Symbionts Of Beetles Research Articles

Characterization of Terpenoids from the Ambrosia Beetle Symbiont and Laurel Wilt Pathogen Harringtonia lauricola.

Little is known concerning terpenoids produced by members of the fungal order Ophiostomales, with the member Harringtonia lauricola having the unique lifestyle of being a beetle symbiont but potentially devastating tree pathogen. Nine known terpenoids, including six labdane diterpenoids (1-6) and three hopane triterpenes (7-9), were isolated from H. lauricola ethyl acetate (EtOAc) extracts for the first time. All compounds were tested for various in vitro bioactivities. Six compounds, 2, 4, 5, 6, 7, and 9, are described functionally. Compounds 2, 4, 5, and 9 expressed potent antiproliferative activity against the MCF-7, HepG2 and A549 cancer cell lines, with half-maximal inhibitory concentrations (IC50s) ~12.54-26.06 μM. Antimicrobial activity bioassays revealed that compounds 4, 5, and 9 exhibited substantial effects against Gram-negative bacteria (Escherichia coli and Ralstonia solanacearum) with minimum inhibitory concentration (MIC) values between 3.13 and 12.50 μg/mL. Little activity was seen towards Gram-positive bacteria for any of the compounds, whereas compounds 2, 4, 7, and 9 expressed antifungal activities (Fusarium oxysporum) with MIC values ranging from 6.25 to 25.00 μg/mL. Compounds 4, 5, and 9 also displayed free radical scavenging abilities towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide (O2-), with IC50 values of compounds 2, 4, and 6 ~3.45-14.04 μg/mL and 22.87-53.31 μg/mL towards DPPH and O2-, respectively. These data provide an insight into the biopharmaceutical potential of terpenoids from this group of fungal insect symbionts and plant pathogens.

Semiochemicals produced by fungal bark beetle symbiont Endoconidiophora rufipennis and the discovery of an anti-attractant for Ips typographus.

Bark beetles vector symbiotic fungal species into their host trees during mass attacks. The symbiotic relationship with blue stain fungi of the Ascomycetes, including genera of Endoconidiophora (syn. = Ceratocystis), promotes successful establishment whereby the microbes help to overcome the host trees' defence and degrade toxic resins. This is the first study to evaluate both the volatile emissions from an insect-associated blue stain fungus over time and the insect response in a field trapping experiment. Volatile emissions from isolates of Endoconidiophora rufipennis (ER) were collected by solid-phase microextraction (SPME) and analysed by gas chromatography-mass spectroscopy (GC-MS) over a period of 30 days. This virulent North American fungus is closely related to E. polonica, a symbiotic fungus known from Eurasian spruce bark beetle Ips typographus.Nine volatiles were emitted by ER in substantial amounts: isoamyl acetate, sulcatone, 2-phenethyl acetate, geranyl acetone, geranyl acetate, citronellyl acetate, (R)- and (S)-sulcatol, and (R)-sulcatol acetate. A late peaking compound was geranyl acetone. In the field trapping experiment, three of the fungal volatiles (geranyl acetone, 2-phenethyl acetate and sulcatone) were tested in combination with a synthetic aggregation pheromone for I. typographus. Traps with geranyl acetone attracted lower numbers of I. typographus compared to traps with 2-phenethyl acetate, sulcatone or the pheromone alone as a control. The results showed that geranyl acetone acts as an anti-attractant and may act naturally on I. typographus as a cue from an associated fungus to signal an overexploited host.

Geosmithia—widespread and abundant but long ignored bark beetle symbionts

Geosmithia—widespread and abundant but long ignored bark beetle symbionts

Flexibility in the ambrosia symbiosis of Xyleborus bispinatus.

