Two new species of Leptographium associated with Tomicus spp. infesting Pinus spp. in Southwestern China.

Leptographium panxianense and L. puerense are proposed as new taxa based on sequence data and morphological characters. The phylogenetic analyses based on ITS2-partial LSU rDNA region, ß-tubulin and elongation factor 1-α genes showed that L. panxianense and L. puerense formed well-supported clades and were closely related to L. yunnanense, L. wushanense and L. conjunctum, and then nested within the L. lundbergii complex. The two species differ in their conidial size and shape. The conidia of L. panxianense are larger than those of L. puerense while the conidial shape of L. puerense is more ovovoid. The optimal growth temperature of both L. panxianense and L. puerense is at 20 °C, which is different from those of L. yunnanense, L. wushanense and L. conjunctum. Comparison of sequence data and morphological characters confirmed the placement of the two undescribed taxa in the genus of Leptographium.

Three new Leptographium spp. (Ophiostomatales) infecting hardwood trees in Norway and Poland.

Species of Leptographium are characterized by mononematous or synnematous conidiophores and are commonly associated with different arthropods. Some of them also produce a sexual state characterised by globose ascomata with elongated necks. Compared to investigations on coniferous trees, the occurrence of Leptographium species on hardwood trees has been poorly studied in Europe. During a survey of ophiostomatoid fungi on various hardwood tree species in Norway and Poland, three unusual species, which fit in the broader morphological description of Leptographium spp., were found in association with Trypodendron domesticum, Trypodendron signatum and Dryocoetes alni, and from wounds on a variety of hardwoods. Phylogenetic analyses of sequence data for six different loci (ITS1-5.8 S-ITS2, ITS2-LSU, ACT, ß-tubulin, CAL, and TEF-1α) showed that these Leptographium species are phylogenetically closely related to the species of the Grosmannia olivacea complex. The first species forms a well-supported lineage that includes Ophiostoma brevicolle, while the two other new taxa resided in a separate lineage; possibly affiliated with Grosmannia francke-grosmanniae. All the new species produce perithecia with necks terminating in ostiolar hyphae and orange-section shaped ascospores with cucullate, gelatinous sheaths. These species also produce dark olivaceous mononematous asexual states in culture. In addition, two of the newly described species have a second type of conidiophore with a short and non-pigmented stipe. The new Leptographium species can be easily distinguished from each other by their appearance and growth in culture. Based on novel morphological characters and distinct DNA sequences, these fungi were recognised as new taxa for which the names Leptographium tardum sp. nov., Leptographium vulnerum sp. nov., and Leptographium flavum sp. nov. are provided.

Two new Leptographium spp. reveal an emerging complex of hardwood-infecting species in the Ophiostomatales.

Species of Leptographium are generally characterized by mononematous conidiophores and are commonly associated with bark beetles and weevils. These species are responsible for sapstain and in some cases serious diseases on a range of primarily coniferous trees. In comparison with coniferous trees, the occurrence of Leptographium species on hardwood trees has been poorly studied in Europe. During a survey of ophiostomatoid fungi on various tree species in Norway and Poland, three unusual species, which fit the broader morphological description of Leptographium spp., were found in association with Scolytus ratzeburgi, Dryocoetes alni and Trypodendron domesticum on a variety of hardwoods, and from wounds on Tilia cordata. Phylogenetic analyses of sequence data for three gene regions (ITS2-LSU, ß-tubulin, and TEF1-α) showed that these Leptographium species are phylogenetically closely related to each other and form a well-supported lineage that included Grosmannia grandifoliae and Leptographium pruni. The first species could be distinguished from the other Leptographium species based on conidiophores arising from spiral hyphae, chlamydospore-like structures and a hyalorhinocladiella-like synanamorph in culture. The second species differs from the previous one by having distinctly shorter conidiophores and smaller conidia. This species also produces a well-developed sporothrix-like synanamorph with denticulate conidiogenous cells. Based on these unusual morphological characteristics and distinct DNA sequences, these fungi were recognised as new taxa for which the names Leptographium trypodendri sp. nov. and L. betulae sp. nov. are provided. The third group of isolates belonged to Grosmannia grandifoliae, representing the first report of this species outside of the USA. The newly defined G. grandifoliae complex is the first species complex in Leptographium s.l. consisting of only hardwood-infecting species.

Wounds on Rapanea melanophloeos provide habitat for a large diversity of Ophiostomatales including four new species.

