Host phylogeny and environment shape the diversity of salamander skin bacterial communities.

The composition and diversity of animal-associated microbial communities are shaped by multiple ecological and evolutionary processes acting at different spatial and temporal scales. Skin microbiomes are thought to be strongly influenced by the environment due to the direct interaction of the host's skin with the external media. As expected, the diversity of amphibian skin microbiomes is shaped by climate and host sampling habitats, whereas phylogenetic effects appear to be weak. However, the relative strength of phylogenetic and environmental effects on salamander skin microbiomes remains poorly understood. Here, we analysed sequence data from 1164 adult salamanders of 44 species to characterise and compare the diversity and composition of skin bacteria. We assessed the relative contribution of climate, host sampling habitat, and host phylogeny to the observed patterns of bacterial diversity. We found that bacterial alpha diversity was mainly associated with host sampling habitat and climate, but that bacterial beta diversity was more strongly associated with host taxonomy and phylogeny. This phylogenetic effect predominantly occurred at intermediate levels of host divergence (0-50 Mya). Our results support the importance of environmental factors shaping the diversity of salamander skin microbiota, but also support host phylogenetic history as a major factor shaping these bacterial communities.

Genomic characterization of antifungal Acinetobacter bacteria isolated from the skin of the frogs Agalychnis callidryas and Craugastor fitzingeri.

Chytridiomycosis, a lethal fungal disease caused by Batrachochytrium dendrobatidis (Bd), is responsible for population declines and extinctions of amphibians worldwide. However, not all amphibian species are equally susceptible to the disease; some species persist in Bd enzootic regions with no population reductions. Recently, it has been shown that the amphibian skin microbiome plays a crucial role in the defense against Bd. Numerous bacterial isolates with the capacity to inhibit the growth of Batrachochytrium fungi have been isolated from the skin of amphibians. Here, we characterized eight Acinetobacter bacteria isolated from the frogs Agalychnis callidryas and Craugastor fitzingeri at the genomic level. A total of five isolates belonged to Acinetobacter pittii,Acinetobacter radioresistens, or Acinetobactermodestus, and three were not identified as any of the known species, suggesting they are members of new species. We showed that seven isolates inhibited the growth of Bd and that all eight isolates inhibited the growth of the phytopathogen fungus Botrytis cinerea. Finally, we identified the biosynthetic gene clusters that could be involved in the antifungal activity of these isolates. Our results suggest that the frog skin microbiome includes Acinetobacter isolates that are new to science and have broad antifungal functions, perhaps driven by distinct genetic mechanisms.

Differential efficiency among DNA extraction methods influences detection of the amphibian pathogen Batrachochytrium dendrobatidis.

Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is responsible for massive declines and extinctions of amphibians worldwide. The most common method for detecting Bd is quantitative polymerase chain reaction (qPCR). qPCR is a highly sensitive detection technique, but its ability to determine the presence and accurately quantify the amount of Bd is also contingent on the efficiency of the DNA extraction method used prior to PCR. Using qPCR, we compared the extraction efficiency of 3 different extraction methods commonly used for Bd detection across a range of zoospore quantities: PrepMan Ultra Reagent, Qiagen DNeasy Blood and Tissue Kit, and Mobio PowerSoil DNA Isolation Kit. We show that not all extraction methods led to successful detection of Bd for the low zoospore quantities and that there was variation in the estimated zoospore equivalents among the methods, which demonstrates that these methods have different extraction efficiencies. These results highlight the importance of considering the extraction method when comparing across studies. The Qiagen DNeasy kit had the highest efficiency. We also show that replicated estimates of less than 1 zoospore can result from known zoospore concentrations; therefore, such results should be considered when obtained from field data. Additionally, we discuss the implications of our findings for interpreting previous studies and for conducting future Bd surveys. It is imperative to use the most efficient DNA extraction method in tandem with the highly sensitive qPCR technique in order to accurately diagnose the presence of Bd as well as other pathogens.