Ambrosia beetles maintain strict associations with specific lineages of fungi. However, anthropogenic introductions of ambrosia beetles into new ecosystems can result in the lateral transfer of their symbionts to other ambrosia beetles. The ability of a Florida endemic ambrosia beetle, Xyleborus bispinatus, to feed and establish persistent associations with two of its known symbionts (Raffaelea subfusca and Raffaelea arxii) and two other fungi (Harringtonia lauricola and Fusarium sp. nov.), which are primary symbionts of invasive ambrosia beetles, was investigated. The stability of these mutualisms and their effect on the beetle's fitness were monitored over five consecutive generations. Surface-disinfested pupae with non-developed mycangia were reared separately on one of the four fungal symbionts. Non-treated beetles (i.e., lab colony) with previously colonized mycangia were used as a control group. Xyleborus bispinatus could exchange its fungal symbionts, survive, and reproduce on different fungal diets, including known fungal associates and phylogenetically distant fungi, which are plant pathogens and primary symbionts of other invasive ambrosia beetles. These changes in fungal diets resulted in persistent mutualisms, and some symbionts even increased the beetle's reproduction. Females that developed on Fusarium sp. nov. had a significantly greater number of female offspring than non-treated beetles. Females that fed solely on Harringtonia or Raffaelea symbionts produced fewer female offspring. Even though some ambrosia beetles like X. bispinatus can partner with different ambrosia fungi, their symbiosis under natural conditions is modulated by their mycangium and possibly other environmental factors. However, exposure to symbionts of invasive beetles can result in stable partnerships with these fungi and affect the population dynamics of ambrosia beetles and their symbionts.

Diversity and Evolution of Entomocorticium (Russulales, Peniophoraceae), a Genus of Bark Beetle Mutualists Derived from Free-Living, Wood Rotting Peniophora.

Symbiosis between insects and fungi arose multiple times during the evolution of both groups, and some of the most biologically diverse and economically important are mutualisms in which the insects cultivate and feed on fungi. Among these are bark beetles, whose ascomycetous cultivars are better known and studied than their frequently-overlooked and poorly understood basidiomycetous partners. In this study, we propose five new species of Entomocorticium, fungal mutualists in the Russulales (Basidiomycota) that are mutualistic symbionts of scolytine beetles. We have isolated these fungi from the beetle mycangia, which are structures adapted for the selective storage and transportation of fungal mutualists. Herein, we present the most complete phylogeny of the closely related genera Entomocorticium and Peniophora and provide insights into how an insect-associated taxon (Entomocorticium) evolved from within a wood-decaying, wind-dispersed lineage (Peniophora). Our results indicate that following a transition from angiosperms to gymnosperms, fungal domestication by beetles facilitated the evolution and diversification of Entomocorticium. We additionally propose four new species: Entomocorticium fibulatum Araújo, Li & Hulcr, sp. nov.; E. belizense Araújo, Li & Hulcr, sp. nov.; E. perryae Araújo, Li & Hulcr, sp. nov.; and E. macrovesiculatum Araújo, Li, Six & Hulcr, sp. nov. Our findings highlight the fact that insect-fungi associations remain an understudied field and that these associations harbor a large reservoir of novel fungal species.

Combined drought and bark beetle attacks deplete non-structural carbohydrates and promote death of mature pine trees.

How carbohydrate reserves in conifers respond to drought and bark beetle attacks are poorly understood. We investigated changes in carbohydrate reserves and carbon-dependent diterpene defences in ponderosa pine trees that were experimentally subjected to two levels of drought stress (via root trenching) and two types of biotic challenge treatments (pheromone-induced bark beetle attacks or inoculations with crushed beetles that include beetle-associated fungi) for two consecutive years. Our results showed that trenching did not influence carbohydrates, whereas both biotic challenges reduced amounts of starch and sugars of trees. However, only the combined trenched-bark beetle attacked trees depleted carbohydrates and died during the first year of attacks. While live trees contained higher carbohydrates than dying trees, amounts of constitutive and induced diterpenes produced did not vary between live and beetle-attacked dying trees, respectively. Based on these results we propose that reallocation of carbohydrates to diterpenes during the early stages of beetle attacks is limited in drought-stricken trees, and that the combination of biotic and abiotic stress leads to tree death. The process of tree death is subsequently aggravated by beetle girdling of phloem, occlusion of vascular tissue by bark beetle-vectored fungi, and potential exploitation of host carbohydrates by bark beetle symbionts as nutrients.

Ethanol-Enriched Substrate Facilitates Ambrosia Beetle Fungi, but Inhibits Their Pathogens and Fungal Symbionts of Bark Beetles.

