Diversification of the gut fungi Smittium and allies (Harpellales) co-occurred with the origin of complete metamorphosis of their symbiotic insect hosts (lower Diptera).

Smittium (Harpellales, Kickxellomycotina) includes fungal symbionts associated with the digestive tracts of immature aquatic stages of various Diptera, including Chironomidae, Culicidae, Dixidae, Simuliidae, Thaumaleidae, and Tipulidae. With 84 species and the largest collection of cultured strains, Smittium has served as a model to understand the biology of these enigmatic trichomycetes gut fungi, from aspects of biodiversity, evolution, genomics, immunology, and physiology. However, evolutionary histories between Smittium species and their hosts are still not firmly established. Robust phylogenies of both Smittium sensu lato (s.l.) and their lower Diptera hosts have been reconstructed separately, facilitating comparative evolutionary studies between the two. The divergence time of the Smittium s.l. clade was estimated for the first time and compared with the evolutionary history of the insect hosts. The insect gut fungi diversified around 272 Ma (204-342 Ma), which co-occurred with the origin of complete metamorphosis of the insect hosts, presumably between 280 Ma and 355 Ma (~270 Ma for Diptera). A co-phylogenetic pattern was recovered for the insects and their symbiotic gut fungi using the statistical method ParaFit. Ancestral state reconstructions of the symbiotic relationship suggest that the ancestor of the Chironomidae may have contributed to the initiation of these insect-fungus symbiotic interactions. Further sampling and sequencing of Smittium and allies as well as their hosts are needed to uncover more patterns and interactions that may occur in this type of symbiosis.