Ambrosia beetle Premnobius cavipennis (Scolytinae: Ipini) carries highly divergent ascomycotan ambrosia fungus,Afroraffaelea ambrosiae gen. nov. et sp. nov. (Ophiostomatales)

Ambrosia beetles and fungi represent an interesting and economically important symbiosis, but the vast majority of ambrosia fungi remain unexplored, hindering research, management of pathogens, and mitigation of invasive species. Beetles in the subtribe Premnobiini are one example of an entire beetle lineage whose fungal symbionts have never been studied. Here, we identify one dominant fungal symbiont of Premnobius cavipennis by using fungus culturing, community sequencing, microtome sectioning and micro-CT scanning of mycangia. Phylogenetic analyses of combined 18S and 28S rDNA and β-tubulin sequences revealed a highly divergent fungal lineage within Ophiostomatales, Afroraffaelea ambrosiae gen. nov. et sp. nov. The newly described fungal lineage represents another origin of the symbiosis within the Kingdom Fungi, adding to our understanding of the geographic ancestry of ambrosia fungi. P. cavipennis possesses pharyngeal mycangia which appear restrictive in fungus selection. This ambrosia beetle-fungus association has remained stable even after invasions into non-native regions.     

The ambrosia symbiosis is specific in some species and promiscuous in others: evidence from community pyrosequencing

Symbioses are increasingly seen as dynamic ecosystems with multiple associates and varying fidelity. Symbiont specificity remains elusive in one of the most ecologically successful and economically damaging eukaryotic symbioses: the ambrosia symbiosis of wood-boring beetles and fungi. We used multiplexed pyrosequencing of amplified internal transcribed spacer II (ITS2) ribosomal DNA (rDNA) libraries to document the communities of fungal associates and symbionts inside the mycangia (fungus transfer organ) of three ambrosia beetle species, Xyleborus affinis, Xyleborus ferrugineus and Xylosandrus crassiusculus. We processed 93 beetle samples from 5 locations across Florida, including reference communities. Fungal communities within mycangia included 14–20 fungus species, many more than reported by culture-based studies. We recovered previously known nutritional symbionts as members of the core community. We also detected several other fungal taxa that are equally frequent but whose function is unknown and many other transient species. The composition of fungal assemblages was significantly correlated with beetle species but not with locality. The type of mycangium appears to determine specificity: two Xyleborus with mandibular mycangia had multiple dominant associates with even abundances; Xylosandrus crassiusculus (mesonotal mycangium) communities were dominated by a single symbiont, Ambrosiella sp. Beetle mycangia also carried many fungi from the environment, including plant pathogens and endophytes. The ITS2 marker proved useful for ecological analyses, but the taxonomic resolution was limited to fungal genus or family, particularly in Ophiostomatales, which are under-represented in our amplicons as well as in public databases. This initial analysis of three beetle species suggests that each clade of ambrosia beetles and each mycangium type may support a functionally and taxonomically distinct symbiosis.     

THE EVOLUTION OF AGRICULTURE IN BEETLES (CURCULIONIDAE: SCOLYTINAE AND PLATYPODINAE)

Abstract Beetles in the weevil subfamilies Scolytinae and Platypodinae are unusual in that they burrow as adults inside trees for feeding and oviposition. Some of these beetles are known as ambrosia beetles for their obligate mutualisms with asexual fungi—known as ambrosia fungi—that are derived from plant pathogens in the ascomycete group known as the ophiostomatoid fungi. Other beetles in these subfamilies are known as bark beetles and are associated with free‐living, pathogenic ophiostomatoid fungi that facilitate beetle attack of phloem of trees with resin defenses. Using DNA sequences from six genes, including both copies of the nuclear gene encoding enolase, we performed a molecular phylogenetic study of bark and ambrosia beetles across these two subfamilies to establish the rate and direction of changes in life histories and their consequences for diversification. The ambrosia beetle habits have evolved repeatedly and are unreversed. The subfamily Platypodinae is derived from within the Scolytinae, near the tribe Scolytini. Comparison of the molecular branch lengths of ambrosia beetles and ambrosia fungi reveals a strong correlation, which a fungal molecular clock suggests spans 60 to 21 million years. Bark beetles have shifted from ancestral association with conifers to angiosperms and back again several times. Each shift to angiosperms is associated with elevated diversity, whereas the reverse shifts to conifers are associated with lowered diversity. The unusual habit of adult burrowing likely facilitated the diversification of these beetle‐fungus associations, enabling them to use the biomass‐rich resource that trees represent and set the stage for at least one origin of eusociality.     

