Ambrosia Beetles Prefer Closed Canopies: A Case Study in Oak Forests in Central Europe

Research Highlights: The percentage of canopy closure was found to be the main factor associated with ambrosia beetle abundance and species richness. The latter two variables increased as canopy closure increased, probably because a high percentage of canopy closure provides a stable and humid environment suitable for the growth of ambrosia fungi. Objectives: Oak is a common host tree for ambrosia beetles (Coleoptera: Curculionidae: Scolytinae), which have independently evolved a nutritional mutualism with fungi. We suspected that ambrosia beetles might have specific habitat preferences that are different from those of other saproxylic beetles and that reflect the specific habitat preferences of their food, i.e., ambrosia fungi. Methods: We assessed ambrosia beetle abundance with ethanol-lured traps in five old-growth oak dominated forests and five managed oak dominated forests (one trap per forest) during the vegetation period in 2020. We determined whether ambrosia beetle abundance and species richness depend on forest type (managed vs. unmanaged), degree of canopy closure, abundance of oak trees, abundance of coarse deadwood, and abundance of dead oak branches. Results: In total, 4137 individuals of six species of ambrosia beetles associated with oaks were captured. The native ambrosia beetle Anisandrus dispar represented the majority of trapped ambrosia bark beetles. A. dispar along with another ambrosia beetle, Xyleborinus saxesenii, represented 99% of all captured beetles. Conclusions: In addition to canopy closure, the abundance of oak trees and the abundance of dead oak branches were significantly associated with ambrosia beetle abundance and species richness. The abundance of A. dispar was mainly correlated with dead oak branch abundance and the degree of canopy closure, whereas the abundances of X. saxesenii and of the invasive species Xyleborinus attenuatus and Cyclorhipidion bodoanum were mainly correlated with the net area occupied by oak trees.

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.

Evidence for Succession and Putative Metabolic Roles of Fungi and Bacteria in the Farming Mutualism of the Ambrosia Beetle Xyleborus affinis

Ambrosia beetles farm their own food fungi within tunnel systems in wood and are among the three insect lineages performing agriculture (the others are fungus-farming ants and termites). In ambrosia beetles, primary ambrosia fungus cultivars have been regarded essential, whereas other microbes have been more or less ignored. Our KEGG analyses suggest so far unknown roles of yeasts and bacterial symbionts, by preparing the tunnel walls for the primary ambrosia fungi. This preparation includes enzymatic degradation of wood, essential amino acid production, and nitrogen fixation. The latter is especially exciting because if it turns out to be present in vivo in ambrosia beetles, all farming animals (including humans) are dependent on atmospheric nitrogen fertilization of their crops. As previous internal transcribed spacer (ITS) metabarcoding approaches failed on covering the primary ambrosia fungi, our 18S metabarcoding approach can also serve as a template for future studies on the ambrosia beetle-fungus symbiosis.

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.

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.