Location Matters: Variations in Cloacal Microbiota Composition of Spatially Separated Freshwater Turtles.

The gut microbiota of vertebrates is malleable and may be shaped by both intrinsic and extrinsic factors. Here, the effect that geography has on the cloacal microbiota of two species of Australian freshwater chelonians, eastern longneck turtle (Chelodina longicollis) and Macquarie River turtle (Emydura macquarii), captured from waterbodies with different levels of anthropogenic pressure was investigated. We analysed the microbiota composition, structure and diversity through 16S rRNA gene amplicon sequencing. It was hypothesised that animals from less disturbed environments would harbour a more diverse cloacal microbial population. The cloacal microbiotas from 93 turtles (C. longicollis n = 78; E. macquarii n = 15), from five locations, were analysed. For both species, the most predominant phylum was Proteobacteria. Cloacal microbiota alpha diversity varied significantly between the C. longicollis from all locations, but no differences were found for E. macquarii. In C. longicollis, turtles from wetlands within the centre of Melbourne had the lowest alpha diversity metrics, while the highest alpha diversity values were seen in turtles captured from an undisturbed rural waterbody. Beta diversity, obtained by weighted UniFrac distance, showed significant differences between locations of capture for both species of turtles in this investigation. For C. longicollis, 87 biomarkers were identified responsible for explaining differences between locations, and in E. macquarii, 42 biomarkers were found. This is the first study to explore the cloacal microbiota composition of the eastern longneck turtle and gives greater insight into microbial community structures in Macquarie River turtles. Our study demonstrated that cloacal microbiota composition of freshwater turtles was significantly influenced by locality and that disrupted environments may reduce microbial diversity in C. longicollis. Interestingly, we discovered that the effects of location contrasted significantly between species for alpha diversity with differences discovered for C. longicollis but not E. macquarii. However, for both species, beta diversity was notably influenced by habitat type. These results highlight the need to interpret chelonian microbiota data in the context of geography and human disturbance of the environment.