Species of Heterobasidion host a diverse pool of partitiviruses with global distribution and interspecies transmission

We investigated the geographic occurrence and genetic diversity of partitiviruses among 247 Heterobasidion specimens representing seven species and originating from Europe, Asia, and North America. Based on sequence analysis, partitiviruses were relatively rare, and occurred only in about 5 % of the Heterobasidion isolates analyzed, constituting a minority (about 28 %) of all virus-infected [double-stranded RNA (dsRNA)-positive] isolates. Altogether ten virus strains were characterized in sequence: one complete genome sequence of 3893 bp, six complete RNA-dependent RNA polymerase sequences of 2000-2033 bp, and three partial polymerase sequences. Based on phylogenetic analysis, the virus strains were assigned into three putative partitivirus species: HetRV1 (Heterobasidion RNA virus 1), HetRV4, and HetRV5. Degenerate consensus primers were designed for RT-PCR detection of these virus species. HetRV1 occurred in five different Heterobasidion species, and resembled the previously described Heterobasidion annosum virus (HaV). Highly similar HetRV1 strains with 98 % nucleotide level similarity were found from H. parviporum (member of the H. annosum species complex) and H. australe (member of the H. insulare complex) growing in the same region in Bhutan. This observation suggests recent virus transmission between these taxonomically distant Heterobasidion species in nature. It was also shown that HetRV1 can be transmitted by mycelial contact between the H. annosum and H. insulare complexes. The two other virus species, HetRV4 and HetRV5, were closely related to the Amasya Cherry Disease-associated mycovirus, to Heterobasidion parviporum partitivirus Fr110B, and also to several plant-infecting alphacryptoviruses. These results are in accordance with the view of a close evolutionary relationship between partitiviruses of plants and fungi.     

Evolutionary history of the conifer root rot fungus Heterobasidion annosum sensu lato

We investigated two hypotheses for the origin of the root rot fungus Heterobasidion annosum species complex: (i) that geology has been an important factor for the speciation (ii) that co-evolutionary processes with the hosts drove the divergence of the pathogen species. The H. annosum species complex consists of five species: three occur in Europe, H. annosum s.s., Heterobasidion parviporum and Heterobasidion abietinum, and two in North America, Heterobasidion irregulare and Heterobasidion occidentale; all with different but partially overlapping host preferences. The evolution of the H. annosum species complex was studied using six partially sequenced genes, between 10 and 30 individuals of each species were analysed. Neighbour-joining trees were constructed for each gene, and a Bayesian tree was built for the combined data set. In addition, haplotype networks were constructed to illustrate the species relationships. For three of the genes, H. parviporum and H. abietinum share haplotypes supporting recent divergence and/or possible gene flow. We propose that the H. annosum species complex originated in Laurasia and that the H. annosum s.s./H. irregulare and H. parviporum/H. abietinum/H. occidentale ancestral species emerged between 45 and 60 Ma in the Palaearctic, well after the radiation of the host genera. Our data imply that H. irregulare and H. occidentale were colonizing North America via different routes. In conclusion, plate tectonics are likely to have been the main factor influencing Heterobasidion speciation and biogeography.     

Inferences on the phylogeography of the fungal pathogen Heterobasidion annosum, including evidence of interspecific horizontal genetic transfer and of human-mediated, long-range dispersal

Fungi in the basidiomycete species complex Heterobasidion annosum are significant root-rot pathogens of conifers throughout the northern hemisphere. We utilize a multilocus phylogenetic approach to examine hypotheses regarding the evolution and divergence of two Heterobasidion taxa associated with pines: the Eurasian H. annosum sensu stricto and the North American H. annosum P intersterility group (ISG). Using DNA sequence information from portions of two nuclear and two mitochondrial loci, we infer phylogenetic relationships via parsimony, Bayesian and median-joining network analysis. Analysis of isolates representative of the entire known geographic range of the two taxa results in monophyletic sister Eurasian and North American lineages, with North America further subdivided into eastern and western clades. Genetically anomalous isolates from the Italian presidential estate of Castelporziano are always part of a North American clade and group with eastern North America, upholding the hypothesis of recent, anthropogenically mediated dispersal. P ISG isolates from Mexico have phylogenetic affinity with both eastern and western North America. Results for an insertion in the mitochondrial rDNA suggest this molecule was obtained from the Heterobasidion S ISG, a taxon sympatric with the P ISG in western North America. These data are compatible with an eastern Eurasian origin of the species, followed by dispersal of two sister taxa into western Eurasia and into eastern North America over a Beringean land bridge, a pattern echoed in the phylogeography of other conifer-associated basidiomycetes.     