Bark beetles (sensu lato) colonize woody tissues like phloem or xylem and are associated with a broad range of micro-organisms. Specific fungi in the ascomycete orders Hypocreales, Microascales and Ophistomatales as well as the basidiomycete Russulales have been found to be of high importance for successful tree colonization and reproduction in many species. While fungal mutualisms are facultative for most phloem-colonizing bark beetles (sensu stricto), xylem-colonizing ambrosia beetles are long known to obligatorily depend on mutualistic fungi for nutrition of adults and larvae. Recently, a defensive role of fungal mutualists for their ambrosia beetle hosts was revealed: Few tested mutualists outcompeted other beetle-antagonistic fungi by their ability to produce, detoxify and metabolize ethanol, which is naturally occurring in stressed and/or dying trees that many ambrosia beetle species preferentially colonize. Here, we aim to test (i) how widespread beneficial effects of ethanol are among the independently evolved lineages of ambrosia beetle fungal mutualists and (ii) whether it is also present in common fungal symbionts of two bark beetle species (Ips typographus, Dendroctonus ponderosae) and some general fungal antagonists of bark and ambrosia beetle species. The majority of mutualistic ambrosia beetle fungi tested benefited (or at least were not harmed) by the presence of ethanol in terms of growth parameters (e.g., biomass), whereas fungal antagonists were inhibited. This confirms the competitive advantage of nutritional mutualists in the beetle’s preferred, ethanol-containing host material. Even though most bark beetle fungi are found in the same phylogenetic lineages and ancestral to the ambrosia beetle (sensu stricto) fungi, most of them were highly negatively affected by ethanol and only a nutritional mutualist of Dendroctonus ponderosae benefited, however. This suggests that ethanol tolerance is a derived trait in nutritional fungal mutualists, particularly in ambrosia beetles that show cooperative farming of their fungi.

Patterns of Diversity in the Symbiotic Mite Assemblage of the Mountain Pine Beetle, Dendroctonus Ponderosae Hopkins

The mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Scolytinae), is an economically important bark beetle species with a wide geographic range spanning from the southwestern United States into northern Canada. This beetle causes extensive tree mortality to 13 pine species. Mites (Acari) are common and abundant symbionts of mountain beetles that may influence their fitness through positive and negative interactions. We present a unique assessment of the mite associates of mountain pine beetles using measures of alpha and beta diversity. We sampled phoretic mites from five beetle populations: Arizona, Colorado, South Dakota, Utah (USA), and Alberta (Canada) that varied in host tree species, local climate, and beetle population level. We collected 4848 mites from 8 genera and 12 species. Fifty to seventy percent of beetles carried mites in flight with the highest mite loads occurring in middle and southern populations; decreasing in northern populations. Mite assemblages (i.e., both richness and composition) varied along a south to north latitudinal gradient and were driven by species turnover (i.e., species replacement). Differences in mite composition increased with distance between populations. We discuss climatic variation, environmental filtering, and host tree differences as factors that could affect differences in mite composition between beetle populations and discuss implications for functional shifts. Our results could represent a model for estimating diversity patterns of mite symbionts associated with other major insect pests in coniferous forest systems.

Insect‐Associated Bacteria Assemble the Antifungal Butenolide Gladiofungin by Non‐Canonical Polyketide Chain Termination

AbstractGenome mining of one of the protective symbionts (Burkholderia gladioli) of the invasive beetle Lagria villosa revealed a cryptic gene cluster that codes for the biosynthesis of a novel antifungal polyketide with a glutarimide pharmacophore. Targeted gene inactivation, metabolic profiling, and bioassays led to the discovery of the gladiofungins as previously‐overlooked components of the antimicrobial armory of the beetle symbiont, which are highly active against the entomopathogenic fungus Purpureocillium lilacinum. By mutational analyses, isotope labeling, and computational analyses of the modular polyketide synthase, we found that the rare butenolide moiety of gladiofungins derives from an unprecedented polyketide chain termination reaction involving a glycerol‐derived C3 building block. The key role of an A‐factor synthase (AfsA)‐like offloading domain was corroborated by CRISPR‐Cas‐mediated gene editing, which facilitated precise excision within a PKS domain.

Insect-Associated Bacteria Assemble the Antifungal Butenolide Gladiofungin by Non-Canonical Polyketide Chain Termination.

Genome mining of one of the protective symbionts (Burkholderia gladioli) of the invasive beetle Lagria villosa revealed a cryptic gene cluster that codes for the biosynthesis of a novel antifungal polyketide with a glutarimide pharmacophore. Targeted gene inactivation, metabolic profiling, and bioassays led to the discovery of the gladiofungins as previously‐overlooked components of the antimicrobial armory of the beetle symbiont, which are highly active against the entomopathogenic fungus Purpureocillium lilacinum. By mutational analyses, isotope labeling, and computational analyses of the modular polyketide synthase, we found that the rare butenolide moiety of gladiofungins derives from an unprecedented polyketide chain termination reaction involving a glycerol‐derived C3 building block. The key role of an A‐factor synthase (AfsA)‐like offloading domain was corroborated by CRISPR‐Cas‐mediated gene editing, which facilitated precise excision within a PKS domain.

