Know When You Are Too Many: Density-Dependent Release of Pheromones During Host Colonisation by the European Spruce Bark Beetle, Ips typographus (L.).

Individuals across various animal species communicate their presence to conspecifics. Especially phytophagous and parasitoid insects with their brood developing on limited resources rely on chemical cues, such as host-marking pheromones, to reduce intraspecific competition. Bark beetles are phytophagous insects with some species being economically and ecologically relevant forest pests. Several of them use the volatile compound verbenone to inhibit attraction and reduce intraspecific competition. However, in the Eurasian spruce bark beetle, Ips typographus (L.), temporal emission patterns did so far not quite support the putative function of verbenone as an indicator of densely colonised host trees. More importantly, it is currently unclear how well verbenone emission is actually related to colonisation density and thus intraspecific competition. Here, we inoculated Norway spruce logs with I. typographus at two defined colonisation densities in the greenhouse and measured the emission of verbenone and its precursors α-pinene and verbenol over time. Verbenone emission was 3-7 times greater from colonised logs compared to decaying logs without beetles during the major part of larval development. Furthermore, our data supports the quantitative hypothesis, that the termination of attack on a tree is mediated by a cessation of the release of verbenol and continuous emission of verbenone. The latter is most likely a passively produced host-marking cue reflecting the actual density of conspecifics since per-beetle emission was unaffected by colonisation density. These findings shed new light on the regulation of bark beetle mass aggregations, which are currently causing previously unseen economic damages in temperate forests.

Evolutionary ecology of the bark beetles Ips typographus and Pityogenes chalcographus.

Ips typographus (L.) and Pityogenes chalcographus (L.) (Coleoptera: Curculionidae) are two common bark beetle species on Norway spruce in Eurasia. Multiple biotic and abiotic factors affect the life cycles of these two beetles, shaping their ecology and evolution. In this article, we provide a comprehensive and comparative summary of selected life-history traits. We highlight similarities and differences in biotic factors, like host range, interspecific competition, host colonization, reproductive behaviour and fungal symbioses. Moreover, we focus on the species' responses to abiotic factors and compare their temperature-dependent development and flight behaviour, cold adaptations and diapause strategies. Differences in biotic and abiotic traits might be the result of recent, species-specific evolutionary histories, particularly during the Pleistocene, with differences in glacial survival and postglacial recolonization. Finally, we discuss future research directions to understand ecological and evolutionary pathways of the two bark beetle species, for both basic research and applied forest management.

First experimental evidence for active farming in ambrosia beetles and strong heredity of garden microbiomes.

Fungal cultivation is a defining feature for advanced agriculture in fungus-farming ants and termites. In a third supposedly fungus-farming group, wood-colonizing ambrosia beetles, an experimental proof for the effectiveness of beetle activity for selective promotion of their food fungi over others is lacking and farming has only been assumed based on observations of social and hygienic behaviours. Here, we experimentally removed mothers and their offspring from young nests of the fruit-tree pinhole borer, Xyleborinus saxesenii. By amplicon sequencing of bacterial and fungal communities of nests with and without beetles we could show that beetles are indeed able to actively shift symbiont communities. Although being consumed, the Raffaelea food fungi were more abundant when beetles were present while a weed fungus (Chaetomium sp.) as well as overall bacterial diversity were reduced in comparison to nests without beetles. Core symbiont communities were generally of low diversity and there were strong signs for vertical transmission not only for the cultivars, but also for secondary symbionts. Our findings verify the existence of active farming, even though the exact mechanisms underlying the selective promotion and/or suppression of symbionts need further investigation.

Symbiont selection via alcohol benefits fungus farming by ambrosia beetles.

