One Health Approach to Globalizing, Accelerating, and Focusing Amphibian and Reptile Disease Research—Reflections and Opinions from the First Global Amphibian and Reptile Disease Conference

The world’s reptiles and amphibians are experiencing dramatic and ongoing losses in biodiversity, changes that can have substantial effects on ecosystems and human health. In 2022, the first Global Amphibian and Reptile Disease Conference was held, using One Health as a guiding principle. The conference showcased knowledge on numerous reptile and amphibian pathogens from several standpoints, including epidemiology, host immune defenses, wild population effects, and mitigation. The conference also provided field experts the opportunity to discuss and identify the most urgent herpetofaunal disease research directions necessary to address current and future threats to reptile and amphibian biodiversity

Host Ecology Rather Than Host Phylogeny Drives Amphibian Skin Microbial Community Structure in the Biodiversity Hotspot of Madagascar

Host-associated microbiotas of vertebrates are diverse and complex communities that contribute to host health. In particular, for amphibians, cutaneous microbial communities likely play a significant role in pathogen defense; however, our ecological understanding of these communities is still in its infancy. Here, we take advantage of the fully endemic and locally species-rich amphibian fauna of Madagascar to investigate the factors structuring amphibian skin microbiota on a large scale. Using amplicon-based sequencing, we evaluate how multiple host species traits and site factors affect host bacterial diversity and community structure. Madagascar is home to over 400 native frog species, all of which are endemic to the island; more than 100 different species are known to occur in sympatry within multiple rainforest sites. We intensively sampled frog skin bacterial communities, from over 800 amphibians from 89 species across 30 sites in Madagascar during three field visits, and found that skin bacterial communities differed strongly from those of the surrounding environment. Richness of bacterial operational taxonomic units (OTUs) and phylogenetic diversity differed among host ecomorphs, with arboreal frogs exhibiting lower richness and diversity than terrestrial and aquatic frogs. Host ecomorphology was the strongest factor influencing microbial community structure, with host phylogeny and site parameters (latitude and elevation) explaining less but significant portions of the observed variation. Correlation analysis and topological congruency analyses revealed little to no phylosymbiosis for amphibian skin microbiota. Despite the observed geographic variation and low phylosymbiosis, we found particular OTUs that were differentially abundant between particular ecomorphs. For example, the genus Pigmentiphaga (Alcaligenaceae) was significantly enriched on arboreal frogs, Methylotenera (Methylophilaceae) was enriched on aquatic frogs, and Agrobacterium (Rhizobiaceae) was enriched on terrestrial frogs. The presence of shared bacterial OTUs across geographic regions for selected host genera suggests the presence of core microbial communities which in Madagascar, might be driven more strongly by a species’ preference for specific microhabitats than by the physical, physiological or biochemical properties of their skin. These results corroborate that both host and environmental factors are driving community assembly of amphibian cutaneous microbial communities, and provide an improved foundation for elucidating their role in disease resistance

Estimating Herd Immunity to Amphibian Chytridiomycosis in Madagascar Based on the Defensive Function of Amphibian Skin Bacteria

For decades, Amphibians have been globally threatened by the still expanding infectious disease, chytridiomycosis. Madagascar is an amphibian biodiversity hotspot where Batrachochytrium dendrobatidis (Bd) has only recently been detected. While no Bd-associated population declines have been reported, the risk of declines is high when invasive virulent lineages become involved. Cutaneous bacteria contribute to host innate immunity by providing defense against pathogens for numerous animals, including amphibians. Little is known, however, about the cutaneous bacterial residents of Malagasy amphibians and the functional capacity they have against Bd. We cultured 3179 skin bacterial isolates from over 90 frog species across Madagascar, identified them via Sanger sequencing of approximately 700 bp of the 16S rRNA gene, and characterized their functional capacity against Bd. A subset of isolates was also tested against multiple Bd genotypes. In addition, we applied the concept of herd immunity to estimate Bd-associated risk for amphibian communities across Madagascar based on bacterial antifungal activity. We found that multiple bacterial isolates (39% of all isolates) cultured from the skin of Malagasy frogs were able to inhibit Bd. Mean inhibition was weakly correlated with bacterial phylogeny, and certain taxonomic groups appear to have a high proportion of inhibitory isolates, such as the Enterobacteriaceae, Pseudomonadaceae, and Xanthamonadaceae (84, 80, and 75% respectively). Functional capacity of bacteria against Bd varied among Bd genotypes; however, there were some bacteria that showed broad spectrum inhibition against all tested Bd genotypes, suggesting that these bacteria would be good candidates for probiotic therapies. We estimated Bd-associated risk for sampled amphibian communities based on the concept of herd immunity. Multiple amphibian communities, including those in the amphibian diversity hotspots, Andasibe and Ranomafana, were estimated to be below the 80% herd immunity threshold, suggesting they may be at higher risk to chytridiomycosis if a lethal Bd genotype emerges in Madagascar. While this predictive approach rests on multiple assumptions, and incorporates only one component of hosts' defense against Bd, their culturable cutaneous bacterial defense, it can serve as a foundation for continued research on Bd-associated risk for the endemic frogs of Madagascar

