Morphological and phylogenetic resolution of (Hypocreales: Clavicipitaceae), a potential biocontrol fungus for in United States Christmas tree production areas.

The entomopathogenic fungus has recently been implicated in natural epizootics among exotic elongate hemlock scale (EHS) insects in Christmas tree farms in the eastern United States. Since 1913, has been reported from various plant feeding Hemiptera in the southeastern United States, but comprehensive morphological and phylogenetic studies of U.S. populations are lacking. The recovery of multiple strains of from mycosed EHS in North Carolina provided an opportunity to conduct pathogenicity assays and morphological and phylogenetic studies to investigate genus- and species-level boundaries among the Clavicipitaceae. Pathogenicity assays confirmed that causes mortality of EHS crawlers, an essential first step in developing this fungus as a biocontrol. Morphological studies revealed that conidia aligned with previous measurements of the -like asexual state of , with conidiophore morphology consistent with historical observations. Additionally, a -like synanamorph was observed in select strains. In both a four-locus, 54-taxon Clavicipitaceae-wide phylogenetic analysis including D1-D2 domains of the nuclear 28S rRNA region (28S), elongation factor 1 alpha (), DNA-directed RNA polymerase II subunit 1 (), and DNA-directed RNA polymerase II subunit 2 () and a two-locus, 38-taxon (28S and ) phylogenetic analysis, all three species were resolved as strongly supported monophyletic lineages across all loci and both methods (maximum likelihood and Bayesian inference) of phylogenetic inference except for 28S for . Despite the strong support for individual species, none of the analyses supported the monophyly of with the inclusion of . Due to the paucity of and sequence data, provided superior delimitation of intraspecies groupings for and should be used in future studies. Further development of as a biocontrol against EHS will require additional surveys across diverse Hemiptera and expanded pathogenicity testing to clarify host range and efficacy of this fungus.