Use of the nuclear gene glyceraldehyde 3-phosphate dehydrogenase for phylogeny reconstruction of recently diverged lineages in Mitthyridium (Musci: Calymperaceae)

A portion of the nuclear gene glyceraldehyde 3-phosphate dehydrogenase (gpd) was sequenced in 26 representatives of the paleotropical moss, Mitthyridium, and a group of 20 outgroup taxa to assess its utility for phylogenetic reconstruction compared with the better understood chloroplast markers, rps4 and trnL. Primers based on plant and fungal sequences were designed to amplify gpd in plants universally with the exclusion of fungal contaminants. The piece amplified spanned 4 introns and 3 of 9 exons, based on comparisons with complete sequence from Arabidopsis. Size variation in gpd ranged from 891 to 1007 bp, in part attributable to 6 indels of variable length found within the introns. Intron 6 contributed most of the length variation and contained a variable purine-repeat motif of possible use as a microsatellite. Phylogenetic analyses of the full gpd amplicon yielded well-resolved trees that were in nearly full accord with the trees derived from the cpDNA partitions for analyses of both the ingroup and ingroup + outgroup taxon sets. Pairwise nucleotide substitution rates of gpd were as much as 2.2 times higher than those in rps4 and 2.8 times higher than in trnL. Excision of the introns left suitable numbers of parsimony informative characters and demonstrated that the full gpd amplicon could be compartmentalized to provide resolution for both shallow and deep phylogenetic branches. Exons of gpd were found to behave in a clock-like fashion for the 26 ingroup taxa and select outgroups. In general, gpd was found to hold great promise not only for improving resolution of chloroplast-derived phylogenies, but also for phylogenetic reconstruction of recent, diversifying lineages.     

Species delimitation in fungal endophyte diversity studies and its implications in ecological and biogeographic inferences

The estimation of species diversity in fungal endophyte communities is based either on species counts or on the assignment of operational taxonomic units (OTUs). Consequently, the application of different species recognition criteria affects not only diversity estimates but also the ecological hypotheses that arise from those observations. The main objective of the study was to examine how the choice and number of genetic markers and species delimitation criteria influence biodiversity estimates. Here, we compare approaches to defining species boundaries in three dominant species complexes of tropical endophytes, specially Colletotrichum gloeosporioides agg., Pestalotiopsis microspora agg. and Trichoderma harzianum agg., from two Amazonian trees: Hevea brasiliensis and H. guianensis. Molecular tools were used to describe and compare the diversity of the different assemblages. Multilocus phylogenetic analyses [gpd, internal transcribed spacer (ITS) and tef1] and modern techniques for phylogenetic species delimitation were overlaid with ecological data to recognize putative species or OTUs. The results demonstrate that ITS alone generally underestimates the number of species predicted by other nuclear loci. These results question the use of ITS and arbitrary divergence thresholds for species delimitation.     

