Three novel Ambrosia Fusarium Clade species producing multiseptate "dolphin-shaped" conidia, and an augmented description of Fusarium kuroshium.

The Ambrosia Fusarium Clade (AFC) is a monophyletic lineage within clade 3 of the Fusarium solani species complex (FSSC) that currently comprises 19 genealogically exclusive species. These fungi are known or predicted to be farmed by adult female Euwallacea ambrosia beetles as a nutritional mutualism (Coleoptera: Scolytinae; Xyleborini). To date, only eight of the 19 AFC species have been described formally with Latin binomials. We describe three AFC species, previously known as AF-8, AF-10, and AF-11, based on molecular phylogenetic analysis of multilocus DNA sequence data and comparative morphological/phenotypic studies. Fusarium duplospermum (AF-8) farmed by E. perbrevis on avocado in Florida, USA, is distinguished by forming two morphologically different types of multiseptate conidia and brownish orange colonies on potato dextrose agar (PDA). Fusarium drepaniforme (AF-10), isolated from an unknown woody host in Singapore and deposited as Herb IMI 351954 in the Royal Botanic Gardens, Kew, UK, under the name F. bugnicourtii, is diagnosed by frequent production of multiseptate sickle-shaped conidia. Fusarium papillatum (AF-11), isolated from mycangia of E. perbrevis infesting tea in Kandy, Sri Lanka, forms multiseptate clavate conidia that possess a papillate apical cell protruding toward the ventral side. Lastly, we prepared an augmented description of F. kuroshium (AF-12), previously isolated from the heads or galleries of E. kuroshio in a California sycamore tree, El Cajon, California, USA, and recently validated nomenclaturally as Fusarium. Conidia formed by F. kuroshium vary widely in size and shape, suggesting a close morphological relationship with F. floridanum, compared with all other AFC species. Maximum likelihood and maximum parsimony analyses of a multilocus data set resolve these three novel AFC species, and F. kuroshium, as phylogenetically distinct based on genealogical concordance. Given the promiscuous nature of several Euwallacea species, and the overlapping geographic range of several AFC species and Euwallacea ambrosia beetles, the potential for symbiont switching among sympatric species is discussed.

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.