Cover Caption

The ambrosia beetle, Xylosandrus discolor (Blandford) (Scolytinae: Curculionidae), is common in southeastern Asia. As this beetle bores into wood and reproduces, its only food source is the white fungi (cover the chamber in the figure) which are cultured by the adult. Xylosandrus ambrosia beetles each have a pronotal mycangia (an open void in the body) which transport ambrosia fungi from their natal gallery to their new gallery. Mycangia are dynamic and their morphological changes correspond to the phases of the symbiosis (see pages 732–742). Photo provided by You Li.     

Mycangia of Ambrosia Beetles Host Communities of Bacteria

The research field of animal and plant symbioses is advancing from studying interactions between two species to whole communities of associates. High-throughput sequencing of microbial communities supports multiplexed sampling for statistically robust tests of hypotheses about symbiotic associations. We focus on ambrosia beetles, the increasingly damaging insects primarily associated with fungal symbionts, which have also been reported to support bacteria. To analyze the diversity, composition, and specificity of the beetles' prokaryotic associates, we combine global sampling, insect anatomy, 454 sequencing of bacterial rDNA, and multivariate statistics to analyze prokaryotic communities in ambrosia beetle mycangia, organs mostly known for transporting symbiotic fungi. We analyze six beetle species that represent three types of mycangia and include several globally distributed species, some with major economic importance (Dendroctonus frontalis, Xyleborus affinis, Xyleborus bispinatus-ferrugineus, Xyleborus glabratus, Xylosandrus crassiusculus, and Xylosandrus germanus). Ninety-six beetle mycangia yielded 1,546 bacterial phylotypes. Several phylotypes appear to form the core microbiome of the mycangium. Three Mycoplasma (originally thought restricted to vertebrates), two Burkholderiales, and two Pseudomonadales are repeatedly present worldwide in multiple beetle species. However, no bacterial phylotypes were universally present, suggesting that ambrosia beetles are not obligately dependent on bacterial symbionts. The composition of bacterial communities is structured by the host beetle species more than by the locality of origin, which suggests that more bacteria are vertically transmitted than acquired from the environment. The invasive X. glabratus and the globally distributed X. crassiusculus have unique sets of bacteria, different from species native to North America. We conclude that the mycangium hosts in multiple vertically transmitted bacteria such as Mycoplasma, most of which are likely facultative commensals or parasites.     

Dynamics of the fungal symbionts in the gallery system and the mycangia of the ambrosia beetle,Xylosandrus mutilatus (Blandford) (Coleoptera: Scolytidae) in relation to its life history

The dynamics of the fungal symbionts in the gallery system and the mycangia of the ambrosia beetle,Xylosandrus mutilatus, were studied in relation to its life history using both isolation experiments and scanning electron microscopy (SEM). In the galleries,Ambrosiella sp. was predominant during the larval stages but its relative dominance gradually decreased during the development of the larvae. In contrast, yeasts (mainlyCandida sp.) andPaecilomyces sp. dominated continuously in the galleries after eclosion.Ambrosiella sp. was consistently stored in the mycangia in all adult stages, except in the teneral and overwintering adults when the other fungi were dominant. No fungal spores occurred in the mycangia of the adult beetles reared under aseptic conditions from the pupal stage, while onlyAmbrosiella sp. was stored in those reared from the teneral-adult stage. These results suggest that: (i) Xmutilatus is associated with at least three fungal species, among whichAmbrosiella sp. is the most essential food resource for development of the broods; (ii) immediately after eclosion, new female adults may take at least four associated fungal species, with no or incomplete selection, into their mycangia from the walls of the cradles; and (iii) conditions may well be produced in the mycangia of both matured and dispersing beetles whereby only the spores ofAmbrosiella sp. can proliferate.     