Iron and reactive oxygen responses in Pinus sylvestris root cortical cells infected with different species of Heterobasidion annosum sensu lato

Defence mechanisms in trees are not well understood. We assessed whether distribution of iron ions and their co-localisation with reactive oxygen species in Pinus sylvestris root cells reflect differential preferences of the pathogens Heterobasidion annosum sensu stricto, H. parviporum and H. abietinum to the host. Strains of H. annosum s.s. characterised by a greater preference for P. sylvestris induced accumulation of superoxide (O(2) (-)) in host cells 6?h after inoculation, whereas two peaks in accumulation of O(2) (-) (after 4 and 48?h) were observed after infection with strains of the pathogens H. parviporum and H. abietinum, which have a lower preference for P. sylvestris. Moreover, strains of H. annosum s.s. caused increased production of hydrogen peroxide (H(2)O(2)) in P. sylvestris cells, in contrast with strains of the other two species (H. parviporum and H. abietinum). Following inoculation with H. annosum s.s. strains, H(2)O(2) was correlated negatively with O(2) (-) and correlated positively with ferrous iron (Fe(2+)). Co-localisation of Fe(3+) with H(2)O(2) may suggest that they are involved in inducing hypersensitive responses and eventually cell death in roots inoculated with H. annosum s.s. strains, in contrast with H. parviporum, in which other mechanisms operate when the host is parasitised.     

Spread of Heterobasidion annosum s.s. and Heterobasidion parviporum in Picea abies 15 years after stump inoculation

The tree pathogenic fungi Heterobasidion annosum s.s. and Heterobasidion parviporum cause root and butt rot in Norway spruce (Picea abies) and produce serious economic losses to the forest sector in Europe. We experimentally studied inter- and intraspecific differences between H. parviporum and H. annosum s.s. in the way they infect stumps and spread into neighbouring trees. Eleven H. parviporum and nine H. annosum s.s. isolates were artificially inoculated on stumps of two spruce stands after first thinning. After 15 years, the same isolates were reisolated from neighbouring trees. Heterobasidion parviporum spread more frequently from the inoculated stumps to the neighbouring trees than H. annosum s.s. The surroundings of H. annosum s.s. stumps that did not spread were often colonized by H. parviporum. Heterobasidion annosum s.s. spread was restricted mainly to the areas of the plot where no other Heterobasidion genotypes had been inoculated. In such cases, H. annosum s.s. tended to develop into bigger genets than H. parviporum. The probability of stump-to-tree spread of H. parviporum depended on the diameter of the stumps, suggesting that H. parviporum spread may relate to the presence of heartwood. Both H. parviporum and H. annosum s.s. proved to be strong pathogens on Norway spruce; however, when competing for the same trees, H. parviporum seemed capable of excluding H. annosum s.s. from the stand.     

Heterologous array analysis in Heterobasidion: hybridisation of cDNA arrays with probe from mycelium of S, P or F-types

Because of the close relatedness between three species of Heterobasidion annosum (P-type), Heterobasidion parviporum (S-type) and Heterobasidion abietinum (F-type), we investigated the possible use of arrays from one species for studies of gene expression in the other. Clones containing partial cDNAs from 94 identifiable genes expressed during spore germination and differentiation in H. parviporum were printed manually in six replications on nylon membranes. The membrane was hybridized with chemifluorescent labelled cDNA from actively growing mycelia of H. parviporum, H. annosum or H. abietinum, cultivated on a non-selective substrate. Product-moment correlation coefficient varied between 0.81 and 0.49. Due to the level of correlation, in the gene expression among the intersterility groups, we concluded that the cDNA array of one can be used to study gene expression in the others.     

New genes for phylogenetic studies of lichenized fungi: glyceraldehyde-3-phosphate dehydrogenase and beta-tubulin genes

