Phylosymbiosis was recently proposed to describe the eco-evolutionary pattern, whereby the ecological relatedness of host-associated microbial communities parallels the phylogeny of related host species. Here, we test the prevalence of phylosymbiosis and its functional significance under highly controlled conditions by characterizing the microbiota of 24 animal species from four different groups (Peromyscus deer mice, Drosophila flies, mosquitoes, and Nasonia wasps), and we reevaluate the phylosymbiotic relationships of seven species of wild hominids. We demonstrate three key findings. First, intraspecific microbiota variation is consistently less than interspecific microbiota variation, and microbiota-based models predict host species origin with high accuracy across the dataset. Interestingly, the age of host clade divergence positively associates with the degree of microbial community distinguishability between species within the host clades, spanning recent host speciation events (~1 million y ago) to more distantly related host genera (~108 million y ago). Second, topological congruence analyses of each group's complete phylogeny and microbiota dendrogram reveal significant degrees of phylosymbiosis, irrespective of host clade age or taxonomy. Third, consistent with selection on host–microbiota interactions driving phylosymbiosis, there are survival and performance reductions when interspecific microbiota transplants are conducted between closely related and divergent host species pairs. Overall, these findings indicate that the composition and functional effects of an animal's microbial community can be closely allied with host evolution, even across wide-ranging timescales and diverse animal systems reared under controlled conditions.

Emerging infectious diseases threaten human and wildlife populations. Altered ecological interactions between mutualistic microbes and hosts can result in disease, but an understanding of interactions between host, microbes and disease-causing organisms may lead to management strategies to affect disease outcomes. Many amphibian species in relatively pristine habitats are experiencing dramatic population declines and extinctions due to the skin disease chytridiomycosis, which is caused by the chytrid fungus Batrachochytrium dendrobatidis. Using a randomized, replicated experiment, we show that adding an antifungal bacterial species, Janthinobacterium lividum, found on several species of amphibians to the skins of the frog Rana muscosa prevented morbidity and mortality caused by the pathogen. The bacterial species produces the anti-chytrid metabolite violacein, which was found in much higher concentrations on frog skins in the treatments where J. lividum was added. Our results show that cutaneous microbes are a part of amphibians' innate immune system, the microbial community structure on frog skins is a determinant of disease outcome and altering microbial interactions on frog skins can prevent a lethal disease outcome. A bioaugmentation strategy may be an effective management tool to control chytridiomycosis in amphibian survival assurance colonies and in nature.

Although the gut microbiome influences numerous aspects of organismal fitness, its role in animal evolution and the origin of new species is largely unknown. Here we present evidence that beneficial bacterial communities in the guts of closely related species of the genus Nasonia form species-specific phylosymbiotic assemblages that cause lethality in interspecific hybrids. Bacterial constituents and abundance are irregular in hybrids relative to parental controls, and antibiotic curing of the gut bacteria significantly rescues hybrid survival. Moreover, feeding bacteria to germ-free hybrids reinstates lethality and recapitulates the expression of innate immune genes observed in conventionally reared hybrids. We conclude that in this animal complex, the gut microbiome and host genome represent a coadapted "hologenome" that breaks down during hybridization, promoting hybrid lethality and assisting speciation.

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Disease has spurred declines in global amphibian populations. In particular, the fungal pathogen Batrachochytrium dendrobatidis has decimated amphibian diversity in some areas unaffected by habitat loss. However, there is little evidence to explain how some amphibian species persist despite infection or even clear the pathogen beyond detection. One hypothesis is that certain bacterial symbionts on the skin of amphibians inhibit the growth of the pathogen. An antifungal strain of Janthinobacterium lividum, isolated from the skin of the red-backed salamander Plethodon cinereus, produces antifungal metabolites at concentrations lethal to B. dendrobatidis. Antifungal metabolites were identified by using reversed phase high performance liquid chromatography, high resolution mass spectrometry, nuclear magnetic resonance, and UV-Vis spectroscopy and tested for efficacy of inhibiting the pathogen. Two metabolites, indole-3-carboxaldehyde and violacein, inhibited the pathogen's growth at relatively low concentrations (68.9 and 1.82 microM, respectively). Analysis of fresh salamander skin confirmed the presence of J. lividum and its metabolites on the skin of host salamanders in concentrations high enough to hinder or kill the pathogen (51 and 207 microM, respectively). These results support the hypothesis that cutaneous, mutualistic bacteria play a role in amphibian resistance to fungal disease. Exploitation of this biological process may provide long-term resistance to B. dendrobatidis for vulnerable amphibians and serve as a model for managing future emerging diseases in wildlife populations.

