Decisive Effects of Life Stage on the Gut Microbiota Discrepancy Between Two Wild Populations of Hibernating Asiatic Toads (Bufo gargarizans)

Until now, the effects of driving factors on the gut microbiota of amphibians are still mostly confounded. Due to a long-term fasting, hibernating amphibians are ideal experimental materials to explore this question. In this study, we characterized the small intestine microbiota of adult hibernating Asiatic toads (Bufo gargarizans) collected from two geographical populations using 16S rRNA amplicon sequencing technique and evaluated the effects of non-dietary factors (e.g., sex and host genetic background). Proteobacteria (0.9196 ± 0.0892) was characterized as the most dominant phylum in the small gut microbiota of hibernating Asiatic toads, among which five core OTUs were identified and three were classified into Pseudomonas. In view of the coincidence between the dominant KEGG pathways (such as the two-component system) and Pseudomonas, Pseudomonas appeared to be a key adaptor for small gut microbiota during hibernation. Furthermore, we detected a greater discrepancy of gut microbiota between geographical populations than between sexes. Both sex and host genetic background showed a minor effect on the gut microbiota variation. Finally, life stage was determined to be the decisive factor driving the gut microbiota discrepancy between populations. However, a large proportion of the gut microbiota variation (∼70%) could not be explained by the measured deterministic factors (i.e., sex, location, body length, and routine blood indices). Therefore, other factors and/or stochastic processes may play key roles in shaping gut bacterial community of hibernating amphibians.

Pseudogenized Amelogenin Reveals Early Tooth Loss in True Toads (Anura: Bufonidae)

Extant anurans (frogs and toads) exhibit reduced dentition, ranging from a lack of mandibular teeth to complete edentulation, as observed in the true toads of the family Bufonidae. The evolutionary timeline of these reductions remains vague due to a poor fossil record. Previous studies have demonstrated an association between the lack of teeth in edentulous vertebrates and the pseudogenization of the major tooth enamel gene amelogenin (AMEL) through accumulation of deleterious mutations and the disruption of its coding sequence. In the present study we have harnessed the pseudogenization of AMEL as a molecular dating tool to correlate loss of dentition with genomic mutation patterns during the rise of the family Bufonidae. Specifically, we have utilized AMEL pseudogenes in three members of the family as a tool to estimate the putative date of edentulation in true toads. Comparison of AMEL sequences from Rhinella marina, Bufo gargarizans and Bufo bufo, with nine extant, dentulous frogs, revealed mutations confirming AMEL inactivation in Bufonidae. AMEL pseudogenes in modern bufonids also exhibited remarkably high 86–93% sequence identity among each other, with only a slight increase in substitution rate and relaxation of selective pressure, in comparison to functional copies in other anurans. Moreover, using selection intensity estimates and synonymous substitution rates, analysis of functional and pseudogenized AMEL resulted in an estimated inactivation window of 46-60 MYA in the lineage leading to modern true toads, a timeline that coincides with the rise of the family Bufonidae.