Host switching by an ambrosia beetle fungal mutualist: Mycangial colonization of indigenous beetles by the invasive laurel wilt fungal pathogen

Ambrosia beetles require their fungal symbiotic partner as their cultivated (farmed) food source in tree galleries. While most fungal-beetle partners do not kill the host trees they inhabit, since their introduction (invasion) into the United states around ~2002, the invasive beetle Xyleborus glabratus has vectored its mutualist partner (but plant pathogenic) fungus, Harringtonia lauricola, resulting in the deaths of over 300 million trees. Concerningly, indigenous beetles have been caught bearing H. lauricola. Here, we show colonization of the mycangia of the indigenous X. affinis ambrosia beetle by H. lauricola. Mycangial colonization occurred within 1 h of feeding, with similar levels seen for H. lauricola as found for the native X. affinis-R. arxii fungal partner. Fungal mycangial occupancy was stable over time and after removal of the fungal source, but showed rapid turnover when additional fungal cells were available. Microscopic visualization revealed two pre-oral mycangial pouches of ~100–200 × 25–50 μm/each, with narrow entry channels of 25–50 × 3–10 μm. Fungi within the mycangia underwent a dimorphic transition from filamentous/blastospore growth to yeast-like budding with alterations to membrane structures. These data identify the characteristics of ambrosia beetle mycangial colonization, implicating turnover as a mechanism for host switching of H. lauricola to other ambrosia beetle species.     

The influence of forest stand and site characteristics on the composition of exotic dominated ambrosia beetle communities (Coleoptera: Curculionidae: Scolytinae)

Economic and biological consequences are associated with exotic ambrosia beetles and their fungal associates. Despite this, knowledge of ambrosia beetles and their ecological interactions remain poorly understood, especially in the oak-hickory forest region. We examined how forest stand and site characteristics influenced ambrosia beetle habitat use as evaluated by species richness and abundance of ambrosia beetles, both the native component and individual exotic species. We documented the species composition of the ambrosia beetle community, flight activity, and habitat use over a 2-yr period by placing flight traps in regenerating clearcuts and older oak-hickory forest stands differing in topographic aspect. The ambrosia beetle community consisted of 20 species with exotic ambrosia beetle species dominating the community. Similar percentages of exotic ambrosia beetles occurred among the four forest habitats despite differences in stand age and aspect. Stand characteristics, such as stand age and forest structure, influenced ambrosia beetle richness and the abundances of a few exotic ambrosia beetle species and the native ambrosia beetle component. Topographic aspect had little influence on ambrosia beetle abundance or species richness. Older forests typically have more host material than younger forests and our results may be related to the amount of dead wood present. Different forms of forest management may not alter the percent contribution of exotic ambrosia beetles to the ambrosia beetle community.     

Identification of the ambrosia fungus of <Emphasis Type="Italic">Xyleborus monographus</Emphasis> and <Emphasis Type="Italic">X. dryographus</Emphasis> (Coleoptera: Curculionidae,Scolytinae)

The scolytid ambrosia beetles Xyleborus monographus and X. dryographus were investigated to identify their nutritional ambrosia fungi. The examination of the oral mycetangia of the beetles, the specialized organs for fungal transport, revealed the dominant occurrence of Raffaelea montetyi, a fungus that was also predominant in the beetle tunnels in the immediate vicinity of the feeding larvae. R. montetyi was previously known only as the ambrosia fungus of the platypodid ambrosia beetle, Platypus cylindrus. These beetle species inhabit the same habitat, mainly trunks of oaks in the Western Palaeartic. The possibility of an exchange of the symbiotic fungus between the ambrosia beetles within their common breeding place is discussed. Consequently, the previous hypothesis of a species-specific association of a single ambrosia fungus with a single beetle species is questioned. A phylogenetic analysis based on DNA sequences classified R. montetyi within the Ophiostomatales of the ascomycetes. The investigation of conidiogenesis of R. montetyi by SEM supported this taxonomic placement and showed the development of the conidia by annellidic percurrent proliferation, identical to the conidiogenesis reported for many anamorph states of the Ophiostomatales.     