IMA Genome - F13

ABSTRACTDraft genomes of the fungal species Ambrosiella cleistominuta, Cercospora brassicicola, C. citrullina, Physcia stellaris, and Teratosphaeria pseudoeucalypti are presented. Physcia stellaris is an important lichen forming fungus and Ambrosiella cleistominuta is an ambrosia beetle symbiont. Cercospora brassicicola and C. citrullina are agriculturally relevant plant pathogens that cause leaf-spots in brassicaceous vegetables and cucurbits respectively. Teratosphaeria pseudoeucalypti causes severe leaf blight and defoliation of Eucalyptus trees. These genomes provide a valuable resource for understanding the molecular processes in these economically important fungi.

A new species of Raffaelea from beetle-infested Leucaena leucocephala

Species of Raffaelea (Ophiostomatales: Ascomycota) are obligate symbionts of ambrosia beetles, some of which pose a substantial threat to forest trees. Leucaena leucocephala is a small mimosoid tree species that is considered as an invasive weed in most of its introduced range globally. During a field expedition on the French island of Réunion, dying L. leucocephala trees were observed. Samples were taken from these trees and isolations made from symptomatic wood tissues that included beetle tunnels, but in the absence of the beetles themselves. Multiple isolates of a fungus resembling a Raffaelea species were obtained from the discoloured wood associated with the beetle tunnels. To determine their identity, microscopic examination was performed and DNA sequences for three gene regions (ITS, LSU, TUB) were obtained. Phylogenetic analyses based on these gene regions revealed that the isolates represent a new species of Raffaelea, described here as R. borbonica sp. nov. A pathogenicity test was conducted with the fungus, which was shown to cause lesions on the inoculated seedlings, but with a low level of aggressiveness.

Fungal symbionts of bark and ambrosia beetles can suppress decomposition of pine sapwood by competing with wood-decay fungi

Bark and ambrosia beetles inoculate dying trees with symbiotic fungi. The effects of these fungi on wood decomposition are poorly understood. We determined the effects of three widespread Ascomycota symbionts and one introduced Basidiomycota symbiont on the decomposition of loblolly pine (Pinus taeda) sapwood. Ascomycetes caused <5% mass loss and no visible structural degradation, whereas the basidiomycete Flavodon ambrosius caused nearly 15% mass loss and visible structural degradation similar to free-living wood-decay fungi. Ophiostoma ips and Raffaelea fusca reduced white- and brown-rot decay through competition with Ganoderma curtisii and Phaeolus schweinitzii, respectively. The inhibitory effects of O. ips and R. fusca on decay were negated when co-inoculated with F. ambrosius suggesting that the spread of this invader could influence forest carbon cycles. In contrast to the predominant forest biology narrative, the common and widespread ophiostomatalean symbionts of bark and ambrosia beetles studied here appear to delay, rather than facilitate tree biomass recycling.

Horizontal Gene Transfer to a Defensive Symbiont with a Reduced Genome in a Multipartite Beetle Microbiome.

Symbiotic mutualisms of bacteria and animals are ubiquitous in nature, running a continuum from facultative to obligate from the perspectives of both partners. The loss of functions required for living independently but not within a host gives rise to reduced genomes in many symbionts. Although the phenomenon of genome reduction can be explained by existing evolutionary models, the initiation of the process is not well understood. Here, we describe the microbiome associated with the eggs of the beetle Lagria villosa, consisting of multiple bacterial symbionts related to Burkholderia gladioli, including a reduced-genome symbiont thought to be the exclusive producer of the defensive compound lagriamide. We show that the putative lagriamide-producing symbiont is the only member of the microbiome undergoing genome reduction and that it has already lost the majority of its primary metabolism and DNA repair pathways. The key step preceding genome reduction in the symbiont was likely the horizontal acquisition of the putative lagriamide lga biosynthetic gene cluster. Unexpectedly, we uncovered evidence of additional horizontal transfers to the symbiont's genome while genome reduction was occurring and despite a current lack of genes needed for homologous recombination. These gene gains may have given the genome-reduced symbiont a selective advantage in the microbiome, especially given the maintenance of the large lga gene cluster despite ongoing genome reduction.IMPORTANCE Associations between microorganisms and an animal, plant, or fungal host can result in increased dependence over time. This process is due partly to the bacterium not needing to produce nutrients that the host provides, leading to loss of genes that it would need to live independently and to a consequent reduction in genome size. It is often thought that genome reduction is aided by genetic isolation-bacteria that live in monocultures in special host organs, or inside host cells, have less access to other bacterial species from which they can obtain genes. Here, we describe exposure of a genome-reduced beetle symbiont to a community of related bacteria with nonreduced genomes. We show that the symbiont has acquired genes from other bacteria despite going through genome reduction, suggesting that isolation has not yet played a major role in this case of genome reduction, with horizontal gene gains still offering a potential route for adaptation.

