Preinvasion Assessment of Exotic Bark Beetle-Vectored Fungi to Detect Tree-Killing Pathogens.

Exotic diseases and pests of trees have caused continental-scale disturbances in forest ecosystems and industries, and their invasions are considered largely unpredictable. We tested the concept of preinvasion assessment of not yet invasive organisms, which enables empirical risk assessment of potential invasion and impact. Our example assesses fungi associated with Old World bark and ambrosia beetles and their potential to impact North American trees. We selected 55 Asian and European scolytine beetle species using host use, economic, and regulatory criteria. We isolated 111 of their most consistent fungal associates and tested their effect on four important southeastern American pine and oak species. Our test dataset found no highly virulent pathogens that should be classified as an imminent threat. Twenty-two fungal species were minor pathogens, which may require context-dependent response for their vectors at North American borders, while most of the tested fungi displayed no significant impact. Our results are significant in three ways; they ease the concerns over multiple overseas fungus vectors suspected of heightened potential risk, they provide a basis for the focus on the prevention of introduction and establishment of species that may be of consequence, and they demonstrate that preinvasion assessment, if scaled up, can support practical risk assessment of exotic pathogens.

Climate change effects on animal ecology: butterflies and moths as a case study.

Butterflies and moths (Lepidoptera) are one of the most studied, diverse, and widespread animal groups, making them an ideal model for climate change research. They are a particularly informative model for studying the effects of climate change on species ecology because they are ectotherms that thermoregulate with a suite of physiological, behavioural, and phenotypic traits. While some species have been negatively impacted by climatic disturbances, others have prospered, largely in accordance with their diversity in life-history traits. Here we take advantage of a large repertoire of studies on butterflies and moths to provide a review of the many ways in which climate change is impacting insects, animals, and ecosystems. By studying these climate-based impacts on ecological processes of Lepidoptera, we propose appropriate strategies for species conservation and habitat management broadly across animals.

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.

A selective fungal transport organ (mycangium) maintains coarse phylogenetic congruence between fungus-farming ambrosia beetles and their symbionts.

Thousands of species of ambrosia beetles excavate tunnels in wood to farm fungi. They maintain associations with particular lineages of fungi, but the phylogenetic extent and mechanisms of fidelity are unknown. We test the hypothesis that selectivity of their mycangium enforces fidelity at coarse phylogenetic scales, while permitting promiscuity among closely related fungal mutualists. We confirm a single evolutionary origin of the Xylosandrus complex-a group of several xyleborine genera that farm fungi in the genus Ambrosiella. Multi-level co-phylogenetic analysis revealed frequent symbiont switching within major Ambrosiella clades, but not between clades. The loss of the mycangium in Diuncus, a genus of evolutionary cheaters, was commensurate with the loss of fidelity to fungal clades, supporting the hypothesis that the mycangium reinforces fidelity. Finally, in vivo experiments tracked symbiotic compatibility throughout the symbiotic life cycle of Xylosandrus compactus and demonstrated that closely related Ambrosiella symbionts are interchangeable, but the probability of fungal uptake in the mycangium was significantly lower in more phylogenetically distant species of symbionts. Symbiont loads in experimental subjects were similar to wild-caught beetles. We conclude that partner choice in ambrosia beetles is achieved in the mycangium, and co-phylogenetic inferences can be used to predict the likelihood of specific symbiont switches.

New Meredithiella species from mycangia of Corthylus ambrosia beetles suggest genus-level coadaptation but not species-level coevolution.

Meredithiella norrisii (Microascales, Ceratocystidaceae) is an ambrosia fungus carried in mycangia of the North American ambrosia beetle, Corthylus punctatissimus. Reports on the identity of the fungal symbionts of other species of Corthylus have been inconsistent. This study tested the hypothesis that Meredithiella spp. are the primary symbionts of Corthylus spp. Cultures and/or internal transcribed spacer (ITS) rDNA barcode sequences of Meredithiella spp. were obtained consistently from beetles and galleries of nine Corthylus spp. The ITS sequences of three putative species of Meredithiella were associated with C. consimilis and C. flagellifer in Mexico and C. calamarius in Costa Rica. The symbiont of C. columbianus in the USA was identified as M. norrisii. Two new Meredithiella spp. are described: M. fracta from C. papulans in Florida and Honduras, and M. guianensis associated with C. crassus and two unidentified Corthylus spp. in French Guyana. The Meredithiella spp. propagate in the mycangia of adult females by thallic-arthric growth, and the ambrosia growth in larval cradles comprises bead-like hyphal swellings or conidiophores, with or without terminal aleurioconidia. Bayesian phylogenetic analysis of a combined 18S and 28S nuc rDNA and translation elongation factor 1-α (TEF1-α) data set demonstrated that Meredithiella is a distinct monophyletic clade within the Ceratocystidaceae, but its phylogenetic placement with regard to the other ambrosial genera in the family remains ambiguous. The mycangia of C. punctatissimus and C. papulans are also compared using light microscopy and micro-computed tomography (micro-CT) imaging, revealing that they differ in both size and shape, but these differences may not correlate with different lineages of Meredithiella.