Rapanea melanophloeos, an important canopy tree in Afromontane forests, is commonly utilised for medicinal bark harvesting. Wounds created from these activities provide entrance for many fungi, including arthropod-associated members of the Ophiostomatales and Microascales (ophiostomatoid fungi). In this study we assessed the diversity of wound-associated Ophiostomatales on storm-damaged R. melanophloeos trees in the Afromontane forests of South Africa. Five species were identified based on micro-morphological and molecular phylogenetic analyses. These included Ophiostoma stenoceras and four newly described taxa Sporothrix itsvo sp. nov., S. rapaneae sp. nov., S. uta sp. nov. and O. noisomeae sp. nov. Four of these are members of the S. schenckii-O. stenoceras complex (O. stenoceras, S. itsvo sp. nov., S. rapaneae sp. nov., S. uta sp. nov.) while O. noisomeae groups basal in the Ophiostomatales alongside the S. lignivora complex and Graphilbum. In addition to other taxa known from this host, the present study shows that there is a rich, yet still poorly explored, diversity of Ophiostomatales associated with R. melanophloeos in Afromontane forests. More taxa are likely to be discovered with increased research effort. These must be assessed in terms of pathogenicity towards this ecologically and economically important tree.

Novel ophiostomatalean fungi from galleries of Cyrtogenius africus (Scolytinae) infesting dying Euphorbia ingens.

Euphorbia ingens trees have been dying in large numbers in the Limpopo Province of South Africa for approximately 15 years. The ambrosia beetle Cyrtogenius africus is often found infesting diseased and dying trees. The aim of this study was to identify the ophiostomatoid fungi occurring in the galleries of C. africus. Logs infested with this beetle were collected from the KwaZulu-Natal, Limpopo, Mpumalanga, and North West Provinces of South Africa. Fungi belonging to the Ophiostomatales were identified based on morphology and comparison of sequence data for the ß-tubulin, ITS1-5.8S-ITS2 and LSU gene regions. A novel species of Ophiostoma and a novel genus in the Ophiostomatales were identified. Inoculation studies with these fungi produced lesions in the branches of healthy E. ingens trees.

Ophiostomatoid fungi associated with mangroves in South Africa, including Ophiostoma palustre sp. nov.

Mangrove trees are continuously under stress due to environmental and/or anthropogenic pressures, which expose them to attack by pathogens, compromising their survival. Ophiostomatoid fungi cause sap stain and diseases of a wide spectrum of tree species globally. These fungi infect trees through natural, insect, animal and/or human made wounds. During routine surveys of mangrove trees in South Africa, wounds on branches and stems of Avicennia marina were regularly monitored for the presence of ophiostomatoid fungi at ten study sites in the country. The stems of four mangrove species, A. marina, Bruguiera gymnorrhiza, Rhizophora mucronata and Barringtonia racemosa were also wounded and evaluated for the appearance of these fungi. Ophiostomatoid fungi were obtained from the mangrove associate B. racemosa, but not from any of the true mangroves. Analyses of DNA sequence data for the internal transcribed spacer, ß-tubulin, calmodulin and translation elongation factor gene regions revealed that the fungi isolated from the wounds on B. racemosa belong to three species in the Ophiostomataceae, including a new taxon described here as Ophiostoma palustre sp. nov. These results suggest that the mangrove associate B. racemosa is more prone to colonization by ophiostomatoid fungi than the true mangroves.

Comparative genomics of the pine pathogens and beetle symbionts in the genus Grosmannia.

Studies on beetle/tree fungal symbionts typically characterize the ecological and geographic distributions of the fungal populations. There is limited understanding of the genome-wide evolutionary processes that act within and between species as such fungi adapt to different environments, leading to physiological differences and reproductive isolation. Here, we assess genomic evidence for such evolutionary processes by extending our recent work on Grosmannia clavigera, which is vectored by the mountain pine beetle and jeffrey pine beetle. We report the genome sequences of an additional 11 G. clavigera (Gc) sensu lato strains from the two known sibling species, Grosmannia sp. (Gs) and Gc. The 12 fungal genomes are structurally similar, showing large-scale synteny within and between species. We identified 103,430 single-nucleotide variations that separated the Grosmannia strains into divergent Gs and Gc clades, and further divided each of these clades into two subclades, one of which may represent an additional species. Comparing variable genes between these lineages, we identified truncated genes and potential pseudogenes, as well as seven genes that show evidence of positive selection. As these variable genes are involved in secondary metabolism and in detoxifying or utilizing host-tree defense chemicals (e.g., polyketide synthases, oxidoreductases, and mono-oxygenases), their variants may reflect adaptation to the specific chemistries of the host trees Pinus contorta, P. ponderosa, and P. jeffreyi. This work provides a comprehensive resource for developing informative markers for landscape population genomics of these ecologically and economically important fungi, and an approach that could be extended to other beetle-tree-associated fungi.