Pathogenicity and Distribution of Fusarium solani Isolates Associated with Erythrina Decline in Japan.

Erythrina spp. trees have been declining since the 2000s worldwide, and fungi belonging to Fusarium solani species complex (FSSC) have been suggested to be a causal factor of decline and mortality of Erythrina variegata trees in Okinawa Island, Japan. In addition to the FSSC isolate grouped as "it-1" based on ITS sequence data (previously called strain A), we conducted an inoculation experiment with two isolates grouped as "it-2" (previously strain B), which is genetically close to it-1. Two it-2 isolates originating from two islands showed pathogenicity to E. variegata with the same symptoms as those caused by it-1 isolate. We also found the isolates of it-1 and it-2 were widely distributed, including on Ishigaki Island, ∼400 km south of Okinawa Island across the ocean. All isolates of it-1 and it-2 belong to the ambrosia Fusarium clade of the FSSC, a group of symbionts of ambrosia beetles, including the pathogens of Fusarium dieback in avocados and teas. The detection of ambrosia beetles Euwallacea spp. from our specimens provided information on the vectors of the pathogens. Our present results suggest the fungi of the FSSC could be responsible for the Erythrina decline in other areas with damage.

Patterns of coevolution between ambrosia beetle mycangia and the Ceratocystidaceae, with five new fungal genera and seven new species.

Ambrosia beetles farm specialised fungi in sapwood tunnels and use pocket-like organs called mycangia to carry propagules of the fungal cultivars. Ambrosia fungi selectively grow in mycangia, which is central to the symbiosis, but the history of coevolution between fungal cultivars and mycangia is poorly understood. The fungal family Ceratocystidaceae previously included three ambrosial genera (Ambrosiella, Meredithiella, and Phialophoropsis), each farmed by one of three distantly related tribes of ambrosia beetles with unique and relatively large mycangium types. Studies on the phylogenetic relationships and evolutionary histories of these three genera were expanded with the previously unstudied ambrosia fungi associated with a fourth mycangium type, that of the tribe Scolytoplatypodini. Using ITS rDNA barcoding and a concatenated dataset of six loci (28S rDNA, 18S rDNA, tef1-α, tub, mcm7, and rpl1), a comprehensive phylogeny of the family Ceratocystidaceae was developed, including Inodoromyces interjectus gen. & sp. nov., a non-ambrosial species that is closely related to the family. Three minor morphological variants of the pronotal disk mycangium of the Scolytoplatypodini were associated with ambrosia fungi in three respective clades of Ceratocystidaceae: Wolfgangiella gen. nov., Toshionella gen. nov., and Ambrosiella remansi sp. nov. Closely-related species that are not symbionts of ambrosia beetles are accommodated by Catunica adiposa gen. & comb. nov. and Solaloca norvegica gen. & comb. nov. The divergent morphology of the ambrosial genera and their phylogenetic placement among non-ambrosial genera suggest three domestication events in the Ceratocystidaceae. Estimated divergence dates for the ambrosia fungi and mycangia suggest that Scolytoplatypodini mycangia may have been the first to acquire Ceratocystidaceae symbionts and other ambrosial fungal genera emerged shortly after the evolution of new mycangium types. There is no evidence of reversion to a non-ambrosial lifestyle in the mycangial symbionts.

Convergent evolution of semiochemicals across Kingdoms: bark beetles and their fungal symbionts

Convergent evolution of semiochemical use in organisms from different Kingdoms is a rarely described phenomenon. Tree-killing bark beetles vector numerous symbiotic blue-stain fungi that help the beetles colonize healthy trees. Here we show for the first time that some of these fungi are able to biosynthesize bicyclic ketals that are pheromones and other semiochemicals of bark beetles. Volatile emissions of five common bark beetle symbionts were investigated by gas chromatography-mass spectrometry. When grown on fresh Norway spruce bark the fungi emitted three well-known bark beetle aggregation pheromones and semiochemicals (exo-brevicomin, endo-brevicomin and trans-conophthorin) and two structurally related semiochemical candidates (exo-1,3-dimethyl-2,9-dioxabicyclo[3.3.1]nonane and endo-1,3-dimethyl-2,9-dioxabicyclo[3.3.1]nonane) that elicited electroantennogram responses in the spruce bark beetle Ips typographus. When grown on malt agar with 13C d-Glucose, the fungus Grosmannia europhioides incorporated 13C into exo-brevicomin and trans-conophthorin. The enantiomeric compositions of the fungus-produced ketals closely matched those previously reported from bark beetles. The production of structurally complex bark beetle pheromones by symbiotic fungi indicates cross-kingdom convergent evolution of signal use in this system. This signaling is susceptible to disruption, providing potential new targets for pest control in conifer forests and plantations.