Animal-microbe mutualisms are typically maintained by vertical symbiont transmission or partner choice. A third mechanism, screening of high-quality symbionts, has been predicted in theory, but empirical examples are rare. Here we demonstrate that ambrosia beetles rely on ethanol within host trees for promoting gardens of their fungal symbiont and producing offspring. Ethanol has long been known as the main attractant for many of these fungus-farming beetles as they select host trees in which they excavate tunnels and cultivate fungal gardens. More than 300 attacks by Xylosandrus germanus and other species were triggered by baiting trees with ethanol lures, but none of the foundresses established fungal gardens or produced broods unless tree tissues contained in vivo ethanol resulting from irrigation with ethanol solutions. More X. germanus brood were also produced in a rearing substrate containing ethanol. These benefits are a result of increased food supply via the positive effects of ethanol on food-fungus biomass. Selected Ambrosiella and Raffaelea fungal isolates from ethanol-responsive ambrosia beetles profited directly and indirectly by (i) a higher biomass on medium containing ethanol, (ii) strong alcohol dehydrogenase enzymatic activity, and (iii) a competitive advantage over weedy fungal garden competitors (Aspergillus, Penicillium) that are inhibited by ethanol. As ambrosia fungi both detoxify and produce ethanol, they may maintain the selectivity of their alcohol-rich habitat for their own purpose and that of other ethanol-resistant/producing microbes. This resembles biological screening of beneficial symbionts and a potentially widespread, unstudied benefit of alcohol-producing symbionts (e.g., yeasts) in other microbial symbioses.

Volatile emission and biosynthesis in endophytic fungi colonizing black poplar leaves.

Plant volatiles play a major role in plant-insect interactions as defense compounds or attractants for insect herbivores. Recent studies have shown that endophytic fungi are also able to produce volatiles and this raises the question of whether these fungal volatiles influence plant-insect interactions. Here, we qualitatively investigated the volatiles released from 13 endophytic fungal species isolated from leaves of mature black poplar (Populus nigra) trees. The volatile blends of these endophytes grown on agar medium consist of typical fungal compounds, including aliphatic alcohols, ketones and esters, the aromatic alcohol 2-phenylethanol and various sesquiterpenes. Some of the compounds were previously reported as constituents of the poplar volatile blend. For one endophyte, a species of Cladosporium, we isolated and characterized two sesquiterpene synthases that can produce a number of mono- and sesquiterpenes like (E)-ß-ocimene and (E)-ß-caryophyllene, compounds that are dominant components of the herbivore-induced volatile bouquet of black poplar trees. As several of the fungus-derived volatiles like 2-phenylethanol, 3-methyl-1-butanol and the sesquiterpene (E)-ß-caryophyllene, are known to play a role in direct and indirect plant defense, the emission of volatiles from endophytic microbial species should be considered in future studies investigating tree-insect interactions.

Ecology and Evolution of Insect-Fungus Mutualisms.

The evolution of a mutualism requires reciprocal interactions whereby one species provides a service that the other species cannot perform or performs less efficiently. Services exchanged in insect-fungus mutualisms include nutrition, protection, and dispersal. In ectosymbioses, which are the focus of this review, fungi can be consumed by insects or can degrade plant polymers or defensive compounds, thereby making a substrate available to insects. They can also protect against environmental factors and produce compounds antagonistic to microbial competitors. Insects disperse fungi and can also provide fungal growth substrates and protection. Insect-fungus mutualisms can transition from facultative to obligate, whereby each partner is no longer viable on its own. Obligate dependency has (a) resulted in the evolution of morphological adaptations in insects and fungi, (b) driven the evolution of social behaviors in some groups of insects, and (c) led to the loss of sexuality in some fungal mutualists.

Pathogen defence is a potential driver of social evolution in ambrosia beetles.

