Exploring the diversity and community structure of the Testudines fecal mycobiome

Most gut microbiome studies have focused on bacteria, leaving a knowledge gap regarding gut associated fungi. We assessed fungal diversity in the gastrointestinal tract of the reptilian order Testudines (turtles and tortoises) using samples from 6 families, 19 genera, and 27 species. A highly diverse community affiliated with 17 phyla and 157 orders was encountered, with four phyla (Neocallimastigomycota, Chytridiomycota, Ascomycota, and Basidiomycota) representing 89.13% of the community. Neocallimastigomycota was identified in host families Testudinidae (land tortoises), Chelidae, Chelydridae, Emydidae, Geoemydidae, and Kinosternidae, with higher relative abundances in Testudinidae (40.18{+/-}37.97%) compared to all other families combined (2.71{+/-}4.04%). Neocallimastigomycota sequences were mostly affiliated with orders Testudinimycetales in the host family Testudinidae and Neocallimastigales in other host families. Chytridiomycota was identified in all host families, but was more ubiquitous and abundant in Kinosternidiae (45.17{+/-}34.12%), and exhibited a high level of variability across samples. Dikarya communities were highly diverse, with 108 orders identified, and, similar to Chytridiomoycota, exhibited a highly stochastic distribution pattern. Representatives of multiple yet-uncultured phyla (Candidatus "Algovoracomycota", "Sedimentomastigomycota", "Tartumycota" and "Cantoromastigomycota") were identified, as well as eight novel orders in Chytridiomycota and Rozellomycota. Deterministic selection shaped community assembly in the host family Testudinidae, while the process was more stochastic in other host families. Distinct community structure was driven by differences in abundance and identity of the Neocallimastigomycota when comparing Testudinidae to. Our results describe a diverse and dynamic fungal community, shaped by the co-occurrence of autochthonous (resident) and transient (allochthonous) members of the gut microbiome. ImportanceFungi are known to inhabit the gastrointestinal tract (GIT) of humans and mammals. However, information on the fungal community in the GIT of reptiles is relatively sparse. We investigated the diversity and community structure of fungi in the reptilian order Testudines. We conducted a culture-independent diversity survey on fecal samples obtained from 27 different host species. We identify representatives of 17 fungal phyla. As well, we demonstrate that the anaerobic gut fungi (phylum Neocallimastigomycota) are not restricted to the family Testudinidae (land tortoises) as previously suggested, but could successfully colonize and inhabit all other testudines families, including those exhibiting a predominantly omnivorous or carnivorous lifestyles. In addition, we expand on the known fungal diversity by identifying additional representatives of multiple recently described yet-uncultured phyla, and describe multiple novel orders and classes within existing phyla. Collectively, this effort adds to the growing body of knowledge of mycobiomes in underexplored animal hosts.