Broadscale Specificity in a Bark Beetle–Fungal Symbiosis: a Spatio-temporal Analysis of the Mycangial Fungi of the Western Pine Beetle

Whether and how mutualisms are maintained through ecological and evolutionary time is a seldom studied aspect of bark beetle–fungal symbioses. All bark beetles are associated with fungi and some species have evolved structures for transporting their symbiotic partners. However, the fungal assemblages and specificity in these symbioses are not well known. To determine the distribution of fungi associated with the mycangia of the western pine beetle (Dendroctonus brevicomis), we collected beetles from across the insect’s geographic range including multiple genetically distinct populations. Two fungi, Entomocorticium sp. B and Ceratocystiopsis brevicomi, were isolated from the mycangia of beetles from all locations. Repeated sampling at two sites in Montana found that Entomocorticium sp. B was the most prevalent fungus throughout the beetle’s flight season, and that females carrying that fungus were on average larger than females carrying C. brevicomi. We present evidence that throughout the flight season, over broad geographic distances, and among genetically distinct populations of beetle, the western pine beetle is associated with the same two species of fungi. In addition, we provide evidence that one fungal species is associated with larger adult beetles and therefore might provide greater benefit during beetle development. The importance and maintenance of this bark beetle–fungus interaction is discussed.     

REPEATED EVOLUTION OF CROP THEFT IN FUNGUS‐FARMING AMBROSIA BEETLES

Ambrosia beetles, dominant wood degraders in the tropics, create tunnels in dead trees and employ gardens of symbiotic fungi to extract nutrients from wood. Specificity of the beetle–fungus relationship has rarely been examined, and simple vertical transmission of a specific fungal cultivar by each beetle species is often assumed in literature. We report repeated evolution of fungal crop stealing, termed mycocleptism, among ambrosia beetles. The mycocleptic species seek brood galleries of other species, and exploit their established fungal gardens by tunneling through the ambient mycelium‐laden wood. Instead of carrying their own fungal sybmbionts, mycocleptae depend on adopting the fungal assemblages of their host species, as shown by an analysis of fungal DNA from beetle galleries. The evidence for widespread horizontal exchange of fungi between beetles challenges the traditional concept of ambrosia fungi as species‐specific symbionts. Fungus stealing appears to be an evolutionarily successful strategy. It evolved independently in several beetle clades, two of which have radiated, and at least one case was accompanied by a loss of the beetles’ fungus‐transporting organs. We demonstrate this using the first robust phylogeny of one of the world's largest group of ambrosia beetles, Xyleborini.     

Relationships among wood‐boring beetles,fungi, and the decomposition of forest biomass

A prevailing paradigm in forest ecology is that wood‐boring beetles facilitate wood decay and carbon cycling, but empirical tests have yielded mixed results. We experimentally determined the effects of wood borers on fungal community assembly and wood decay within pine trunks in the southeastern United States. Pine trunks were made either beetle‐accessible or inaccessible. Fungal communities were compared using culturing and high‐throughput amplicon sequencing (HTAS) of DNA and RNA. Prior to beetle infestation, living pines had diverse fungal endophyte communities. Endophytes were displaced by beetle‐associated fungi in beetle‐accessible trees, whereas some endophytes persisted as saprotrophs in beetle‐excluded trees. Beetles increased fungal diversity several fold. Over forty taxa of Ascomycota were significantly associated with beetles, but beetles were not consistently associated with any known wood‐decaying fungi. Instead, increasing ambrosia beetle infestations caused reduced decay, consistent with previous in vitro experiments that showed beetle‐associated fungi reduce decay rates by competing with decay fungi. No effect of bark‐inhabiting beetles on decay was detected. Platypodines carried significantly more fungal taxa than scolytines. Molecular results were validated by synthetic and biological mock communities and were consistent across methodologies. RNA sequencing confirmed that beetle‐associated fungi were biologically active in the wood. Metabarcode sequencing of the LSU/28S marker recovered important fungal symbionts that were missed by ITS2, though community‐level effects were similar between markers. In contrast to the current paradigm, our results indicate ambrosia beetles introduce diverse fungal communities that do not extensively decay wood, but instead reduce decay rates by competing with wood decay fungi.     