No impact of a short-term climatic "El Niño" fluctuation on gut microbial diversity in populations of the Galápagos marine iguana (Amblyrhynchus cristatus)

Gut microorganisms are crucial for many biological functions playing a pivotal role in the host's well-being. We studied gut bacterial community structure of marine iguana populations across the Galápagos archipelago. Marine iguanas depend heavily on their specialized gut microbiome for the digestion of dietary algae, a resource whose growth was strongly reduced by severe "El Niño"-related climatic fluctuations in 2015/2016. As a consequence, marine iguana populations showed signs of starvation as expressed by a poor body condition. Body condition indices (BCI) varied between island populations indicating that food resources (i.e., algae) are affected differently across the archipelago during 'El Niño' events. Though this event impacted food availability for marine iguanas, we found that reductions in body condition due to "El Niño"-related starvation did not result in differences in bacterial gut community structure. Species richness of gut microorganisms was instead correlated with levels of neutral genetic diversity in the distinct host populations. Our data suggest that marine iguana populations with a higher level of gene diversity and allelic richness may harbor a more diverse gut microbiome than those populations with lower genetic diversity. Since low values of these diversity parameters usually correlate with small census and effective population sizes, we use our results to propose a novel hypothesis according to which small and genetically less diverse host populations might be characterized by less diverse microbiomes. Whether such genetically depauperate populations may experience additional threats from reduced dietary flexibility due to a limited intestinal microbiome is currently unclear and calls for further investigation

Diversity, elevational variation, and phylogeographic origin of stump-toed frogs (Microhylidae: Cophylinae: Stumpffia) on the Marojejy massif, northern Madagascar

Stump-toed frogs (genus Stumpffia Boettger, 1881) are a diverse group of small-bodied frogs endemic to Madagascar. Seven species of this genus occur on Marojejy, a steep massif in northeastern Madagascar. Here we examine the elevational distribution, phylogenetic position, biogeographic origin, and genetic differentiation of this Stumpffia assemblage. We show that none of these species is another’s closest relative, but rather they are all independent lineages that probably colonised the Marojejy Massif through repeated immigration events. All of the lineages on Marojejy are most closely related to species south and southwest of the massif, except one lineage, formerly known as Stumpffia sp. Ca07, but here assigned to S. sorata as a deep conspecific lineage (and referred to as S. cf. sorata), which occurs also in Sorata, 90 km north of Marojejy. The species on Marojejy are typically restricted to narrow elevational ranges, but at least two species, S. cf. sorata and S. tridactyla, occur over elevations spanning 1000 metres. We assessed the genetic variability of these populations, and found considerable haplotype separation in fragments of the mitochondrial 16S rRNA and nuclear Rag-1 genes, suggesting some disruption of gene flow associated with elevation. We discuss the biogeographic implications of our findings and, based on previously published data, the evolution of non-overlapping bioacoustic parameters among the diverse assemblage of Stumpffia species on the Marojejy massif

Species complexes and the importance of Data Deficient classification in Red List assessments: The case of Hylobatrachus frogs

This work is licensed under a Creative Commons Attribution 4.0 International License.Taxonomy is the cornerstone of extinction risk assessments. Currently, the IUCN Red List treats species complexes either under a single overarching species name—resulting in an unhelpfully broad circumscription and underestimated threat assessment that does not apply to any one species lineage—or omits them altogether—resulting in the omission of species that should be assessed. We argue that taxonomic uncertainty alone, as in species complexes, should be grounds for assessment as Data Deficient (DD). Yet, use of the DD category is currently discouraged, resulting in assessments based on poor data quality and dismissal of the importance of taxonomic confidence in conservation. This policy may be leading to volatile and unwarranted assessments of hundreds of species across the world, and needs to be revised. To illustrate this point, we here present a partial taxonomic revision of torrent frogs from eastern Madagascar in the Mantidactylus subgenus Hylobatrachus. Two named species, Mantidactylus (Hylobatrachus) lugubris and M. (H.) cowanii, and several undescribed candidate species are recognised, but the application of the available names has been somewhat ambiguous. In a recent re-assessment of its conservation status, M. (H.) lugubris was assessed including all complex members except M. (H.) cowanii within its distribution, giving it a status of Least Concern and distribution over most of eastern Madagascar. After describing two of the unnamed lineages as Mantidactylus (Hylobatrachus) atsimo sp. nov. (from southeastern Madagascar) and Mantidactylus (Hylobatrachus) petakorona sp. nov. (from the Marojejy Massif in northeastern Madagascar), we show that Mantidactylus (Hylobatrachus) lugubris is restricted to the central east of Madagascar, highlighting the inaccuracy of its current Red List assessment. We propose to re-assess its status under a more restrictive definition that omits well-defined candidate species, thus representing the actual species to which its assessment refers, to the best of current knowledge. We recommend that for species complexes in general, (1) nominal lineages that can be confidently restricted should be assessed under the strict definition, (2) non-nominal species-level lineages and ambiguous names should be prioritised for taxonomic research, and (3) ambiguous names should be assessed as DD to highlight the deficiency in data on their taxonomic status, which is an impediment to their conservation. This would reduce ambiguity and underestimation of threats involved in assessing species complexes, and place the appropriate emphasis on the importance of taxonomy in anchoring conservation.Deutscher Akademischer Austauschdienst fellowshipDeutsche Forschungsgemeinschaft (VE247/13-1 and 15-1

