Mitochondrial introgression, color pattern variation, and severe demographic bottlenecks in three species of Malagasy poison frogs, genus Mantella

Madagascar is a biodiversity hotspot particularly rich in amphibian diversity and only a few charismatic Malagasy amphibians have been investigated for their population-level differentiation. The Mantella madagascariensis group is composed of two rainforest and three swamp forest species of poison frogs. We first confirm the monophyly of this clade using DNA sequences of three nuclear and four mitochondrial genes, and subsequently investigate the population genetic differentiation and demography of the swamp forest species using one mitochondrial, two nuclear and a set of nine microsatellite markers. Our results confirm the occurrence of two main mitochondrial lineages, one dominated by Mantella aurantiaca (a grouping supported also by our microsatellite-based tree) and the other by Mantella crocea + Mantella milotympanum. These two main lineages probably reflect an older divergence in swamp Mantella. Widespread mitochondrial introgression suggests a fairly common occurrence of inter-lineage gene flow. However, nuclear admixture seems to play only a limited role in this group, and the analyses of the RAG-1 marker points to a predominant incomplete lineage sorting scenario between all five species of the group, which probably diverged relatively recently. Our demographic analyses show a common, severe and recent demographic contraction, inferred to be in temporal coincidence with the massive deforestation events that took place in the past 1000 years. Current data do not allow to conclusively delimit independent evolutionary units in these frogs, and we therefore refrain to suggest any taxonomic changes

Anthropogenic landscape change and amphibian diversity in tropical montane biodiversity hotspots: insights from satellite remote sensing in the Madagascar highlands

The magnitude of anthropogenic landscape change in tropical montane biodiversity hotspots and its relationship with biodiversity is a global issue that remains ‘locked-in’ in the broad narrative of tropical change in Africa. Over a montane biodiversity hotspot of Madagascar highlands (Ankaratra Massif), we conducted analysis on land cover change with Landsat satellite sensor data to identify the magnitude of change (1995–2016) and on the habitat change–amphibian diversity relationship to understand links with biodiversity. The results evidenced that 17.8% of the biodiversity hotspot experienced change in land cover in only 20 years. That pressured the already threatened forests, particularly since 2005. Of the total forest area in 1995 (2062.7 ha), 21.5% was cleared by 2016 (1618.3 ha). Changes in forest cover followed a bidirectional pattern. While in the period 1995–2005, forests expanded at a rate of 2.0% year−1 (from 2062.7 to 2524.8 ha), the area declined between 2005 and 2016 at a rate of − 4.1% year−1, fourfold the rate reported nationally for Madagascar (− 1.1% year−1). Forest-to-shrubland transitions emerged as being of increasing concern to forest integrity. We identified a significant link between habitat change and amphibian diversity, but only for species richness. Counter to expectations, no significant relationship was found between species richness and deforestation rates, and between microendemism rates and any of the habitat change variables. Species richness responded to the spatiotemporal variability in vegetation dynamics represented by the standard deviation of the Normalized Differenced Vegetation Index (NDVI_std). Species richness was strongly negatively related to NDVI_std in the short-term (R2 = 0.91, p = 0.003) and long-term (R2 = 0.69, p = 0.03), increasing where the spatiotemporal variability in NDVI was lower. The magnitude of changes in this biodiversity hotspot suggests that region-specific assessments are necessary in the context of the tropical change narrative in Africa and should consider conservation policies tailored for local conditions. Reducing deforestation and land conversion rates through a management plan codesigned with local communities is urgent. Habitat change appears to impact on amphibian diversity by altering the functional attributes of the habitat and not just by reducing habitat extent. NDVI_std seems a relevant indirect metric for monitoring such change although other biophysical attributes obtained from satellite sensor data should be integrated and explored.info:eu-repo/semantics/acceptedVersio

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

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

Antifungal isolates database of amphibian skin-associated bacteria and function against emerging fungal pathogens