Taxonomy and phylogeny of the Leptographium procerum complex, including Leptographium sinense sp. nov. and Leptographium longiconidiophorum sp. nov.

Leptographium procerum (Ophiostomatales, Ascomycota) is a well-known fungal associate of pine root-infesting bark beetles and weevils, occurring in several countries of the world. The fungus is not a primary pathogen but has been associated with white pine root decline in the USA and with serious damage caused by the introduced red turpentine beetle (RTB) Dendroctonus valens in China. Several species closely related to L. procerum have been described during the past decade. The aim of this study was to reevaluate species boundaries in the L. procerum complex using multigene phylogenetic analyses and morphological comparisons. Phylogenetic analyses of seven gene regions (ITS2-LSU, actin, ß-tubulin, calmodulin, translation elongation factor 1-α, and the mating type genes MAT1-1-3 and MAT1-2-1) distinguished between nine species in the complex. These included L. procerum, L. bhutanense, L. gracile, L. profanum, L. pini-densiflorae, L. sibiricum, L. sinoprocerum, as well as two new species described here as Leptographium sinense sp. nov. from Hylobitelus xiaoi on Pinus elliottii in China, and Leptographium longiconidiophorum sp. nov. from Pinus densiflora in Japan. Leptographium latens is reduced to synonymy with L. gracile, and an epitype is designated for L. procerum, because a living culture associated with the holotype of L. procerum did not exist. Amplification patterns of the mating type genes suggest that all known species in the L. procerum complex are heterothallic, although sexual states have not been observed for any of the species. The results also suggest that Eastern Asia is most probably the centre of species diversity for the L. procerum complex.

New species of Ophiostomatales from Scolytinae and Platypodinae beetles in the Cape Floristic Region, including the discovery of the sexual state of Raffaelea.

Olea capensis and Rapanea melanophloeos are important canopy trees in South African Afromontane forests. Dying or recently dead individuals of these trees are often infested by Scolytinae and Platypodinae (Curculionidae) beetles. Fungi were isolated from the surfaces of beetles emerging from wood samples and their galleries. Based on micro-morphological and phylogenetic analyses, four fungal species in the Ophiostomatales were isolated. These were Sporothrix pallida and three taxa here newly described as Sporothrix aemulophila sp. nov., Raffaelea vaginata sp. nov. and Raffaelea rapaneae sp. nov. This study represents the first collection of S. pallida, a species known from many environmental samples from across the world, from Scolytinae beetles. S. aemulophila sp. nov. is an associate of the ambrosia beetle Xyleborinus aemulus. R. rapaneae sp. nov. and R. vaginata sp. nov. were associated with a Lanurgus sp. and Platypodinae beetle, respectively, and represent the first Raffaelea spp. reported from the Cape Floristic Region. Of significance is that R. vaginata produced a sexual state analogous with those of Ophiostoma seticolle and O. deltoideosporum that also grouped in our analyses in Raffaelea s. str., to date considered an asexual genus. The morphology of the ossiform ascospores and anamorphs of the three species corresponded and the generic circumscription of Raffaelea is thus emended to accommodate sexual states. The two known species are provided with new combinations, namely Raffaelea seticollis (R.W. Davidson) Z.W. de Beer and T.A. Duong comb. nov. and Raffaelea deltoideospora (Olchow. and J. Reid) Z.W. de Beer and T.A. Duong comb. nov.

Ophiostomatoid fungi including two new fungal species associated with pine root-feeding beetles in northern Spain.