Abstract:Primers for amplification and sequencing of partial glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were designed for lichenized fungi. The 5′ gpd primer is most probably fungal specific, since a BLAST search in GenBank found identical sequences only from ascomycetous taxa, whereas the 3′ gpd primer was more universal. Utility of the gpd primers and previously designed beta-tubulin primers was tested in nine lichen taxa. Both the gpd and beta-tubulin primer pairs amplified in most of the taxa examined: the gpd primers generated a c. 1100 nucleotide fragment, whereas the PCR product obtained from the beta-tubulin primers was c. 900 nucleotides long. The gpd amplification products of Cladonia arbuscula and C. rangiferina were sequenced and both were found to contain three introns, the length of which varied between 49 to 83 nucleotides. To examine the applicability of gpd sequences in resolving relationships within Ascomycota, trees were calculated from 22 fungal gpd sequences obtained from GenBank together with the twoCladonia sequences using parsimony jackknifing. The gpd tree was compared with the SSU rDNA tree of the respective species (or genera). A similar analysis of the beta-tubulin gene was not performed, because only a few beta-tublin sequences from the same taxa were available in GenBank. The gpd tree was well resolved but in conflict with the SSU rDNA tree. In contrast to the SSU rDNA tree, the gpd tree did not support the monophyly of the Ascomycota. Analysis of the combined data set produced a tree very similar to that of the SSU rDNA data. However, the relationship of Lecanorales to the other orders remained unresolved. Even though gpd and beta-tubulin are highly conserved proteins, the third codon positions and introns are variable and both genes have the potential for inferring phylogenetic relationships at the lower taxonomic levels in the lichenized fungi. The two genes may be useful even below species level, depending on the species investigated.     

Significant diversity and potential problems associated with inferring population structure within the Cenococcum geophilum species complex

Cenococcum geophilum is perhaps the most widely distributed and most recognized ectomycorrhizal fungus with a host range of more than 200 tree species from 40 genera of both angiosperms and gymnosperms. We conducted a phylogenetic analysis on a large collection of isolates (n=74) from North America and Europe based on glyceraldehyde 3-phosphate dehydrogenase (gpd). A subset of isolates (n=22) also was analyzed with the more conservative LSU-rDNA locus. Significant nucleotide diversity was detected (approximately 20%) in the gpd region and the LSU-rDNA analysis supported that the C. geophilum isolates studied were monophyletic but distinct from two isolates, Am5-1 and N2-10, which previously were used in population genetic studies of this species. These results suggest that Am5-1 and N2-10 are likely two undescribed species or even genera. Our results suggest that C. geophilum sensu lato is a species complex and support previous molecular, physiological and morphological studies that have shown significant diversity in C. geophilum. This study also revealed that caution is advised when conducting population genetic studies in C. geophilum due to the possibility of pooling unrelated isolates. This potential problem also has implications for other fungal taxa because cryptic species routinely have been found in recent years based on molecular data.     

The phylogenetic position of an <Emphasis Type="Italic">Armillaria</Emphasis> species from Amami-Oshima,a subtropical island of Japan,based on elongation factor and ITS sequences

An undetermined Armillaria species was collected on Amami-Oshima, a subtropical island of Japan. The phylogenetic position of the Armillaria sp. was determined using sequences of the elongation factor-1α (EF-1α) gene and the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of ribosomal DNA (rDNA). The phylogenetic analyses based on EF-1α and ITS sequences showed that this species differs from known Japanese taxa of Armillaria. The sequences of this species and A. novae-zelandiae from Southeast Asia were contained in a strongly supported clade, which was adjacent to a well-supported sister clade containing A. novae-zelandiae from Australia and New Zealand.     

Stability, ephemerality and dispersal ability: microarthropod assemblages on fungal sporophores

A comprehensive analysis of the microarthropod fauna from fungal sporophores revealed a series of recurrent patterns demonstrating the non-random structure of these assemblages. Despite the existence of a strong species-area relationship from sporophores of certain fungi, particularly among perennial species, several small sporophores always maintained a more diverse microarthropod fauna than fewer large sporophores of equivalent total area. A consistendy high degree of overlap in microarthropod species occurrence between larger and smaller sporophores was indicated by the presence of highly significant nested subset structure. The fauna from Hetervbasidion annosum sporophores was the most nested, followed by Hypholoma fasciculate and a collection of pooled agarics, respectively. When the fauna was split into functional groups, microphytophages were always significandy nested in their distributions but, when significant, macrophytophages and panphytophages had stronger nested hierarchies. Non-random organization was least evident among predatory species. Micro-arthropods had significandy ordered distributions on sporophores of various fungi. Many species occurred on perennial H. annosum sporophores of all sizes while others had more restricted distributions. Most species fi/om very ephemeral agarics, including those which were widely distributed on H. annosum, were restricted to a small number oflarger sporophores. The pattern from H. fasciculare was intermediate; most species had narrow distributions and were present only on larger sporophores, except a small number of more widely distributed species.     