Multiple evolutionary origins lead to diversity in the metabolic profiles of ambrosia fungi

Abstract Ambrosia fungi are an ecological assemblage cultivated by ambrosia beetles as required nutrient sources. This mutualism evolved in multiple beetle and fungus lineages. Whether convergence in ecology led to convergent metabolism in ambrosia fungi is unknown. We compared the assimilation of 190 carbon sources in five independent pairs of ambrosia fungi and closely related, non-ambrosial species. Ecological convergence versus phylogenetic divergence in carbon source use was tested using variation partitioning. We found no convergence in carbon utilization capacities. Instead, metabolic variation was mostly explained by phylogenetic relationships. In addition, carbon usage in ambrosia fungi was equally diverse as that in non-ambrosial species. Thus, carbon metabolism of each ambrosia fungus is determined by its inherited metabolism, not the transition towards symbiosis. In contrast to other fungus-farming systems of termites and attine ants, the fungal symbionts of ambrosia beetles are functionally diverse, reflecting their independent evolutionary origins.

Defense Responses and Changes in Symbiotic Gut Microflora in the Colorado Potato Beetle Leptinotarsa decemlineata under the Effect of Endophytic Bacteria from the Genus Bacillus

Phytophagous insects and host plants have a complex of microsymbionts and make up a united co-evolving system with them. Microsymbiotic complexes are actively involved in stress responses of macrosymbionts. We established that a treatment of potato plants with endophytic bacterial strains Bacillus thuringiensis var. thuringiensis-5689, B. th. var. kurstaki-5351, and Bacillus subtilis 26D decreased the survival rate of the plant feeder, Colorado potato beetle Leptinotarsa decemlineata Say. The B. th. strains suppressed phenoloxidase and acetylcholinesterase activities in the beetle hemolymph. An antagonistic relationship was found between endophytic bacteria B. subtilis 26D and beetle symbiotic bacteria from the genera Acinetobacter and Enterobacter, with the former being able to suppress the growth of endophytic colonies. The recombinant B. subtilis strain 26D Cry, containing the B. th. var. kurstaki δ-endotoxin cry1Ia gene, combined the ability of the original B. subtilis 26D strain to suppress the development of beetle symbionts and immune responses with a production of the Cry toxin, thus leading to a high mortality of the phytophage.

Detecting Symbioses in Complex Communities: the Fungal Symbionts of Bark and Ambrosia Beetles Within Asian Pines.

Separating symbioses from incidental associations is a major obstacle in symbiosis research. In this survey of fungi associated with Asian bark and ambrosia beetles, we used quantitative culture and DNA barcode identification to characterize fungal communities associated with co-infesting beetle species in pines (Pinus) of China and Vietnam. To quantitatively discern likely symbioses from coincidental associations, we used multivariate analysis and multilevel pattern analysis (a type of indicator species analysis). Nearly half of the variation in fungal community composition in beetle galleries and on beetle bodies was explained by beetle species. We inferred a spectrum of ecological strategies among beetle-associated fungi: from generalist multispecies associates to highly specialized single-host symbionts that were consistently dominant within the mycangia of their hosts. Statistically significant fungal associates of ambrosia beetles were typically only found with one beetle species. In contrast, bark beetle-associated fungi were often associated with multiple beetle species. Ambrosia beetles and their galleries were frequently colonized by low-prevalence ambrosia fungi, suggesting that facultative ambrosial associations are commonplace, and ecological mechanisms such as specialization and competition may be important in these dynamic associations. The approach used here could effectively delimit symbiotic interactions in any system where symbioses are obscured by frequent incidental associations. It has multiple advantages including (1) powerful statistical tests for non-random associations among potential symbionts, (2) simultaneous evaluation of multiple co-occurring host and symbiont associations, and (3) identifying symbionts that are significantly associated with multiple host species.