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.

Wood decay fungus Flavodon ambrosius (Basidiomycota: Polyporales) is widely farmed by two genera of ambrosia beetles.

The ambrosia fungus Flavodon ambrosius is the primary nutritional mutualist of ambrosia beetles Ambrosiodmus and Ambrosiophilus in North America. F. ambrosius is the only known ambrosial basidiomycete, unique in its efficient lignocellulose degradation. F. ambrosius is associated with both native American beetle species and species introduced from Asia. It remains unknown whether F. ambrosius is strictly a North American fungus, or whether it is also associated with these ambrosia beetle genera on other continents. We isolated fungi from the mycangia and galleries of ambrosia beetles Ambrosiodmus rubricollis, Ambrosiodmus minor, Ambrosiophilus atratus, and Ambrosiophilus subnepotulus in China, South Korea, and Vietnam. Phylogenetic analyses suggest that all Asian and North American isolates represent a single haplotype. These results confirm Flavodon ambrosius as the exclusive mutualistic fungus of multiple Ambrosiodmus and Ambrosiophilus beetle species around the world, making it the most widespread known ambrosia fungus species, both geographically and in terms of the number of beetle species. The Flavodon-beetle symbiosis appears to employ an unusually strict mechanism for maintaining fidelity, compared to the symbioses of the related Xyleborini beetles, which mostly vector more dynamic fungal communities.

PCR Multiplexes Discriminate Fusarium Symbionts of Invasive Euwallacea Ambrosia Beetles that Inflict Damage on Numerous Tree Species Throughout the United States.

Asian Euwallacea ambrosia beetles vector Fusarium mutualists. The ambrosial fusaria are all members of the ambrosia Fusarium clade (AFC) within the Fusarium solani species complex (FSSC). Several Euwallacea-Fusarium mutualists have been introduced into nonnative regions and have caused varying degrees of damage to orchard, landscape, and forest trees. Knowledge of symbiont fidelity is limited by current identification methods, which typically requires analysis of DNA sequence data from beetles and the symbionts cultured from their oral mycangia. Here, polymerase chain reaction (PCR)-based diagnostic tools were developed to identify the six Fusarium symbionts of exotic Euwallacea spp. currently known within the United States. Whole-genome sequences were generated for representatives of six AFC species plus F. ambrosium and aligned to the annotated genome of F. euwallaceae. Taxon-specific primer-annealing sites were identified that rapidly distinguish the AFC species currently within the United States. PCR specificity, reliability, and sensitivity were validated using a panel of 72 Fusarium isolates, including 47 reference cultures. Culture-independent multiplex assays accurately identified two AFC fusaria using DNA isolated from heads of their respective beetle partners. The PCR assays were used to show that Euwallacea validus is exclusively associated with AF-4 throughout its sampled range within eastern North America. The rapid assay supports federal and state agency efforts to monitor spread of these invasive pests and mitigate further introductions.

New Raffaelea species (Ophiostomatales) from the USA and Taiwan associated with ambrosia beetles and plant hosts.

Raffaelea (Ophiostomatales) is a genus of more than 20 ophiostomatoid fungi commonly occurring in symbioses with wood-boring ambrosia beetles. We examined ambrosia beetles and plant hosts in the USA and Taiwan for the presence of these mycosymbionts and found 22 isolates representing known and undescribed lineages in Raffaelea. From 28S rDNA and ß-tubulin sequences, we generated a molecular phylogeny of Ophiostomatales and observed morphological features of seven cultures representing undescribed lineages in Raffaelea s. lat. From these analyses, we describe five new species in Raffaelea s. lat.: R. aguacate, R. campbellii, R. crossotarsa, R. cyclorhipidia, and R. xyleborina spp. nov. Our analyses also identified two plant-pathogenic species of Raffaelea associated with previously undocumented beetle hosts: (1) R. quercivora, the causative agent of Japanese oak wilt, from Cyclorhipidion ohnoi and Crossotarsus emancipatus in Taiwan, and (2) R. lauricola, the pathogen responsible for laurel wilt, from Ambrosiodmus lecontei in Florida. The results of this study show that Raffaelea and associated ophiostomatoid fungi have been poorly sampled and that future investigations on ambrosia beetle mycosymbionts should reveal a substantially increased diversity.