Social immunity-the collective behavioural defences against pathogens-is considered a crucial evolutionary force for the maintenance of insect societies. It has been described and investigated primarily in eusocial insects, but its role in the evolutionary trajectory from parental care to eusociality is little understood. Here, we report on the existence, plasticity, effectiveness and consequences of social pathogen defence in experimental nests of cooperatively breeding ambrosia beetles. After an Aspergillus spore buffer solution or a control buffer solution had been injected in laboratory nests, totipotent adult female workers increased their activity and hygienic behaviours like allogrooming and cannibalism. Such social immune responses had not been described for a non-eusocial, cooperatively breeding insect before. Removal of beetles from Aspergillus-treated nests in a paired experimental design revealed that the hygienic behaviours of beetles significantly reduced pathogen prevalence in the nest. Furthermore, in response to pathogen injections, female helpers delayed dispersal and thus prolonged their cooperative phase within their mother's nest. Our findings of appropriate social responses to an experimental immune challenge in a cooperatively breeding beetle corroborate the view that social immunity is not an exclusive attribute of eusocial insects, but rather a concomitant and presumably important feature in the evolutionary transitions towards complex social organization.

Defensive symbioses of animals with prokaryotic and eukaryotic microorganisms.

Many organisms team up with microbes for defense against predators, parasites, parasitoids, or pathogens. Here we review the described protective symbioses between animals (including marine invertebrates, nematodes, insects, and vertebrates) and bacteria, fungi, and dinoflagellates. We focus on associations where the microbial natural products mediating the protective activity have been elucidated or at least strong evidence for the role of symbiotic microbes in defense is available. In addition to providing an overview of the known defensive animal-microbe symbioses, we aim to derive general patterns on the chemistry, ecology, and evolution of such associations.

Larval helpers and age polyethism in ambrosia beetles.

Division of labor among the workers of insect societies is a conspicuous feature of their biology. Social tasks are commonly shared among age groups but not between larvae and adults with completely different morphologies, as in bees, wasps, ants, and beetles (i.e., Holometabola). A unique yet hardly studied holometabolous group of insects is the ambrosia beetles. Along with one tribe of ants and one subfamily of termites, wood-dwelling ambrosia beetles are the only insect lineage culturing fungi, a trait predicted to favor cooperation and division of labor. Their sociality has not been fully demonstrated, because behavioral observations have been missing. Here we present behavioral data and experiments from within nests of an ambrosia beetle, Xyleborinus saxesenii. Larval and adult offspring of a single foundress cooperate in brood care, gallery maintenance, and fungus gardening, showing a clear division of labor between larval and adult colony members. Larvae enlarge the gallery and participate in brood care and gallery hygiene. The cooperative effort of adult females in the colony and the timing of their dispersal depend on the number of sibling recipients (larvae and pupae), on the presence of the mother, and on the number of adult workers. This suggests that altruistic help is triggered by demands of brood dependent on care. Thus, ambrosia beetles are not only highly social but also show a special form of division of labor that is unique among holometabolous insects.

Highly diverse cuticular hydrocarbon profiles but no evidence for aggression towards non-kin in the ambrosia beetle Xyleborinus saxesenii.

Animal societies use nestmate recognition to protect against social cheaters and parasites. In most social insect societies, individuals recognize and exclude any non-nestmates and the roles of cuticular hydrocarbons as recognition cues are well documented. Some ambrosia beetles live in cooperatively breeding societies with farmed fungus cultures that are challenging to establish, but of very high value once established. Hence, social cheaters that sneak into a nest without paying the costs of nest foundation may be selected. Therefore, nestmate recognition is also expected to exist in ambrosia beetles, but so far nobody has investigated this behavior and its underlying mechanisms. Here we studied the ability for nestmate recognition in the cooperatively breeding ambrosia beetle Xyleborinus saxesenii, combining behavioural observations and cuticular hydrocarbon analyses. Laboratory nests of X. saxesenii were exposed to foreign adult females from the same population, another population and another species. Survival as well as the behaviours of the foreign female were observed. The behaviours of the receiving individuals were also observed. We expected that increasing genetic distance would cause increasing distance in chemical profiles and increasing levels of behavioural exclusion and possibly mortality. Chemical profiles differed between populations and appeared as variable as in other highly social insects. However, we found only very little evidence for the behavioural exclusion of foreign individuals. Interpopulation donors left nests at a higher rate than control donors, but neither their behaviours nor the behaviours of receiver individuals within the nest showed any response to the foreign individual in either of the treatments. These results suggest that cuticular hydrocarbon profiles might be used for communication and nestmate recognition, but that behavioural exclusion of non-nestmates is either absent in X. saxesenii or that agonistic encounters are so rare or subtle that they could not be detected by our method. Additional studies are needed to investigate this further.