Long-Term Habitat Fragmentation Is Associated With Reduced MHC IIB Diversity and Increased Infections in Amphibian Hosts

Habitat fragmentation and wildlife disease are two widespread drivers of biodiversity loss, yet few empirical studies have explored their interactions. In this study, we utilized a naturally fragmented island system to examine the impacts of fragmentation on genetic diversity and amphibian infection dynamics. We determined the impacts of fragmentation on genetic diversity at the immunity locus MHC IIB, a hypothesized predictor of disease susceptibility. Contrary to the expectation that MHC diversity would remain high due to balancing selection, island populations lost genetic diversity at this locus while simultaneously experiencing positive selection at MHC IIB. We then used Next-Generation Sequencing to identify a variety of potential eukaryotic parasites from amphibian skin swabs. Island populations exhibited higher potential parasite richness (proportion of eukaryotic microbe operational taxonomic units or OTUs from parasitic taxa) relative to mainland populations. MHC homozygotes hosted a lower diversity of potential parasites, and population-level MHC diversity was negatively associated with parasite richness. Our results show that genetic erosion can occur at the MHC IIB locus following fragmentation, which may contribute to increased susceptibility to parasites

Amphibian skin-associated Pigmentiphaga: Genome sequence and occurrence across geography and hosts

The bacterial communities colonizing amphibian skin have been intensively studied due to their interactions with pathogenic chytrid fungi that are causing drastic amphibian population declines. Bacteria of the family Alcaligenaceae, and more specifically of the genus Pigmentiphaga, have been found to be associated specifically to arboreal frogs. Here we analyze their occurrence in a previously assembled global skin microbiome dataset from 205 amphibian species. Pigmentiphaga made up about 5% of the total number of reads in this global dataset. They were mostly found in unrelated arboreal frogs from Madagascar (Mantellidae and Hyperoliidae), but also occurred at low abundances on Neotropical frogs. Based on their 16S sequences, most of the sequences belong to a clade within Pigmentiphaga not assignable to any type strains of the five described species of the genus. One isolate from Madagascar clustered with Pigmentiphaga aceris (>99% sequence similarity on 16S rRNA gene level). Here, we report the full genome sequence of this bacterium which, based on 16S sequences of >97% similarity, has previously been found on human skin, floral nectar, tree sap, stream sediment and soil. Its genome consists of a single circular chromosome with 6,165,255 bp, 5,300 predicted coding sequences, 57 tRNA genes, and three rRNA operons. In comparison with other known Pigmentiphaga genomes it encodes a higher number of genes associated with environmental information processing and cellular processes. Furthermore, it has a biosynthetic gene cluster for a nonribosomal peptide syntethase, and bacteriocin biosynthetic genes can be found, but clusters for β-lactones present in other comparative Pigmentiphaga genomes are lacking

A distinctive new frog species (Anura, Mantellidae) supports the biogeographic linkage of two montane rainforest massifs in northern Madagascar

We describe a new species of the genus Gephyromantis, subgenus Vatomantis (Mantellidae, Mantellinae), from moderately high elevation (1164–1394 m a.s.l.) on the Marojejy, Sorata, and Andravory Massifs in northern Madagascar. The new species, Gephyromantis (Vatomantis) lomorina sp. n. is highly distinct from all other species, and was immediately recognisable as an undescribed taxon upon its discovery. It is characterised by a granular, mottled black and green skin, reddish eyes, paired subgular vocal sacs of partly white colour, bulbous femoral glands present only in males and consisting of three large granules, white ventral spotting, and a unique, amplitude-modulated advertisement call consisting of a series of 24–29 rapid, quiet notes at a dominant frequency of 5124–5512 Hz. Genetically the species is also strongly distinct from its congeners, with uncorrected pairwise distances ≥10 % in a fragment of the mitochondrial 16S rRNA gene to all other nominal Gephyromantis species. A molecular phylogeny based on 16S sequences places it in a clade with species of the subgenera Laurentomantis and Vatomantis, and we assign it to the latter subgenus based on its morphological resemblance to members of Vatomantis. We discuss the biogeography of reptiles and amphibians across the massifs of northern Madagascar, the evidence for a strong link between Marojejy and Sorata, and the role of elevation in determining community sharing across this landscape