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 (<i>Trypodendron, Indocryphalus</i>, and <i>Xyloterinus politus</i>) and characterized their ambrosia fungi in pure culture and by DNA sequencing. The prothoracic mycangia of seven <i>Trypodendron</i> species all yielded <i>Phialophoropsis</i> (Microascales) ambrosia fungi, including three new species, although these relationships were not all species specific. <i>Indocryphalus</i> mycangia are characterized for the first time in the Asian <i>I. pubipennis</i>. They comprise triangular prothoracic cavities substantially smaller than those of <i>Trypodendron</i> and unexpectedly carry an undescribed species of <i>Toshionella</i> (Microascales), which are otherwise ambrosia fungi of Asian <i>Scolytoplatypus</i> (Scolytoplatypodini). <i>Xyloterinus politus</i> has two different mycangia, each with a different ambrosia fungus: <i>Raffaelea</i> cf. <i>canadensis</i> RNC5 (Ophiostomatales) in oral mycangia of both sexes and <i>Kaarikia abrahamsonii</i> (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, <i>Trypodendron</i> and <i>X. politus</i> harbor a surprising diversity of facultative symbionts in their galleries, including <i>Raffaelea</i>. 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.