Metabarcoding reveals a hidden endophytic stage of the 'zombie-ant' fungus in Amazonian mosses

Hypocrealean fungi include numerous entomopathogens, notably the hyperdiverse genus Ophiocordyceps , in which lineages manipulate insect hosts to die in specific microhabitats, such as mosses, which may facilitate spore dispersal. A dual endophyte-entomopathogen lifestyle has been proposed for Ophiocordyceps from temperate and subtropical systems; however, megadiverse tropical ecosystems remain poorly explored. Our study aims to characterize fungal communities in central Amazonian bryophytes from the Adolpho Ducke Forest Reserve in order to investigate the alleged dual lifestyle within a hyperdiverse tropical environment. Four sample types were analyzed: 1) Ophiocordyceps from the host insect; 2) bryophyte leaves at the host insect biting site; 3) bryophyte leaves adjacent to (≥ 5 cm) the biting site; 4) and bryophyte leaves (≥ 10 m) from the same locality within the study site. Total genomic DNA was extracted, the TEF1-α region was amplified, and amplicons were sequenced using Oxford Nanopore technology. Metabarcoding of 45 libraries revealed structured fungal communities within Amazonian bryophytes, resulting in the identification of 2,277 fungal taxa. Across all bryophyte samples, we report a significant detection of entomopathogenic fungi, mostly comprising ant-specific Ophiocordyceps (e.g., O. camponoti-nidulantis and O. kniphofioides ). Together, these results support the hypothesis of a ubiquitous endophytic stage of myrmecophilous Ophiocordyceps among Amazonian bryophytes, indicating a closer link between host manipulation and plant association than previously recognized.