Fidelity or love the one you're with? Biotic complexity and tradeoffs can drive strategy and specificity in beetle-fungus by-product mutualisms.

By-product mutualisms are ubiquitous yet seldom considered in models of mutualism. Most models represent conditional mutualisms that shift between mutualism and antagonism in response to shifts in costs and benefits resulting from changes in environmental quality. However, in by-product mutualisms, benefits arise as a part of normal life processes that may be costly to produce but incur little-to-no additional costs in response to the interaction. Without costs associated with the interaction, they do not have antagonistic alternate states. Here, we present a conceptual model that differs from traditional conditional models in three ways: (1) partners exchange by-product benefits, (2) interactions do not have alternate antagonistic states, and (3) tradeoffs are allowed among factors that influence environmental quality (rather than all factors that contribute to environmental quality being combined into a single gradient ranging from high to low). We applied this model to bark and ambrosia beetles (Curculionidae: Scolytinae), a diverse group that associates with fungi and that has repeatedly developed two distinct pathways to by-product mutualism. We used independent axes for each major factor influencing environmental quality in these systems, including those that exhibit tradeoffs (tree defense and nutritional quality). For these symbioses, tradeoffs in these two factors are key to which mutualism pathway is taken.

Comparing the succession of microbial communities throughout development in field and laboratory nests of the ambrosia beetle Xyleborinus saxesenii.

Some fungus-farming ambrosia beetles rely on multiple nutritional cultivars (Ascomycota: Ophiostomatales and/or yeasts) that seem to change in relative abundance over time. The succession of these fungi could benefit beetle hosts by optimal consumption of the substrate and extended longevity of the nest. However, abundances of fungal cultivars and other symbionts are poorly known and their culture-independent quantification over development has been studied in only a single species. Here, for the first time, we compared the diversity and succession of both fungal and bacterial communities of fungus gardens in the fruit-tree pinhole borer, Xyleborinus saxesenii, from field and laboratory nests over time. By amplicon sequencing of probed fungus gardens of both nest types at three development phases we showed an extreme reduction of diversity in both bacterial and fungal symbionts in laboratory nests. Furthermore, we observed a general transition from nutritional to non-beneficial fungal symbionts during beetle development. While one known nutritional mutualist, Raffaelea canadensis, was occurring more or less stable over time, the second mutualist R. sulphurea was dominating young nests and decreased in abundance at the expense of other secondary fungi. The quicker the succession proceeded, the slower offspring beetles developed, suggesting a negative role of these secondary symbionts. Finally, we found signs of transgenerational costs of late dispersal for daughters, possibly as early dispersers transmitted and started their own nests with less of the non-beneficial taxa. Future studies should focus on the functional roles of the few bacterial taxa that were present in both field and laboratory nests.

Volatiles of fungal cultivars act as cues for host-selection in the fungus-farming ambrosia beetle Xylosandrus germanus.

