Anaerobic gut fungi (AGF, Neocallimastigomycota) are a group of microscopic fungi that live in the gastrointestinal tracts of herbivores. While relatively well-studied in mammals, there is little evidence for their occurrence in non-mammals. Our group wanted to assess the diversity of anaerobic gut fungi in tortoises, and in doing so identified novel deep-branching tortoise-associated genera. We sought to further study these genera by evaluating evolutionary divergence time, carbohydrate metabolism, and horizontal gene transfer relative to their mammalian counterparts.

Sampled tortoises harbored distinct, less diverse AGF communities

Fecal samples were collected from nine zoo-housed tortoise species in Oklahoma. Using the D2 LSU region, we characterized the AGF communities in these samples and found that three uncultured genera were prevalent. These uncultured genera are a rare and extremely minor component of mammalian AGF communities, so we consider them “tortoise-associated AGF”. The tortoises we sampled harbored distinct, less diverse AGF communities compared to mammals, with differences associated with the presence of these tortoise-associated AGF.

Tortoise-associated AGF predate the K-Pg extinction event

We cultured twenty-nine isolates from the tortoise fecal samples encompassing each of the three tortoise-associated AGF genera. Isolates of NY54 and NY36 were maintained and characterized as the novel genera Testudinimyces and Astrotestudinimyces. We performed phylogenomic and molecular dating analyses based on transcriptomes from these genera and found that they diverged 104-112 million years ago. This pushes the origins of AGF-host symbiosis from the late to early Cretaceous period and predates the evolution of both herbivorous mammals and grasses, previously thought to be critical for AGF divergence into a distinct phylum.

Tortoise-associated AGF have a limited capacity for carbohydrate metabolism

Based on our transcriptomics data, we found that our tortoise-associated AGF genera lacked many mammal-associated AGF gene clusters encoding metabolic functions, especially carbohydrate metabolism. We found clear differences in the relative composition of carbohydrate-active enzymes between tortoise- and mammal-associated AGF genera. These enzymes are colocalized by extracellular multienzyme complexes called cellulosomes. We found that in tortoise-associated AGF a smaller percentage of total transcripts were attributed to cellulosome parts, which we confirmed with proteomics.

Tortoise-associated AGF exhibit less horizontal gene transfer 

Many of the carbohydrate-active enzyme families missing or reduced in tortoise-associated AGF have been previously shown as acquired by AGF via horizontal gene transfer. We found that there were far fewer events of horizontal gene transfer in tortoise- vs mammal-associated AGF (35 vs 277). Most of the events in tortoise-associated AGF were previously identified in mammal-associated AGF and related to metabolic functions like enabling anaerobiosis and acquiring carbohydrate-active enzymes. This finding indicates an ancient acquisition of these genes preceding the tortoise- and mammal-associated AGF split, after which only mammal-associated AGF underwent a wave of gene acquisition and gained their powerful plant biomass degrading machinery.

Conclusion

This investigation of AGF communities in tortoises revealed that three genera represented the majority of AGF in most of our samples, contrasting their low abundance in sampled mammals. Evolutionary timing estimates for these tortoise-associated AGF genera predate all other known AGF genera and the divergence of current mammalian hosts. Finally, the limited capacity for carbohydrate metabolism displayed by tortoise-associated AGF suggests they contribute minimally to substrate depolymerization and leaves the ecological role of tortoise-associated AGF unclear. Regardless of this uncertainty, what is clear is that the presence of AGF in non-mammalian hosts requires us to rethink what we know about the evolution of the anaerobic fungi, AGF-host symbiosis, and their ecological roles.