Many bark beetles live in a symbiosis with ophiostomatoid fungi but very little is known regarding these fungi in Spain. In this study, we considered the fungi associated with nine bark beetle species and one weevil infesting two native tree species (Pinus sylvestris and Pinus nigra) and one non-native (Pinus radiata) in Cantabria (Northern Spain). This included examination of 239 bark beetles or their galleries. Isolations yielded a total of 110 cultures that included 11 fungal species (five species of Leptographium sensu lato including Leptographium absconditum sp. nov., five species of Ophiostoma sensu lato including Ophiostoma cantabriense sp. nov, and one species of Graphilbum). The most commonly encountered fungal associates of the bark beetles were Grosmannia olivacea, Leptographium procerum, and Ophiostoma canum. The aggressiveness of the collected fungal species was evaluated using inoculations on two-year-old P. radiata seedlings. Leptographium wingfieldii, Leptographium guttulatum, and Ophiostoma ips were the only species capable of causing significant lesions.

Multigene phylogenies of Ophiostomataceae associated with Monterey pine bark beetles in Spain reveal three new fungal species.

Ophiostoma species, some of which cause sapstain in timber and/or are mild pathogens, are common fungal associates of bark beetles (Coleoptera: Scolytinae). Three new Ophiostomataceae from Spain are recognized in the present study based on comparisons of sequence data for three gene regions as well as morphological characteristics. The new taxa are described as Ophiostoma nebulare sp. nov., Ophiostoma euskadiense sp. nov. and Graphilbum crescericum sp. nov.

The mtDNA rns gene landscape in the Ophiostomatales and other fungal taxa: twintrons, introns, and intron-encoded proteins.

Comparative sequence analysis of the mitochondrial small subunit ribosomal RNA (rns) gene among species of Ophiostoma, Grosmannia, Ceratocystiopsis and related taxa provides an overview of the types of introns that have invaded this gene within the ophiostomatoid fungi. The rns gene appears to be a reservoir for a number of group I and group II introns along with intron-associated open reading frames such as homing endonucleases and reverse transcriptases. This study uncovered two twintrons, one at position mS917 where a group ID intron encoding a LAGLIDADG ORF invaded another ORF-less group ID intron. Another twintron complex was detected at position mS1247 here a group IIA1 intron invaded the open reading frame embedded within a group IC2 intron. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent losses. Results of this study will make a significant contribution to the understanding of the complexity of the mitochondrial intron landscape, and offer a resource to those annotating mitochondrial genomes. It will also serve as a resource to those that bioprospect for ribozymes and homing endonucleases.

The relative abundance of mountain pine beetle fungal associates through the beetle life cycle in pine trees.

The mountain pine beetle (MPB) is a native bark beetle of western North America that attacks pine tree species, particularly lodgepole pine. It is closely associated with the ophiostomatoid ascomycetes Grosmannia clavigera, Leptographium longiclavatum, Ophiostoma montium, and Ceratocystiopsis sp.1, with which it is symbiotically associated. To develop a better understanding of interactions between beetles, fungi, and host trees, we used target-specific DNA primers with qPCR to assess the changes in fungal associate abundance over the stages of the MPB life cycle that occur in galleries under the bark of pine trees. Multivariate analysis of covariance identified statistically significant changes in the relative abundance of the fungi over the life cycle of the MPB. Univariate analysis of covariance identified a statistically significant increase in the abundance of Ceratocystiopsis sp.1 through the beetle life cycle, and pair-wise analysis showed that this increase occurs after the larval stage. In contrast, the abundance of O. montium and Leptographium species (G. clavigera, L. longiclavatum) did not change significantly through the MPB life cycle. From these results, the only fungus showing a significant increase in relative abundance has not been formally described and has been largely ignored by other MPB studies. Although our results were from only one site, in previous studies we have shown that the fungi described were all present in at least ten sites in British Columbia. We suggest that the role of Ceratocystiopsis sp.1 in the MPB system should be explored, particularly its potential as a source of nutrients for teneral adults.

Grosmannia and Leptographium spp. associated with conifer-infesting bark beetles in Finland and Russia, including Leptographium taigense sp. nov.

Species of Grosmannia with Leptographium anamorphs include important forest pathogens and agents of blue stain in timber. They are commonly found in association with forest pests, such as bark beetles. During a survey of ophiostomatoid fungi in eastern parts of Finland and neighboring Russia, species belonging to the genus Grosmannia were isolated from 12 different bark beetle species infesting Picea abies and Pinus sylvestris, the most economically important conifers in the region. Identification of these fungi was based on morphology, DNA sequence comparisons for three gene regions and phylogenetic analyses. A total of ten taxa were identified. These belonged to six different species complexes in Grosmannia. The phylogenetic analyses provided an opportunity to redefine the G. galeiformis-, L. procerum-, L. lundbergii-, G. piceiperda-, G. olivacea- and G. penicillata-complexes, and to consider the species emerging from the survey within the context of these complexes. The species included G. galeiformis, G. olivacea, L. chlamydatum, L. lundbergii, L. truncatum and a novel taxon, described here as L. taigense sp. nov. In addition, species closely related to G. cucullata, G. olivaceapini comb. nov., G. piceiperda and L. procerum were isolated but their identity could not be resolved. The overall results indicate that the diversity of Grosmannia species in the boreal forests remains poorly understood and that further studies are needed to clarify the status of several species or species complexes.