Phylogenetic study and identification of human pathogenic Vibrio species based on partial hsp60 gene sequences

The use of hsp60 gene sequences for phylogenetic study and identification of pathogenic marine vibrios was investigated. A 600-bp partial hsp60 gene was amplified by PCR and sequenced from 29 strains representing 15 Vibrio species within the family Vibrionaceae. Sequence comparison of the amplified partial hsp60 gene revealed 71-82% sequence identity among different Vibrio species and 96-100% sequence identity among epidemiologically distinct strains with the same species designation. This degree of discrimination allows unambiguous differentiation of all Vibrio species included in the current study from each other, as well as from Aeromonas hydrophila and Plesiomonas shigelloides, which are often misidentified as Vibrio species by conventional biochemical methods. Based on the hsp60 gene sequences, two previously unidentified shrimp isolates were found to be more closely related to Vibrio alginolyticus (93-94% sequence identity) than to Vibrio parahaemolyticus (89% sequence identity), whereas 16S rRNA gene analysis was unable to differentiate among these closely related species (95-97% sequence identity). Our results indicate that the hsp60 gene may be a useful alternative target for phylogenetic analysis and species identification of marine Vibrios to complement more conventional identification systems.     

Molecular phylogeny and divergence time of the water penny genus Eubrianax (Coleoptera: Psephenidae) in Japan

The phylogenetic relationships among the Japanese members of the genus Eubrianax (Coleoptera: Psephenidae) were examined using the mitochondrial cytochrome oxidase subunit I (COI) gene and nuclear 28S rRNA gene sequences. Based on the molecular phylogeny as well as morphological features, the species status of Eubrianax brunneicornis Nakane, 1952 was proposed. The phylogenetic analyses recovered monophyly of the previously proposed pellucidus species group with four Japanese species, whereas a single Japanese species of the granicollis group was included in the lineage of the ramicornis group with five Japanese species. The divergence times of the species were estimated by dating the phylogenetic tree against the fossil record and a molecular clock based on the COI gene. The divergence of the Japanese species was inferred to have occurred during the Pliocene epoch.     

A re-examination of the phylogenetic relationship between the causal agents of carrot black rot, Alternaria radicina and A. carotiincultae

The phylogenetic relationship between Alternaria radicina and A. carotiincultae was reexamined based on morphology, sequence analysis of rDNA (ITS and mitochondrial small subunit [mtSSU]), protein coding genes (actin [ACT], beta-tubulin, chitin synthase [CHS], translation elongation factor [EF-1a], Alternaria allergen a1 [Alt a1], and glyceraldehyde-3-phosphate dehydrogenase [gpd]), and RAPD and ISSR analysis of total genomic DNA. Although some morphological characters overlapped to a degree, with A. radicina isolates expressing moderate variation and A. carotiincultae isolates being highly uniform, A. carotiincultae could be differentiated from A. radicina based on significantly greater growth rate on potato dextrose agar (PDA) or acidified PDA (APDA) and average number of transverse septa per conidium. Sequence of rDNA and two protein coding genes, ACT and CHS, were invariant between species. However polymorphism with the EF-1a, beta-tubulin, and Alt a1 gene strictly separated the population of A. radicina and A. carotiincultae as distinct lineages, as did RAPD and ISSR analysis. The polymorphic gpd gene did not strictly separate the two species. However isolates of A. radicina encompassed several haplotypes, one of which was the exclusive haplotype possessed by A. carotiincultae isolates, suggesting evidence of incomplete lineage sorting. The results suggest that A. carotiincultae is closely related to A. radicina but is a recently divergent and distinct lineage, which supports its status as a separate species.     