Fungal Associates of the Xylosandrus compactus (Coleoptera: Curculionidae, Scolytinae) Are Spatially Segregated on the Insect Body.

Studies of symbioses have traditionally focused on explaining one-to-one interactions between organisms. In reality, symbioses are often much more dynamic. They can involve many interacting members, and change depending on context. In studies of the ambrosia symbiosis-the mutualism between wood borer beetles and fungi-two variables have introduced uncertainty when explaining interactions: imprecise symbiont identification, and disregard for anatomical complexity of the insects. The black twig borer, Xylosandrus compactus Eichhoff, is a globally invasive ambrosia beetle that infests >200 plant species. Despite many studies on this beetle, reports of its primary symbionts are conflicting. We sampled adult X. compactus and infested plant material in central Florida to characterize the fungal symbiont community using dilution series, beetle partitioning, and DNA-based identification. X. compactus was consistently associated with two fungal taxa, Fusarium spp. and Ambrosiella xylebori Multivariate analyses revealed that A. xylebori was strongly associated with the beetle mycangium while Fusarium spp. were associated with the abdomen and external surfaces. The Fusarium spp. carried by X. compactus are not members of the Ambrosia Fusarium Clade, and are probably not mutualists. Fungal community composition of the mycangium was less variable than external body surfaces, thus providing a more consistent fungal inoculum. This is the first report of spatial partitioning as a mechanism for maintenance of a multimember ambrosia fungus community. Our results provide an explanation for discrepancies among previous reports, and suggest that conflicting results are not due to differences in symbiont communities, but due to inconsistent and incomplete sampling.

Identification, pathogenicity and abundance of Paracremonium pembeum sp. nov. and Graphium euwallaceae sp. nov.--two newly discovered mycangial associates of the polyphagous shot hole borer (Euwallacea sp.) in California.

Fusarium euwallaceae is a well-characterized fungal symbiont of the exotic ambrosia beetle Euwallacea sp. (polyphagous shot hole borer [PSHB]), together inciting Fusarium dieback on many host plants in Israel and California. Recent discoveries of additional fungal symbionts within ambrosia beetle mycangia suggest these fungi occur as communities. Colony-forming units of Graphium euwallaceae sp. nov. and Paracremonium pembeum sp. nov., two novel fungal associates of PSHB from California, grew from 36 macerated female heads and 36 gallery walls collected from Platanus racemosa, Acer negundo, Persea americana and Ricinus communis. Fungi were identified based on micromorphology and phylogenetic analyses of the combined internal transcribed spacer region (nuc rDNA ITS1-5.8S-ITS2 [ITS barcode]), elongation factor (EF 1-α), small subunit (18S rDNA) sequences for Graphium spp., ITS, EF 1-α, calmodulin (cmdA), large subunit of the ATP citrate lyase (acl1), ß-tubulin (tub2), RNA polymerase II second largest subunit (rpb2) and large subunit (28S rDNA) sequences for Paracremonium spp. Other Graphium spp. recovered from PSHB in Vietnam, Euwallacea fornicatus in Thailand, E. validus in Pennsylvania and Paracremonium sp. recovered from PSHB in Vietnam were identified. F. euwallaceae was recovered from mycangia at higher frequencies and abundances in all hosts except R. communis, in which those of F. euwallaceae and P. pembeum were equal. P. pembeum was relatively more abundant within gallery walls of A. negundo and R. communis. In all hosts combined F. euwallaceae was relatively more abundant within PSHB heads than gallery walls. All three fungi grew at different rates and colonized inoculated excised stems of P. americana and A. negundo. P. pembeum produced longer lesions than F. euwallaceae and G. euwallaceae on inoculated avocado shoots. Results indicate PSHB is associated with a dynamic assemblage of mycangial fungal associates that pose additional risk to native and nonnative hosts in California.