Landscape‐scale genetic differentiation of a mycangial fungus associated with the ambrosia beetle,Xylosandrus germanus (Blandford) (Curculionidae:Scolytinae) in Japan

In this study, we examined the genetic structures of the ambrosia fungus isolated from mycangia of the scolytine beetle, Xylosandrus germanus to understand their co‐evolutionary relationships. We analyzed datasets of three ambrosia fungus loci (18S rDNA, 28S rDNA, and the β‐tubulin gene) and a X. germanus locus dataset (cytochrome c oxidase subunit 1 (COI) mitochondrial DNA). The ambrosia fungi were separated into three cultural morphptypes, and their haplotypes were distinguished by phylogenetic analysis on the basis of the three loci. The COI phylogenetic analysis revealed three distinct genetic lineages (clades A, B, and C) within X. germanus, each of which corresponded to specific ambrosia fungus cultural morphptypes. The fungal symbiont phylogeny was not concordant with that of the beetle. Our results suggest that X. germanus may be unable to exchange its mycangial fungi, but extraordinary horizontal transmission of symbiotic fungi between the beetle's lineages occurred at least once during the evolutionary history of this symbiosis.     

Plasticity of mycangia in Xylosandrus ambrosia beetles

Insects that depend on microbial mutualists evolved a variety of organs to transport the microsymbionts while dispersing. The ontogeny and variability of such organs is rarely studied, and the microsymbiont*s effects on the animal tissue development remain unknown in most cases. Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae or Platypodinae) and their mutualistic fungi are an ideal system to study the animalfungus interactions. While the interspecific diversity of their fungus transport organ一 mycangia—is well-known, their developmental plasticity has been poorly described. To determine the ontogeny of the mycangium and the influence of the symbiotic fungus on the tissue development, we dissected by hand or scanned with micro-CT the mycangia in various developmental stages in five Xylosandrus ambrosia beetle species that possess a large, mesonotal mycangium: Xylosandrus amputatus. Xylosandrus compactus, Xylosandrus crassiusculus, Xylosandrus discolor, and Xylosandrus germanus. We processed 181 beetle samples from the United States and China. All five species displayed three stages of the mycangium development:(1) young teneral adults had an empty, deflated and cryptic mycangium without fungal mass;(2) in fully mature adults during dispersal, the promesonotal membrane was inflated, and most individuals developed a mycangium mostly filled with the symbiont, though size and symmetry varied;and (3) after successful establishment of their new galleries, most females discharged the bulk of the fun gal inoculum and deflated the mycangium. Experimental aposymbiotic individuals demonstrated that the pronotal membrane invaginated independently of the presence of the fungus, but the fungus was required for inflation. Mycangia are more dynamic than previously thought, and their morphological changes correspond to the phases of the symbiosis. Importantly, studies of the fungal symbionts or plant pathogen transmission in ambrosia beetles need to consider which developmental stage to sample. We provide illustrations of the different stages, including microphotography of dissections and micro-CT scans.     

New Fungus-Insect Symbiosis: Culturing,Molecular, and Histological Methods Determine Saprophytic Polyporales Mutualists of Ambrosiodmus Ambrosia Beetles

Ambrosia symbiosis is an obligate, farming-like mutualism between wood-boring beetles and fungi. It evolved at least 11 times and includes many notorious invasive pests. All ambrosia beetles studied to date cultivate ascomycotan fungi: early colonizers of recently killed trees with poor wood digestion. Beetles in the widespread genus Ambrosiodmus, however, colonize decayed wood. We characterized the mycosymbionts of three Ambrosiodmus species using quantitative culturing, high-throughput metabarcoding, and histology. We determined the fungi to be within the Polyporales, closely related to Flavodon flavus. Culture-independent sequencing of Ambrosiodmus minor mycangia revealed a single operational taxonomic unit identical to the sequences from the cultured Flavodon. Histological sectioning confirmed that Ambrosiodmus possessed preoral mycangia containing dimitic hyphae similar to cultured F. cf. flavus. The Ambrosiodmus-Flavodon symbiosis is unique in several aspects: it is the first reported association between an ambrosia beetle and a basidiomycotan fungus; the mycosymbiont grows as hyphae in the mycangia, not as budding pseudo-mycelium; and the mycosymbiont is a white-rot saprophyte rather than an early colonizer: a previously undocumented wood borer niche. Few fungi are capable of turning rotten wood into complete animal nutrition. Several thousand beetle-fungus symbioses remain unstudied and promise unknown and unexpected mycological diversity and enzymatic innovations.     

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

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.     