Microbial symbionts of vertebrate skin have an important function in defense of the host against pathogens. In particular, the emerging chytrid fungus Batrachochytrium dendrobatidis, causes widespread disease in amphibians but can be inhibited via secondary metabolites produced by many different skin-associated bacteria. Similarly, the fungal pathogens of terrestrial salamander eggs Mariannaea elegans and Rhizomucor variabilis are also inhibited by a variety of skin-associated bacteria. Indeed, probiotic therapy against fungal diseases is a recent approach in conservation medicine with growing experimental support. We present a comprehensive Antifungal Isolates Database of amphibian skin-associated bacteria that have been cultured, isolated, and tested for antifungal properties. At the start, this database includes nearly 2000 cultured bacterial isolates from 37 amphibian host species across 18 studies on five continents: Africa, Oceania, Europe, and North and South America. As the research community gathers information on additional isolates, the database will be updated periodically. The resulting database can serve as a conservation tool for amphibians and other organisms, and provides empirical data for comparative and bioinformatic studies. The database consists of a FASTA file containing 16S rRNA gene sequences of the bacterial isolates, and a metadata file containing information on the host species, life-stage, geographic region, and antifungal capacity and taxonomic identity of the isolate

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi

T.W.J.G., M.C.F., D.S.S., A.L., E.C., F.C.C., J.B., A.A.C., C.M., F.S., B.R.S., S.O., were supported through the Biodiversa project RACE: Risk Assessment of Chytridiomycosis to European Amphibian Biodiversity (NERC standard grant NE/K014455/1 and NE/E006701/1; ANR-08-BDVA-002-03). M.C.F., J.S., C.W., P.G. were supported by the Leverhulme Trust (RPG-2014-273), M.C.F., A.C., C.W. were supported by the Morris Animal Foundation. J.V. was supported by the Bolyai János Research Grant of the Hunagrian Academy of Sciences (BO/00597/14). F.G. and D.G. were supported by the Conservation Leadership Programme Future Conservationist Award. C.S.A. was supported by Fondecyt (No. 1181758). M.C.F. and A.C. were supported by. Mohamed bin Zayed Species Conservation Fund Project (152510704). GMR held a doctoral scholarship (SFRH/BD/69194/2010) from Fundação para a Ciência e a Tecnologia. L.F.T., C.L., L.P.R. K.R.Z., T.Y.J., T.S.J. were supported by São Paulo Research Foundation (FAPESP #2016/25358-3), the National Counsel of Technological and Scientific Development (CNPq #300896/2016–6) and a Catalyzing New International Collaborations grant from the United States NSF (OISE-1159513). C.S.A. was supported by Fondecyt (No. 1181758). T.M.D. was supported by the Royal Geographical Society and the Royal Zoological Society of Scotland. B.W. was supported by the National Research Foundation of Korea (2015R1D1A1A01057282).Peer reviewedPublisher PD

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi

© The Author(s) 2018.Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.T.W.J.G., M.C.F., D.S.S., A.L., E.C., F.C.C., J.B., A.A.C., C.M., F.S., B.R.S., S.O., were supported through the Biodiversa project RACE: Risk Assessment of Chytridiomycosis to European Amphibian Biodiversity (NERC standard grant NE/K014455/1 and NE/E006701/1; ANR-08-BDVA-002-03). M.C.F., J.S., C.W., P.G. were supported by the Leverhulme Trust (RPG-2014-273), M.C.F., A.C., C.W. were supported by the Morris Animal Foundation. J.V. was supported by the Bolyai János Research Grant of the Hunagrian Academy of Sciences (BO/00597/14). F.G. and D.G. were supported by the Conservation Leadership Programme Future Conservationist Award. C.S.A. was supported by Fondecyt (No. 1181758). M.C.F. and A.C. were supported by. Mohamed bin Zayed Species Conservation Fund Project (152510704). GMR held a doctoral scholarship (SFRH/ BD/69194/2010) from Fundação para a Ciência e a Tecnologia. L.F.T., C.L., L.P.R. K.R.Z., T.Y.J., T.S.J. were supported by São Paulo Research Foundation (FAPESP #2016/25358-3), the National Counsel of Technological and Scientifc Development (CNPq #300896/2016–6) and a Catalyzing New International Collaborations grant from the United States NSF (OISE-1159513). C.S.A. was supported by Fondecyt (No. 1181758). T.M.D. was supported by the Royal Geographical Society and the Royal Zoological Society of Scotland. B.W. was supported by the National Research Foundation of Korea (2015R1D1A1A01057282).Peer Reviewe