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.

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.

Discordant phylogenies suggest repeated host shifts in the Fusarium-Euwallacea ambrosia beetle mutualism.

The mutualism between xyleborine beetles in the genus Euwallacea (Coleoptera: Curculionidae: Scolytinae) and members of the Ambrosia Fusarium Clade (AFC) represents one of 11 known evolutionary origins of fungiculture by ambrosia beetles. Female Euwallacea beetles transport fusarial symbionts in paired mandibular mycangia from their natal gallery to woody hosts where they are cultivated in galleries as a source of food. Native to Asia, several exotic Euwallacea species were introduced into the United States and Israel within the past two decades and they now threaten urban landscapes, forests and avocado production. To assess species limits and to date the evolutionary diversification of the mutualists, we reconstructed the evolutionary histories of key representatives of the Fusarium and Euwallacea clades using maximum parsimony and maximum likelihood methods. Twelve species-level lineages, termed AF 1-12, were identified within the monophyletic AFC and seven among the Fusarium-farming Euwallacea. Bayesian diversification-time estimates placed the origin of the Euwallacea-Fusarium mutualism near the Oligocene-Miocene boundary ∼19-24 Mya. Most Euwallacea spp. appear to be associated with one species of Fusarium, but two species farmed two closely related fusaria. Euwallacea sp. #2 in Miami-Dade County, Florida cultivated Fusarium spp. AF-6 and AF-8 on avocado, and Euwallacea sp. #4 farmed Fusarium ambrosium AF-1 and Fusarium sp. AF-11 on Chinese tea in Sri Lanka. Cophylogenetic analyses indicated that the Euwallacea and Fusarium phylogenies were largely incongruent, apparently due to the beetles switching fusarial symbionts (i.e., host shifts) at least five times during the evolution of this mutualism. Three cospeciation events between Euwallacea and their AFC symbionts were detected, but randomization tests failed to reject the null hypothesis that the putative parallel cladogenesis is a stochastic pattern. Lastly, two collections of Euwallacea sp. #2 from Miami-Dade County, Florida shared an identical cytochrome oxidase subunit 1 (CO1) allele with Euwallacea validus, suggesting introgressive hybridization between these species and/or pseudogenous nature of this marker. Results of the present study highlight the importance of understanding the potential for and frequency of host-switching between Euwallacea and members of the AFC, and that these shifts may bring together more aggressive and virulent combinations of these invasive mutualists.

An inordinate fondness for Fusarium: phylogenetic diversity of fusaria cultivated by ambrosia beetles in the genus Euwallacea on avocado and other plant hosts.

Ambrosia beetle fungiculture represents one of the most ecologically and evolutionarily successful symbioses, as evidenced by the 11 independent origins and 3500 species of ambrosia beetles. Here we document the evolution of a clade within Fusarium associated with ambrosia beetles in the genus Euwallacea (Coleoptera: Scolytinae). Ambrosia Fusarium Clade (AFC) symbionts are unusual in that some are plant pathogens that cause significant damage in naïve natural and cultivated ecosystems, and currently threaten avocado production in the United States, Israel and Australia. Most AFC fusaria produce unusual clavate macroconidia that serve as a putative food source for their insect mutualists. AFC symbionts were abundant in the heads of four Euwallacea spp., which suggests that they are transported within and from the natal gallery in mandibular mycangia. In a four-locus phylogenetic analysis, the AFC was resolved in a strongly supported monophyletic group within the previously described Clade 3 of the Fusarium solani species complex (FSSC). Divergence-time estimates place the origin of the AFC in the early Miocene ∼21.2 Mya, which coincides with the hypothesized adaptive radiation of the Xyleborini. Two strongly supported clades within the AFC (Clades A and B) were identified that include nine species lineages associated with ambrosia beetles, eight with Euwallacea spp. and one reportedly with Xyleborus ferrugineus, and two lineages with no known beetle association. More derived lineages within the AFC showed fixation of the clavate (club-shaped) macroconidial trait, while basal lineages showed a mix of clavate and more typical fusiform macroconidia. AFC lineages consisted mostly of genetically identical individuals associated with specific insect hosts in defined geographic locations, with at least three interspecific hybridization events inferred based on discordant placement in individual gene genealogies and detection of recombinant loci. Overall, these data are consistent with a strong evolutionary trend toward obligate symbiosis coupled with secondary contact and interspecific hybridization.