The genus Ambrosiella accommodates species of Ceratocystidaceae (Microascales) that are obligate, mutualistic symbionts of ambrosia beetles, but the genus appears to be polyphyletic and more diverse than previously recognized. In addition to Ambrosiella xylebori, Ambrosiella hartigii, Ambrosiella beaveri, and Ambrosiella roeperi, three new species of Ambrosiella are described from the ambrosia beetle tribe Xyleborini: Ambrosiella nakashimae sp. nov. from Xylosandrus amputatus, Ambrosiella batrae sp. nov. from Anisandrus sayi, and Ambrosiella grosmanniae sp. nov. from Xylosandrus germanus. The genus Meredithiella gen. nov. is created for symbionts of the tribe Corthylini, based on Meredithiella norrisii sp. nov. from Corthylus punctatissimus. The genus Phialophoropsis is resurrected to accommodate associates of the Xyloterini, including Phialophoropsis trypodendri from Trypodendron scabricollis and Phialophoropsis ferruginea comb. nov. from Trypodendron lineatum. Each of the ten named species was distinguished by ITS rDNA barcoding and morphology, and the ITS rDNA sequences of four other putative species were obtained with Ceratocystidaceae-specific primers and template DNA extracted from beetles or galleries. These results support the hypothesis that each ambrosia beetle species with large, complex mycangia carries its own fungal symbiont. Conidiophore morphology and phylogenetic analyses using 18S (SSU) rDNA and TEF1α DNA sequences suggest that these three fungal genera within the Ceratocystidaceae independently adapted to symbiosis with the three respective beetle tribes. In turn, the beetle genera with large, complex mycangia appear to have evolved from other genera in their respective tribes that have smaller, less selective mycangia and are associated with Raffaelea spp. (Ophiostomatales).

Isolations from the granulate ambrosia beetle, Xylosandrus crassiusculus (Coleoptera: Curculionidae: Scolytinae: Xyleborini), collected in Georgia, South Carolina, Missouri and Ohio, yielded an undescribed species of Ambrosiella in thousands of colony-forming units (CFU) per individual female. Partial sequences of ITS and 28S rDNA regions distinguished this species from other Ambrosiella spp., which are asexual symbionts of ambrosia beetles and closely related to Ceratocystis spp. Ambrosiella roeperi sp. nov. produces sporodochia of branching conidiophores with disarticulating swollen cells, and the branches are terminated by thick-walled aleurioconidia, similar to the conidiophores and aleurioconidia of A. xylebori, which is the mycangial symbiont of a related ambrosia beetle, X. compactus. Microscopic examinations found homogeneous masses of arthrospore-like cells growing in the mycangium of X. crassiusculus, without evidence of other microbial growth. Using fungal-specific primers, only the ITS rDNA region of A. roeperi was amplified and sequenced from DNA extractions of mycangial contents, suggesting that it is the primary or only mycangial symbiont of this beetle in USA.

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.

Genera of ambrosia beetles in the tribe Xyleborini with large, mesonotal mycangia host unique fungal symbionts in the genus Ambrosiella. The symbiont of a recent invasive to the USA from Asia, Anisandrus maiche Stark, had not been previously characterized. We found the mycangium anatomy of An. maiche collected in Ohio to be similar to that of Anisandrus dispar and consistently isolated a novel fungus, Ambrosiella cleistominuta sp. nov., from An. maiche mycangia and galleries. The fungus was distinguished from other named Ambrosiella by morphological characters and DNA sequences (ITS rDNA and tef-1α). The mycangial symbionts of ambrosia beetles had been assumed to be strictly asexual, but A. cleistominuta produces cleistothecious ascomata with ascospores in beetle galleries and in culture. In contrast to ascomata of other Ceratocystidaceae, the relatively small ascomata of A. cleistominuta are neckless and without ostioles. The ascospores are relatively large, and single-ascospore colonies produced ascomata and ascospores in culture, showing that A. cleistominuta is homothallic.

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.