Molecular markers reveal genetic isolation and phylogeography of the S and F intersterility groups of the wood-decay fungus Heterobasidion annosum

The root-rot fungus Heterobasidion annosum (Fr.) Bref. species complex consists of three intersterility groups (S, F, and P), separated by their host affinity. The phylogenetic relationship of the species complex was studied, with the focus on the S and F groups, by comparing DNA sequences of four nuclear gene fragments: calmodulin, glyceraldehyde 3-phosphate dehydrogenase, heat stress protein 80-1, and elongation factor 1-alpha, and one anonymous locus, from 29 fungal isolates originating from Europe, Asia, and North America. The phylogeny of each separate gene locus as well as the combined dataset consisted of three main clades: European F group isolates, Euroasian S group isolates, and North American S group isolates, suggesting them to be separated into phylogenetic species. The results also support the hypothesis of an early separation between the S and F groups, indicating that their distribution have followed their host tree species for a considerable time period.     

B型烟粉虱和温室白粉虱热激蛋白90基因（hsp90）的全长cDNA克隆与系统发育分析

B型烟粉虱Bemisia tabaci (Gennadius) biotype B和温室白粉虱Trialeurodes vaporariorum均为全球普遍发生的重要害虫。本研究以其他昆虫热激蛋白90基因（hsp90）保守区域设计兼并引物扩增两种粉虱hsp90中间片段, 然后利用RACE技术获得全长cDNA。温室白粉虱hsp90全长cDNA的开放性阅读框2 166 bp, 编码722个氨基酸; 烟粉虱hsp90全长cDNA的开放性阅读框2 160 bp, 编码720个氨基酸。两种粉虱HSP90的完整氨基酸序列相似性高达92.94％, 并均具有定义HSP90家族签名序列的5个氨基酸保守区域和末尾基序“MEEVD”。通过real-time PCR技术, 探测到两个基因在mRNA水平上皆能高温诱导表达。采用昆虫纲所有完整HSP90氨基酸序列进行Kimura双参数遗传距离分析并构建NJ进化树, 结果显示hsp90在昆虫纲低级阶元水平和高级阶元水平系统进化上能得到一个较理想结果。本研究结果为B型烟粉虱和温室白粉虱抗逆适应性研究提供基础, 并进一步验证保守的功能基因hsp90可以作为研究生物系统发育的手段之一     

The Heat-Shock Gene hsp83 of Drosophila auraria: Genomic Organization, Nucleotide Sequence, and Long Antiparallel Coupled ORFs (LAC ORFs)

The genomic organization of the hsp83 gene of Drosophila auraria, a far-eastern endemic species belonging to the montium subgroup of the melanogaster species group, is presented here. Based on in situ hybridization on polytene chromosomes, cDNA and genomic clone mapping, nucleotide sequencing, and genomic Southern analysis, hsp83 is shown to be present as a single-copy gene at locus 64B on the 3L chromosome arm in D. auraria. This gene is organized into two exons separated by a 929-bp intron. The first exon represents the mRNA leader sequence and is not translated, while the coding region, having a length of 2,151 bp, is solely included in the second exon. Nucleotide sequence comparisons of D. auraria hsp83 with homologous sequences from other organisms show high conservation of the coding region (88–92% identity) in the genus Drosophila, in addition to the conserved genomic organization of two-exons–one-intron, of comparable size and arrangement. A phylogenetic tree based on the protein sequences of homologous genes from representative organisms is in accord with the accredited phylogenetic position of D. auraria. In the hsp83 gene region, a second case of long antiparallel coupled open reading frames (LAC ORFs) for this species was found. The antiparallel to the hsp83 gene ORF is 1,554 bases long, while the two ORFs overlap has a size of 1,548 bp. The anti-hsp83 ORF does not show significant homology to any known gene sequences. In addition, no similar LAC ORF structures were found in homologous gene regions of other organisms. Received: 18 April 1997 / Accepted: 1 August 1997