Many wood-boring insects use aggregation pheromones during mass colonization of host trees. Bark beetles (Curculionidae: Scolytinae) are a model system, but much less is known about the role of semiochemicals during host selection by ambrosia beetles. As an ecological clade within the bark beetles, ambrosia beetles are obligately dependent on fungal mutualists for their sole source of nutrition. Mass colonization of trees growing in horticultural settings by exotic ambrosia beetles can occur, but aggregation cues have remained enigmatic. To elucidate this mechanism, we first characterized the fungal associates of the exotic, mass-aggregating ambrosia beetle Xylosandrus germanus in Southern Germany. Still-air olfactometer bioassays documented the attraction of X. germanus to its primary nutritional mutualist Ambrosiella grosmanniae and to a lesser extent another common fungal isolate (Acremonium sp.). During two-choice bioassays, X. germanus was preferentially attracted to branch sections (i.e., bolts) that were either pre-colonized by conspecifics or pre-inoculated with A. grosmanniae. Subsequent analyses identified microbial volatile organic compounds (MVOCs) that could potentially function as aggregation pheromones for X. germanus. To our knowledge, this is the first evidence for fungal volatiles as attractive cues during host selection by X. germanus. Adaptive benefits of responding to fungal cues associated with an infestation of conspecifics could be a function of locating a suitable substrate for cultivating fungal symbionts and/or increasing the likelihood of mating opportunities with the flightless males. However, this requires solutions for evolutionary conflict arising due to potential mixing of vertically transmitted and horizontally acquired symbiont strains, which are discussed.

Erwiniaceae bacteria play defensive and nutritional roles in two widespread ambrosia beetles.

Ambrosia beetles are fungal-growing insects excavating galleries deep inside the wood. Their success as invaders increased scientific interest towards them. However, most studies on their microbiota targeted their fungal associates whereas the role of bacterial associates is understudied. To explore the role of abundant microbial associates, we isolated bacteria from active galleries of two widespread ambrosia beetles, Xylosandrus crassiusculus and X. germanus. These isolates were classified within the Erwiniaceae family and through a phylogenetic analysis including isolates from other insects we showed that they clustered with isolates obtained from ambrosia and bark beetles, including Erwinia typographi. The whole genome analysis of the isolate from active galleries of X. crassiusculus suggested that this bacterium plays both a nutritional role, by providing essential amino acids and enzymes for the hydrolysis of plant biomass, and a defensive role, by producing antibiotics. This defensive role was also tested in vitro against fungi, including mutualists, common associates, and parasites. The bacteria inhibited the growth of some of the common associates and parasites but did not affect mutualists. Our study supported the hypothesis of a mutualist role of Erwiniaceae bacteria in ambrosia beetles and highlighed the importance of bacteria in maintaining the symbiosis of their host with nutritional fungi.

Patterns of functional enzyme activity in fungus farming ambrosia beetles.

INTRODUCTION: In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. RESULTS: We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-ß-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-ß-1,4-xylanase activity was exclusively detected in larvae. CONCLUSION: Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray-parenchyma cells in the wood xylem. Furthermore, the detection of xylanolytic enzymes exclusively in larvae (which feed on fungus colonized wood) and not in adults (which feed only on fungi) indicates that only larvae (pre-) digest plant cell wall structures. This implies that in X. saxesenii and likely also in many other ambrosia beetles, adults and larvae do not compete for the same food within their nests - in contrast, larvae increase colony fitness by facilitating enzymatic wood degradation and fungus cultivation.

Genome sequence of Streptomyces griseus strain XylebKG-1, an ambrosia beetle-associated actinomycete.

Streptomyces griseus strain XylebKG-1 is an insect-associated strain of the well-studied actinobacterial species S. griseus. Here, we present the genome of XylebKG-1 and discuss its similarity to the genome of S. griseus subsp. griseus NBRC13350. XylebKG-1 was isolated from the fungus-cultivating Xyleborinus saxesenii system. Given its similarity to free-living S. griseus subsp. griseus NBRC13350, comparative genomics will elucidate critical components of bacterial interactions with insects.

Nutrient-Poor Breeding Substrates of Ambrosia Beetles Are Enriched With Biologically Important Elements.