Phylogeny and taxonomy of species in the Grosmannia serpens complex.

Grosmannia serpens was first described from pine in Italy in 1936 and it has been recorded subsequently from many countries in both the northern and southern hemispheres. The fungus is vectored primarily by root-infesting bark beetles and has been reported to contribute to pine-root diseases in Italy and South Africa. The objective of this study was to consider the identity of a global collection of isolates not previously available and using DNA sequence-based comparisons not previously applied to most of these isolates. Phylogenetic analyses of the ITS2-LSU, actin, beta-tubulin, calmodulin and translation elongation factor-1 alpha sequences revealed that these morphologically similar isolates represent a complex of five cryptic species. Grosmannia serpens sensu stricto thus is redefined and comprises only isolates from Italy including the ex-type isolate. The ex-type isolate of Verticicladiella alacris was shown to be distinct from G. serpens, and a new holomorphic species, G. alacris, is described. The teleomorph state of G. alacris was obtained through mating studies in the laboratory, confirming that this species is heterothallic. Most of the available isolates, including those from South Africa, USA, France, Portugal and some from Spain, represent G. alacris. The remaining three taxa, known only in their anamorph states, are described as the new species Leptographium gibbsii for isolates from the UK, L. yamaokae for isolates from Japan and L. castellanum for isolates from Spain and the Dominican Republic.

Comparative phylogeography, genetic differentiation and contrasting reproductive modes in three fungal symbionts of a multipartite bark beetle symbiosis.

Multipartite symbioses are complex symbiotic relationships involving multiple interacting partners. These types of partnerships provide excellent opportunities in which to apply a comparative approach to identify common historical patterns of population differentiation and species-specific life history traits. Using three symbiotic blue-stain fungal species (Ophiostomatacea) associated with outbreaking populations of the mountain pine beetle (Dendroctonus ponderosae Hopkins) in western Canada, we applied phylogenetic, population genetic and demographic approaches to clarify phylogeographic patterns among the three fungal species. Broadly, the three species showed significant population differentiation, forming northern and southern populations, despite dramatic differences in haplotype diversity. Finer structuring and population demographic patterns were less consistent, showing some interspecific incongruence. By contrasting these species simultaneously, we were able to identify differences in recombination rate and ecological traits that can explain the observed patterns of incongruence among the fungal species. By applying a comparative approach to partners of a multipartite symbiosis, we were able to distinguish congruent population structuring and species-specific differences that help us to understand the complexity and evolution of this symbiotic system.

Gene genealogies reveal cryptic species and host preferences for the pine fungal pathogen Grosmannia clavigera.

Grosmannia clavigera is a fungal pathogen of pine forests in western North America and a symbiotic associate of two sister bark beetles: Dendroctonus ponderosae and D. jeffreyi. This fungus and its beetle associate D. ponderosae are expanding in large epidemics in western North America. Using the fungal genome sequence and gene annotations, we assessed whether fungal isolates from the two beetles inhabiting different species of pine in epidemic regions of western Canada and the USA, as well as in localized populations outside of the current epidemic, represent different genetic lineages. We characterized nucleotide variations in 67 genomic regions and selected 15 for the phylogenetic analysis. Using concordance of gene genealogies and distinct ecological characteristics, we identified two sibling phylogenetic species: Gc and Gs. Where the closely related Pinus ponderosa and P. jeffreyi are infested by localized populations of their respective beetles, Gc is present. In contrast, Gs is an exclusive associate of D. ponderosae mainly present on its primary host-tree P. contorta; however, in the current epidemic areas, it is also found in other pine species. These results suggest that the host-tree species and the beetle population dynamics may be important factors associated with the genetic divergence and diversity of fungal partners in the beetle-tree ecosystems. Gc represents the original G. clavigera holotype, and Gs should be described as a new species.