Given the complexity of host-microbiota symbioses, scientists and philosophers are asking questions at new biological levels of hierarchical organization—what is a holobiont and hologenome? When should this vocabulary be applied? Are these concepts a null hypothesis for host-microbe systems or limited to a certain spectrum of symbiotic interactions such as host-microbial coevolution? Critical discourse is necessary in this nascent area, but productive discourse requires that skeptics and proponents use the same lexicon.

The comparative structure of bacterial communities among closely related host species remains relatively unexplored. For instance, as speciation events progress from incipient to complete stages, does divergence in the composition of the species' microbial communities parallel the divergence of host nuclear genes? To address this question, we used the recently diverged species of the parasitoid wasp genus Nasonia to test whether the evolutionary relationships of their bacterial microbiotas recapitulate the Nasonia phylogenetic history. We also assessed microbial diversity in Nasonia at different stages of development to determine the role that host age plays in microbiota structure. The results indicate that all three species of Nasonia share simple larval microbiotas dominated by the γ-proteobacteria class; however, bacterial species diversity increases as Nasonia develop into pupae and adults.Finally, under identical environmental conditions, the relationships of the microbial communities reflect the phylogeny of the Nasonia host species at multiple developmental stages, which suggests that the structure of an animal's microbial community is closely allied with divergence of host genes. These findings highlight the importance of host evolutionary relationships on microbiota composition and have broad implications for future studies of microbial symbiosis and animal speciation.

The disease chytridiomycosis, which is caused by the chytrid fungus Batrachochytrium dendrobatidis, is associated with recent declines in amphibian populations. Susceptibility to this disease varies among amphibian populations and species, and resistance appears to be attributable in part to the presence of antifungal microbial species associated with the skin of amphibians. The betaproteobacterium Janthinobacterium lividum has been isolated from the skins of several amphibian species and produces the antifungal metabolite violacein, which inhibits B. dendrobatidis. In this study, we added J. lividum to red-backed salamanders (Plethodon cinereus) to obtain an increased range of violacein concentrations on the skin. Adding J. lividum to the skin of the salamander increased the concentration of violacein on the skin, which was strongly associated with survival after experimental exposure to B. dendrobatidis. As expected from previous work, some individuals that did not receive J. lividum and were exposed to B. dendrobatidis survived. These individuals had concentrations of bacterially produced violacein on their skins that were predicted to kill B. dendrobatidis. Our study suggests that a threshold violacein concentration of about 18 M on a salamander's skin prevents mortality and morbidity caused by B. dendrobatidis. In addition, we show that over one-half of individuals in nature support antifungal bacteria that produce violacein, which suggests that there is a mutualism between violaceinproducing bacteria and P. cinereus and that adding J. lividum is effective for protecting individuals that lack violacein-producing skin bacteria.The amphibian fungal pathogen Batrachochytrium dendrobatidis causes a lethal skin disease that has caused substantial declines in amphibian populations (18). However, some species, such as the bullfrog (Rana catesbeiana) and the tiger salamander (Ambystoma tigrinum), are relatively asymptomatic when they are infected with this pathogen (4, 5). Variation in survival among species has been attributed to differences in innate immune factors, such as antimicrobial peptides (20) and skin-associated microbial species (8-11), as well as behavior (16). The presence of antifungal microbes is of particular interest because it suggests that these organisms are mutualistic associates of amphibian species. In addition, augmentation of the cutaneous microbial community by adding species of bacteria that inhibit B. dendrobatidis has the potential to provide resistance to chytridiomycosis (9).We have identified a number of bacteria associated with the skin of amphibians that inhibit B. dendrobatidis in vitro via secretion of antifungal metabolites (2,3,10,11). The bacterial species used in this study, Janthinobacterium lividum, produces the anti-B. dendrobatidis metabolites violacein and indole-3-carboxaldehyde (MIC, 1.82 M and 69 M, respectively) (3). We have shown that violacein inhibits B. dendrobatidis in laboratory assays (3) and is strongly correlated with survival in vivo of the frog species Ra...