Identification of the genes underlying adaptation sheds light on the biological functions targeted by natural selection. Searches for footprints of positive selection, in the form of rapid amino acid substitutions, and the identification of species-specific genes have proved to be powerful approaches to identifying the genes involved in host specialization in plant-pathogenic fungi. We used an evolutionary comparative genomic approach to identify genes underlying host adaptation in the ant-infecting genus Ophiocordyceps, which manipulates ant behaviour. A comparison of the predicted genes in the genomes of species from three species complexes-O. unilateralis, O. australis and O. subramanianii-revealed an enrichment in pathogenesis-associated functions, including heat-labile enterotoxins, among species-specific genes. Furthermore, these genes were overrepresented among those displaying significant footprints of positive selection. Other categories of genes suspected to be important for virulence and pathogenicity in entomopathogenic fungi (e.g., chitinases, lipases, proteases, core secondary metabolism genes) were much less represented, although a few candidate genes were found to evolve under positive selection. An analysis including orthologs from other entomopathogenic fungi in a broader context showed that positive selection on enterotoxins was specific to the ant-infecting genus Ophiocordyceps. Together with previous studies reporting the overexpression of an enterotoxin during behavioural manipulation in diseased ants, our findings suggest that heat-labile enterotoxins are important effectors in host adaptation and co-evolution in the Ophiocordyceps entomopathogenic fungi.

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The identification and delimitation of species boundaries are essential for understanding speciation and adaptation processes and for the management of biodiversity as well as development for applications. Ophiocordyceps unilateralis sensu lato is a complex of fungal pathogens parasitizing Formicine ants, inducing zombie behaviors in their hosts. Previous taxonomic works with limited numbers of samples and markers led to the "one ant-one fungus" paradigm, resulting in the use of ant species as a proxy for fungal identification. Here, a population genomic study with sampling on three ant species across Thailand supported the existence of host-specific species in O. unilateralis s.l. with no footprints of long term introgression despite occasional host shifts and first-generation hybrids. We further detected genetic clusters within the previously delimited fungal species, with each little footprints of recombination, suggesting high levels of inbreeding. The clusters within each of O. camponoti-leonardi and O. camponoti-saundersi were supported by differentiation throughout the genome, suggesting they may constitute further cryptic species parasitizing the same host, challenging the one ant-one fungus paradigm. These genetic clusters had different geographical ranges, supporting different biogeographic influences between the north/center and the south of Thailand, reinforcing the scenario in which Thailand endured compartmentation during the latest Pleistocene glacial cycles.