi; thus, we suggest the ecological mechanism proven here to reinforce isolation among Heterobasidion spp. may be generally valid for host-specialized fungi. The validity of this generalization is supported by the low number of known fungal hybrids and by their distinctive feature of being found in substrates different from those colonized by parental species.     

Phylogenetic and morphological analysis of Aspergillus ochraceoroseus

Aspergillus ochraceoroseus produces the yellow-gold conidia and other characteristics of Aspergillus subgenus Circumdati section Circumdati. However, this species produces aflatoxin, a secondary metabolite characteristic of some members of subgenus Circumdati section Flavi and sterigmatocystin, a related secondary metabolite usually associated with subgenus Nidulantes sections Nidulantes and Versicolores, as well as members of several other genera. Our morphological data support the placement of A. ochraceoroseus in subgenus Circumdati. Sequence data from A. ochraceoroseus aflatoxin and sterigmatocystin genes aflR and nor-1/stcE, as well as 5.8S ITS and beta tubulin genes, were compared to those of aspergilli in sections Circumdati, Flavi, Nidulantes and Versicolores. In the sequence comparisons, A. ochraceoroseus was related more closely to the species in subgenus Nidulantes than to species from subgenus Circumdati.     

ITS1 versus ITS2 as DNA metabarcodes for fungi

The nuclear ribosomal Internal Transcribed Spacer ITS region is widely used as a DNA metabarcoding marker to characterize the diversity and composition of fungal communities. In amplicon pyrosequencing studies of fungal diversity, one of the spacers ITS1 or ITS2 of the ITS region is normally used. In this methodological study we evaluate the usability of ITS1 vs. ITS2 as a DNA metabarcoding marker for fungi. We analyse three data sets: two comprising ITS1 and ITS2 sequences of known taxonomic affiliations and a third comprising ITS1 and ITS2 environmental amplicon pyrosequencing data. Clustering analyses of sequences with known taxonomy using the bioinformatics pipeline ClustEx revealed that a 97% similarity cut‐off represent a reasonable threshold for estimating the number of known species in the data sets for both ITS1 and ITS2. However, no single threshold value worked well for all fungi at the same time within the curated UNITE database, and we found that the Operational Taxonomic Unit (OTU) concept is not easily translated into the level of species because many species are distributed over several clusters. Clustering analyses of the 134 692 ITS1 and ITS2 pyrosequences using a 97% similarity cut‐off revealed a high similarity between the two data sets when it comes to taxonomic coverage. Although some groups are under‐ or unrepresented in the two data sets due to, e.g. primer mismatches, our results indicate that ITS1 and ITS2 to a large extent yield similar results when used as DNA metabarcodes for fungi.     

Secondary contact zones of closely‐related Erebia butterflies overlap with narrow phenotypic and parasitic clines

Zones of secondary contact between closely related taxa are a common legacy of the Quaternary ice ages. Despite their abundance, the factors that keep species apart and prevent hybridization are often unknown. Here, we study a very narrow contact zone between three closely related butterfly species of the Erebia tyndarus species complex. Using genomic data, we first determined whether gene flow occurs and then assessed whether it might be hampered by differences in chromosome number between some species. We found interspecific gene flow between sibling species that differ in karyotype by one chromosome. Conversely, only F1 hybrids occurred between two species that have the same karyotype, forming a steep genomic cline. In a second step, we fitted clines to phenotypic, ecological and parasitic data to identify the factors associated with the genetic cline. We found clines for phenotypic data and the prevalence of the endosymbiont parasite Wolbachia to overlap with the genetic cline, suggesting that they might be drivers for separating the two species. Overall, our results highlight that some gene flow is possible between closely related species despite different chromosome numbers, but that other barriers restrict such gene flow.     

In vitro interactions between Fusarium verticillioides and Ustilago maydis through real-time PCR and metabolic profiling

The goal of this research was to determine mechanisms of interaction between endophytic strains of Fusarium verticillioides (Sacc.) Nirenberg and the pathogen, Ustilago maydis (DC) (Corda). Endophytic strains of the fungus F. verticillioides are commonly found in association with maize (Zea mays) and when co-inoculated with U. maydis, often lead to decreased disease severity caused by the pathogen. Here, we developed methods (liquid chromatography-mass spectrometry) to evaluate changes in relative concentration of metabolites produced during in vitro interactions between the endophyte and pathogen. Fungi were grown on two different media, in single and in confronted cultures. We used real-time PCR (qPCR) assays to measure relative changes in fungal biomass, that occurred in confronted cultures compared to single cultures. The results showed that most secondary metabolites are constitutively produced by each species. Metabolite profiles are complex for U. maydis (twenty chromatographic peaks detected) while relatively fewer compounds were detected for F. verticillioides (six chromatographic peaks). In confronted cultures, metabolite ratio (metabolite concentration/biomass) generally increases for U. maydis metabolites while no significant changes were observed for most F. verticillioides metabolites. The results show that F. verticillioides is a strong antagonist of U. maydis as its presence leads to large reductions in U. maydis biomass. We infer that few U. maydis metabolites likely serve antibiotic functions against F. verticillioides. The methods described here are sufficiently sensitive to detect small changes in biomass and metabolite concentration associated with differing genotypes of the interacting species.     

Predicting Plastid Marker Variation: Can Complete Plastid Genomes from Closely Related Species Help?

Rapidly evolving non-coding plastid regions (NCPs) are currently widely used in evolutionary biology especially in plant systematic studies where NCPs have become one of the most commonly used tools in clarifying species relationships. Currently, the generally small amount of sequence variation provided by NCPs compared to nuclear regions makes plastid phylogeny reconstruction challenging at the species-level, especially so in species rich clades such as Solanum with c. 1,200 species. Previous studies have established that the set of most highly variable NCPs vary between major plant families, and here we explore whether this variation extends beyond family level to genera and major clades within genera. Using full plastome data, we identify the most highly variable plastid markers in the Potato clade of Solanum. We then compare sequence variation between the Potato and the closely related Morelloid clade. Results show that whilst a narrow set of NCPs show consistently high variation, levels of sequence variation in most NCPs differ greatly between the two closely related clades. The high variation detected between closely related groups implies that repeated screening studies will be needed for individual projects despite the potential availability of results from closely related taxa, and indicates a narrower applicability of family-specific screening studies than previously thought.     

Phylogenetic and trait-based assembly of arbuscular mycorrhizal fungal communities

Both competition and environmental filtering are expected to influence the community structure of microbes, but there are few tests of the relative importance of these processes because trait data on these organisms is often difficult to obtain. Using phylogenetic and functional trait information, we tested whether arbuscular mycorrhizal (AM) fungal community composition in an old field was influenced by competitive exclusion and/or environmental filtering. Communities at the site were dominated by species from the most speciose family of AM fungi, the Glomeraceae, though species from two other lineages, the Acaulosporaceae and Gigasporaceae were also found. Despite the dominance of species from a single family, AM fungal species most frequently co-existed when they were distantly related and when they differed in the ability to colonize root space on host plants. The ability of AM fungal species to colonize soil did not influence co-existence. These results suggest that competition between closely related and functionally similar species for space on plant roots influences community assembly. Nevertheless, in a substantial minority of cases communities were phylogenetically clustered, indicating that closely related species could also co-occur, as would be expected if i) the environment restricted community membership to single functional type or ii) competition among functionally similar species was weak. Our results therefore also suggest that competition for niche space between closely related fungi is not the sole influence of mycorrhizal community structure in field situations, but may be of greater relative importance than other ecological mechanisms.