Non‐destructive observation of the mycangia of Euwallacea interjectus (Blandford) (Coleoptera: Curculionidae: Scolytinae) using X‐ray computed tomography

X‐ray microtomography has been applied successfully to obtain reliable microstructural information of many insect species. Nonetheless, the technique has not been widely applied to ambrosia beetles. The ambrosia beetle Euwallacea interjectus (Blandford) was first recorded as a vector of plant pathogenic fungus Ceratocystis ficicola Kajitani & Masuya, which has caused serious wilt disease in many fig orchards in Japan since 1999. Previous studies of E. interjectus have not described the mycangia (fungus‐storing organ) in detail. In this study, we non‐destructively examined the internal structure of an adult female of E. interjectus through computed microtomography scans. Paired mycangia were observed on typical computed tomography cross‐sections of the head. Each mycangium, ovoid in shape, was located in tissues just posterior to emarginated notch of eyes, adjacent to pharynx. Three dimensions (length × width × depth) of the mycangia were measured on stereography. We confirmed the absence of mycangia in the other body parts, such as elytra, prothorax and coxa of legs.     

A mid-Cretaceous ambrosia fungus, Paleoambrosia entomophila gen. nov. et sp. nov. (Ascomycota: Ophiostomatales) in Burmese (Myanmar) amber,and evidence for a femoral mycangium

An ambrosia fungus is described from filamentous sporodochia adjacent to a wood–boring ambrosia beetle (Coleoptera: Curculionidae: Platypodinae) in mid-Cretaceous Burmese amber. Yeast-like propagules and hyphal fragments of Paleoambrosia entomophila gen. nov. et sp. nov. occur in glandular sac mycangia located inside the femur of the beetle. This is the first record of a fossil ambrosia fungus, showing that symbiotic associations between wood–boring insects and ectosymbiotic fungi date back some 100 million years ago. The present finding moves the origin of fungus-growing by insects from the Oligocene to the mid-Cretaceous and suggests a Gondwanan origin.     

Ambrosia fungi in the insect-fungi symbiosis in relation to cork oak decline

Ambrosia fungi live associated with beetles (Scolytidae and Platypodidae) in host trees and act as a food source for the insects. The symbiotic relation is important to the colonizing strategies of host trees by beetles. Ambrosia fungi are dimorphic: they grow as ambrosial form and as mycelium. The fungi are highly specialized, adapted to a specific beetle and to the biotope where they both live. In addition other fungi have been found such as tree pathogenic fungi that may play a role in insects host colonization success. Saprophytic fungi are also present in insects galleries. These may decompose cellulose and/or be antagonistic to other less beneficial fungi. This paper summarizes the importance of ambrosia fungi and the interaction with insects and hosts. The possibility of the transport of pathogenic fungi by Platypus cylindrus to cork oak thus contributing for its decline is discussed.     

Evidence for a new lineage of primary ambrosia fungi in Geosmithia Pitt (Ascomycota: Hypocreales)

Geosmithia is a genus of mitosporic filamentous fungi typically associated with phloeophagous bark beetles world-wide. During this study, the fungal associates of ambrosia beetles Cnesinus lecontei, Eupagiocerus dentipes, and Microcorthylus sp. from Costa Rica, were studied using morphology and DNA sequences. Fungal associates belonged to four undescribed Geosmithia species. Geosmithia eupagioceri sp. nov. and G. microcorthyli sp. nov. are evidently primary ambrosia fungi of their respective vectors E. dentipes and Microcorthylus species. They both have convergently evolved distinct morphological adaptations including the production of large, solitary and globose conidia, and yeast-like cells. Tunnels of C. lecontei contained an undescribed Geosmithia species, but its nutritional importance for its vector is unclear. An auxiliary ambrosia fungus, Geosmithia rufescens sp. nov., was found associated with both G. eupagioceri and the Geosmithia species associated with C. lecontei. G. microcorthyli is genetically quite similar to the phloem-associated Geosmithia sp. 8 from Europe. Large differences in morphology between these two species suggest the rapid co-evolution resulting from the close symbiosis of the former with its beetle host. The ITS rDNA sequences of G. microcorthyli and Geosmithia sp. 8 were not diagnostic, suggesting that alternative markers such as EF-1α, IGS rDNA or β-tubulin should be used, together with morphological and ecological data, for species delimitation in this genus. The primary ambrosia fungi described here are derived from phloem-associated ancestors, and represent two independent lineages of ambrosia fungi in the Hypocreales and a new ecological strategy within Geosmithia.