Fungus-farming within galleries in the xylem of trees has evolved independently in at least twelve lineages of weevils (Curculionidae: Scolytinae, Platypodinae) and one lineage of ship-timber beetles (Lymexylidae). Jointly these are termed ambrosia beetles because they actively cultivate nutritional "ambrosia fungi" as their main source of food. The beetles are obligately dependent on their ambrosia fungi as they provide them a broad range of essential nutrients ensuring their survival in an extremely nutrient-poor environment. While xylem is rich in carbon (C) and hydrogen (H), various elements essential for fungal and beetle growth, such as nitrogen (N), phosphorus (P), sulfur (S), potassium (K), calcium (Ca), magnesium (Mg), and manganese (Mn) are extremely low in concentration. Currently it remains untested how both ambrosia beetles and their fungi meet their nutritional requirements in this habitat. Here, we aimed to determine for the first time if galleries of ambrosia beetles are generally enriched with elements that are rare in uncolonized xylem tissue and whether these nutrients are translocated to the galleries from the xylem by the fungal associates. To do so, we examined natural galleries of three ambrosia beetle species from three independently evolved farming lineages, Xyleborinus saxesenii (Scolytinae: Xyleborini), Trypodendron lineatum (Scolytinae: Xyloterini) and Elateroides dermestoides (Lymexylidae), that cultivate unrelated ambrosia fungi in the ascomycete orders Ophiostomatales, Microascales, and Saccharomycetales, respectively. Several elements, in particular Ca, N, P, K, Mg, Mn, and S, were present in high concentrations within the beetles' galleries but available in only very low concentrations in the surrounding xylem. The concentration of elements was generally highest with X. saxesenii, followed by T. lineatum and E. dermestoides, which positively correlates with the degree of sociality and productivity of brood per gallery. We propose that the ambrosia fungal mutualists are translocating essential elements through their hyphae from the xylem to fruiting structures they form on gallery walls. Moreover, the extremely strong enrichment observed suggests recycling of these elements from the feces of the insects, where bacteria and yeasts might play a role.

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.

Four mycangium types and four genera of ambrosia fungi suggest a complex history of fungus farming in the ambrosia beetle tribe Xyloterini.

Ambrosia beetles farm fungal cultivars (ambrosia fungi) and carry propagules of the fungal mutualists in storage organs called mycangia, which occur in various body parts and vary greatly in size and complexity. The evolution of ambrosia fungi is closely tied to the evolution and development of the mycangia that carry them. The understudied ambrosia beetle tribe Xyloterini included lineages with uncharacterized ambrosia fungi and mycangia, which presented an opportunity to test whether developments of different mycangium types in a single ambrosia beetle lineage correspond with concomitant diversity in their fungal mutualists. We collected representatives of all three Xyloterini genera (Trypodendron, Indocryphalus, and Xyloterinus politus) and characterized their ambrosia fungi in pure culture and by DNA sequencing. The prothoracic mycangia of seven Trypodendron species all yielded Phialophoropsis (Microascales) ambrosia fungi, including three new species, although these relationships were not all species specific. Indocryphalus mycangia are characterized for the first time in the Asian I. pubipennis. They comprise triangular prothoracic cavities substantially smaller than those of Trypodendron and unexpectedly carry an undescribed species of Toshionella (Microascales), which are otherwise ambrosia fungi of Asian Scolytoplatypus (Scolytoplatypodini). Xyloterinus politus has two different mycangia, each with a different ambrosia fungus: Raffaelea cf. canadensis RNC5 (Ophiostomatales) in oral mycangia of both sexes and Kaarikia abrahamsonii (Sordariomycetes, genus incertae sedis with affinity for Distoseptisporaceae), a new genus and species unrelated to other known ambrosia fungi, in shallow prothoracic mycangia of females. In addition to their highly adapted mycangial mutualists, Trypodendron and X. politus harbor a surprising diversity of facultative symbionts in their galleries, including Raffaelea. A diversity of ambrosia fungi and mycangia suggest multiple ancestral cultivar captures or switches in the history of tribe Xyloterini, each associated with unique adaptations in mycangium anatomy. This further supports the theory that developments of novel mycangium types are critical events in the evolution of ambrosia beetles and their coadapted fungal mutualists.