# The oral eukaryotic microbiome of Melanophryniscus admirabilis, a microendemic and critically endangered toad

## TL;DR

This study explores the oral fungal community of a critically endangered toad species in Brazil, finding a diverse microbiome that may help protect against fungal pathogens.

## Contribution

The first characterization of the oral eukaryotic microbiome in Melanophryniscus admirabilis, revealing a potentially protective role of Malassezia fungi.

## Key findings

- The oral microbiome of M. admirabilis is dominated by the fungal genus Malassezia, which may compete with the pathogen Batrachochytrium dendrobatidis.
- Functional predictions suggest the presence of saprotrophic activity and metabolic contributions to host-associated niches.
- A high proportion of unclassified sequences highlights gaps in reference databases for amphibian-associated eukaryotes.

## Abstract

The oral eukaryotic microbiome of amphibians remains largely unexplored, despite its potential importance for host health and resistance to fungal pathogens such as Batrachochytrium dendrobatidis (Bd). Melanophryniscus admirabilis, a critically endangered red-belly toad species endemic to Brazil and restricted to a 700-m stretch of the Forqueta River, offers unique insights into host-microbiome interactions within highly specialized and threatened neotropical environments. While its narrow distribution limits broader applications, the genus Melanophryniscus is widely distributed across South America, potentially serving as a broader model for comparative microbiome research across varied ecological contexts.

We analyzed the oral eukaryotic microbiota of ten wild M. admirabilis using 18S rRNA gene amplicon sequencing, with the Illumina MiSeq platform. Taxonomic assignments were performed at the phylum, class, and genus levels. Microbial community structure was assessed via hierarchical clustering and non-metric multidimensional scaling (NMDS) method based on Bray-Curtis dissimilarity. In addition, functional profiles were inferred from taxonomic data using PICRUSt2 to explore the potential ecological roles of the detected taxa.

Excluding host-derived reads, the predominant fungal phyla identified were Ascomycota and Basidiomycota. Among them, the genus Malassezia was present across all samples, suggesting a potentially host-adapted association. Given its known adaptation to mucosal environments and consistent abundance in our dataset, we hypothesize that Malassezia may compete with the fungal pathogen Bd, potentially acting as a natural microbial protector. Other fungal genera, including Phlebia, Microdochium, Fusarium, and Rhodotorula, were detected at lower abundance and may reflect a mixture of commensal, environmental, or opportunistic fungi. Functional prediction analyses revealed signatures of saprotrophic activity and suggested potential metabolic contributions to host-associated niches. The high proportion of unclassified and multi-affiliated sequences highlights the current limitations of reference databases for amphibian-associated eukaryotes, and underscores the value of this study in providing a novel community-level description of oral fungi in a neotropical anuran species.

This study provides the first characterization of the oral eukaryotic microbiome of M. admirabilis, revealing a diverse and structured fungal community dominated by Malassezia, with predicted functions related to environmental adaptation and fungal competition. These findings suggest that the oral cavity of amphibians harbors functionally active microbial communities that may play a role in pathogen resistance and host-microbe symbiosis.