Spatial community structure of mountain pine beetle fungal symbionts across a latitudinal gradient.

Symbiont redundancy in obligate insect-fungal systems is thought to buffer the insect host against symbiont loss and to extend the environmental conditions under which the insect can persist. The mountain pine beetle is associated with at least three well-known and putatively obligate ophiostomatoid fungal symbionts that vary in their environmental tolerances. To better understand the spatial variation in beetle-fungal symbiotic associations, we examined the community composition of ophiostomatoid fungi associated with the mountain pine beetle as a function of latitude and elevation. The region investigated represents the leading edge of a recent outbreak of mountain pine beetle in western Canada. Using regression and principal components analysis, we identified significant spatial patterns in fungal species abundances that indicate symmetrical replacement between two of the three fungi along a latitudinal gradient and little variation in response to elevation. We also identified significant variation in the prevalence of pair-wise species combinations that occur within beetle galleries. Frequencies of pair-wise combinations were significantly different from what was expected given overall species abundances. These results suggest that complex processes of competitive exclusion and coexistence help determine fungal community composition and that the consequences of these processes vary spatially. The presence of three fungal symbionts in different proportions and combinations across a wide range of environmental conditions may help explain the success of mountain pine beetle attacks across a broad geographic range.

Fungal associates of the lodgepole pine beetle, Dendroctonus murrayanae.

Bark beetles are well known vectors of ophiostomatoid fungi including species of Ophiostoma, Grosmannia and Ceratocystis. In this study, the most common ophiostomatoid fungi associated with the lodgepole pine beetle, Dendroctonus murrayanae, were characterized. Pre-emergent and post-attack adult beetles were collected from lodgepole pines at four sites in British Columbia, Canada. Fungi were isolated from these beetles and identified using a combination of morphology and DNA sequence comparisons of five gene regions. In all four populations, Grosmannia aurea was the most common associate (74-100% of all beetles) followed closely by Ophiostoma abietinum (29-75%). Other fungi isolated, in order of their relative prevalence with individual beetles were an undescribed Leptographium sp. (0-13%), Ophiostoma ips (0-15%), Ophiostoma piliferum (0-11%), a Pesotum sp. (0-11%) and Ophiostoma floccosum (0-1%). Comparisons of the DNA sequences of Leptographium strains isolated in this study, with ex-type isolates of G. aurea, Grosmannia robusta, Leptographium longiclavatum, and Leptographium terebrantis, as well as with sequences from GenBank, revealed a novel lineage within the Grosmannia clavigera complex. This lineage included some of the D. murrayane isolates as well as several isolates from previous studies referred to as L. terebrantis. However, the monophyly of this lineage is not well supported and a more comprehensive study will be needed to resolve its taxonomic status as one or more novel taxa.

Isolations from the redbay ambrosia beetle, Xyleborus glabratus, confirm that the laurel wilt pathogen, Raffaelea lauricola, originated in Asia.

The laurel wilt pathogen Raffaelea lauricola was hypothesized to have been introduced to the southeastern USA in the mycangium of the redbay ambrosia beetle, Xyleborus glabratus, which is native to Asia. To test this hypothesis adult X. glabratus were trapped in Taiwan and on Kyushu Island, Japan, in 2009, and dead beetles were sent to USA for isolation of fungal symbionts. Individual X. glabratus were macerated in glass tissue grinders, and the slurry was serially diluted and plated onto malt agar medium amended with cycloheximide, a medium semiselective for Ophiostoma species and their anamorphs, including members of Raffaelea. R. lauricola was isolated from 56 of 85 beetles in Taiwan and 10 of 16 beetles in Japan at up to an estimated 10 000 CFUs per beetle. The next most commonly isolated species was R. ellipticospora, which also has been recovered from X. glabratus trapped in the USA, as were two other fungi isolated from beetles in Taiwan, R. fusca and R. subfusca. Three unidentified Raffaelea spp. and three unidentified Ophiostoma spp. were isolated rarely from X. glabratus collected in Taiwan. Isolations from beetles similarly trapped in Georgia, USA, yielded R. lauricola and R. ellipticospora in numbers similar to those from beetles trapped in Taiwan and Japan. The results support the hypothesis that R. lauricola was introduced into the USA in mycangia of X. glabratus shipped to USA in solid wood packing material from Asia. However differences in the mycangial mycoflora of X. glabratus in Taiwan, Japan and USA suggest that the X. glabratus population established in USA originated in another part of Asia.