Throughout evolution, entomopathogenic (insect-pathogenic) fungi have played a pivotal role in regulating insect populations. However, little is known about ancient entomopathogenic fungi due to the scarcity of fossils displaying typical pathogenic structures on their presumed hosts. Here, we report two new fungi, Paleoophiocordyceps gerontoformicae sp. nov. and Paleoophiocordyceps ironomyiae sp. nov., from mid-Cretaceous Kachin amber (approx. 99 million years old). They share common traits with Ophiocordyceps and are associated with an ant pupa and a fly, respectively. These fossils are among the oldest fossil records of animal-pathogenic fungi. In addition, we performed a divergence time estimation analysis showing that Ophiocordyceps likely originated during the Early Cretaceous. We further compiled the hosts of extant Ophiocordyceps and inferred the evolution of host associations within the genus based on ancestral character state reconstruction. Our results suggest that Ophiocordyceps made a host jump from Coleoptera to Lepidoptera and Hymenoptera during the Cretaceous, and its subsequent speciation was probably related to the increase in diversity and abundance of its moth and ant hosts. Our results not only highlight the ecological significance of pathogenic fungi in Mesozoic terrestrial ecosystems, but also provide new insights into the coevolution between entomopathogenic fungi and host insects.

Botanic Garden Meise Meise Flanders Belgium

Department of Geological Survey and Mineral Exploration Myanmar Gems Museum Ministry of Natural Resources and Environmental Conservation Nay Pyi Taw Myanmar

Faculty of Science University of South Bohemia in Ceske Budejovice Ceske Budejovice Czech Republic

Institute of Entomology Biology Centre Czech Academy of Sciences Ceske Budejovice Czech Republic

MEC International Joint Laboratory for Palaeobiology and Palaeoenvironment Yunnan University Kunming Yunnan People's Republic of China

Natural History Museum of Denmark University of Copenhagen Copenhagen Denmark

Research Group Mycology Department of Biology Ghent University Ghent Belgium

School of Life Science Yunnan University Kunming Yunnan People's Republic of China

Southwest United Graduate School Kunming Yunnan People's Republic of China

State Key Laboratory of Genetic Resources and Evolution Kunming Institute of Zoology Chinese Academy of Sciences Kunming Yunnan People's Republic of China

State Key Laboratory of Mycology Institute of Microbiology Chinese Academy of Sciences Beijing People's Republic of China

State Key Laboratory of Palaeobiology and Stratigraphy Nanjing Institute of Geology and Palaeontology Chinese Academy of Sciences Nanjing People's Republic of China

University of Chinese Academy of Sciences Beijing People's Republic of China

Yunnan Herbal Laboratory College of Ecology and Environmental Sciences Yunnan University Kunming Yunnan People's Republic of China

Yunnan Key Laboratory for Palaeobiology Institute of Palaeontology Yunnan University Kunming Yunnan People's Republic of China

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Cretaceous entomopathogenic fungi illuminate the early evolution of insect-fungal associations