An expanded concept of Ceratocystis manginecans and five new species in the Latin American clade of Ceratocystis.

The genus Ceratocystis contains a number of emerging plant pathogens, mostly members of the Latin American Clade (LAC), in which there are several unresolved taxonomic controversies. Among the most important are Brazilian pathogens in the C. fimbriata complex, C. manginecans and C. eucalypticola. Representatives of C. manginecans and C. eucalypticola from India and China, respectively, were shown to be fully interfertile in laboratory matings, and hybrids between the putative species were identified on Punica in India. An Indian tester strain was sexually compatible with representatives of what has been considered C. fimbriata on numerous hosts across Brazil. In this revision of the LAC, the name C. fimbriata is restricted to the widely dispersed Ipomoea strain, and C. manginecans is recognized as a Brazilian species that is important on Mangifera, Eucalyptus, and many other crops. C. mangivora and C. mangicola are also considered synonyms of C. manginecans. Based on phylogenetics and mating studies, two other Brazilian species are recognized: C. atlantica, sp. nov., and C. alfenasii, sp. nov., each with wide host ranges. Three new Caribbean species are recognized based on phylogenetics and earlier inoculation studies: C. costaricensis, sp. nov., on Coffea, C. cubensis, sp. nov., on Spathodea, and C. xanthosomatis, sp. nov., on the vegetatively propagated aroids Xanthosoma and Syngonium. Some of the other Ceratocystis species were based primarily on unique internal transcribed spacer (ITS) rDNA sequences, but the unreliability of rDNA sequences was demonstrated when intraspecific crossing of isolates with differing ITS sequences generated single-ascospore progeny with intragenomic variation in ITS sequences and others with new ITS sequences. Species recognition in Ceratocystis should use phenotype, including intersterility tests, to help identify which lineages are species. Although some species remain under-studied, we recognize 16 species in the LAC, all believed to be native to Latin America, the Caribbean region, or eastern USA.

Recent and Ongoing Horizontal Transfer of Mitochondrial Introns Between Two Fungal Tree Pathogens.

Two recently introduced fungal plant pathogens (Ceratocystis lukuohia and Ceratocystis huliohia) are responsible for Rapid 'ohi'a Death (ROD) in Hawai'i. Despite being sexually incompatible, the two pathogens often co-occur in diseased 'ohi'a sapwood, where genetic interaction is possible. We sequenced and annotated 33 mitochondrial genomes of the two pathogens and related species, and investigated 35 total Ceratocystis mitogenomes. Ten mtDNA regions [one group I intron, seven group II introns, and two autonomous homing endonuclease (HE) genes] were heterogeneously present in C. lukuohia mitogenomes, which were otherwise identical. Molecular surveys with specific primers showed that the 10 regions had uneven geographic distribution amongst populations of C. lukuohia. Conversely, identical orthologs of each region were present in every studied isolate of C. huliohia regardless of geographical origin. Close relatives of C. lukuohia lacked or, rarely, had few and dissimilar orthologs of the 10 regions, whereas most relatives of C. huliohia had identical or nearly identical orthologs. Each region included or worked in tandem with HE genes or reverse transcriptase/maturases that could facilitate interspecific horizontal transfers from intron-minus to intron-plus alleles. These results suggest that the 10 regions originated in C. huliohia and are actively moving to populations of C. lukuohia, perhaps through transient cytoplasmic contact of hyphal tips (anastomosis) in the wound surface of 'ohi'a trees. Such contact would allow for the transfer of mitochondria followed by mitochondrial fusion or cytoplasmic exchange of intron intermediaries, which suggests that further genomic interaction may also exist between the two pathogens.

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.

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.

Three genera in the Ceratocystidaceae are the respective symbionts of three independent lineages of ambrosia beetles with large, complex mycangia.

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).