Much remains unknown regarding speciation. Host-pathogen interactions are a major driving force for diversification, but the genomic basis for speciation and host shifting remains unclear. The fungal genus Metarhizium contains species ranging from specialists with very narrow host ranges to generalists that attack a wide range of insects. By genomic analyses of seven species, we demonstrated that generalists evolved from specialists via transitional species with intermediate host ranges and that this shift paralleled insect evolution. We found that specialization was associated with retention of sexuality and rapid evolution of existing protein sequences whereas generalization was associated with protein-family expansion, loss of genome-defense mechanisms, genome restructuring, horizontal gene transfer, and positive selection that accelerated after reinforcement of reproductive isolation. These results advance understanding of speciation and genomic signatures that underlie pathogen adaptation to hosts.peciation is a central component of biological diversification and is increasingly viewed as a continuum or process rather than an event. However, failure to identify transitional species has hindered progress in understanding genomic patterns of divergence along the speciation continuum (1). Plant or animal pathogenic fungi are genetically tractable models for the study of speciation due to their diverse lifestyles and the occurrence of sibling species that differ from each other principally in host specificity (2, 3). However, fundamental questions remain, including whether generalization or specialization to particular hosts is the ancestral condition, whether we can identify the existence of transitional forms, and what are the underlying molecular mechanisms driving speciation (4).We exploited the ascomycete genus Metarhizium, a radiating lineage of insect pathogens that are frequently used as biological insecticides (5, 6) and for genomic studies into the nature of adaptive differences by which novel pathogens emerge and form new species. Besides the previously sequenced Metarhizium robertsii (abbreviated as MAA) and Metarhizium acridum (MAC) (7), five new species were sequenced: Metarhizium album (MAM), Metarhizium majus (MAJ), Metarhizium guizhouense (MGU), Metarhizium brunneum (MBR), and Metarhizium anisopliae (MAN) (Dataset S1, Table S1). MAM is specific for hemipteran insects (8) whereas MAJ and MGU have intermediate host ranges as they are predominately associated with coleopteran insects but can also infect lepidopterans (9). Like MAA, MBR and MAN are generalists parasitizing a broad range of insects representing more than seven orders (10,11). Generalist species such as MAA and MBR can also colonize the roots of plants (12), consistent with increased phenotypic flexibility.Our analyses revealed that the evolutionary trajectory of Metarhizium spp. was from specialists via intermediate host range species to generalists that coincided with host insect diversification and availability. This host adaptati...