Microbial ecology provides insights into the ecological and evolutionary dynamics of microbial communities underpinning every ecosystem on Earth. Microbial communities can now be investigated in unprecedented detail, although there is still a wealth of open questions to be tackled. Here we identify 50 research questions of fundamental importance to the science or application of microbial ecology, with the intention of summarising the field and bringing focus to new research avenues. Questions are categorised into seven themes: host–microbiome interactions; health and infectious diseases; human health and food security; microbial ecology in a changing world; environmental processes; functional diversity; and evolutionary processes. Many questions recognise that microbes provide an extraordinary array of functional diversity that can be harnessed to solve real-world problems. Our limited knowledge of spatial and temporal variation in microbial diversity and function is also reflected, as is the need to integrate micro- and macro-ecological concepts, and knowledge derived from studies with humans and other diverse organisms. Although not exhaustive, the questions presented are intended to stimulate discussion and provide focus for researchers, funders and policy makers, informing the future research agenda in microbial ecology.

Emerging infectious diseases in wildlife are responsible for massive population declines. In amphibians, chytridiomycosis caused by Batrachochytrium dendrobatidis, Bd, has severely affected many amphibian populations and species around the world. One promising management strategy is probiotic bioaugmentation of antifungal bacteria on amphibian skin. In vivo experimental trials using bioaugmentation strategies have had mixed results, and therefore a more informed strategy is needed to select successful probiotic candidates. Metagenomic, transcriptomic, and metabolomic methods, colloquially called "omics", are approaches that can better inform probiotic selection and optimize selection protocols. The integration of multiple omic data using bioinformatic and statistical tools and in silico models that link bacterial community structure with bacterial defensive function can allow the identification of species involved in pathogen inhibition. We recommend using 16S rRNA gene amplicon sequencing and methods such as indicator species analysis, the K-S Measure, and co-occurrence networks to identify bacteria that are associated with pathogen resistance in field surveys and experimental trials. In addition to 16S amplicon sequencing, we recommend approaches that give insight into symbiont function such as shotgun metagenomics, metatranscriptomics or metabolomics to maximize the probability of finding effective probiotic candidates, which can then be isolated in culture and tested in persistence and clinical trials. An effective mitigation strategy to ameliorate chytridiomycosis and other emerging infectious diseases is necessary; the advancement of omic methods and the integration of multiple omic data provide a promising avenue toward conservation of imperiled species.

Amphibians support symbiotic bacterial communities on their skin that protect against a range of infectious pathogens, including the amphibian chytrid fungus. The conditions under which amphibians are maintained in captivity (e.g. diet, substrate, enrichment) in ex situ conservation programmes may affect the composition of the bacterial community. In addition, ex situ amphibian populations may support different bacterial communities in comparison to in situ populations of the same species. This could have implications for the suitability of populations intended for reintroduction, as well as the success of probiotic bacterial inoculations intended to provide amphibians with a bacterial community that resists invasion by the chytrid fungus. We aimed to investigate the effect of a carotenoid-enriched diet on the culturable bacterial community associated with captive red-eyed tree frogs (Agalychnis callidryas) and make comparisons to bacteria isolated from a wild population from the Chiquibul Rainforest in Belize. We successfully showed carotenoid availability influences the overall community composition, species richness and abundance of the bacterial community associated with the skin of captive frogs, with A. callidryas fed a carotenoid-enriched diet supporting a greater species richness and abundance of bacteria than those fed a carotenoid-free diet. Our results suggest that availability of carotenoids in the diet of captive frogs is likely to be beneficial for the bacterial community associated with the skin. We also found wild A. callidryas hosted more than double the number of different bacterial species than captive frogs with very little commonality between species. This suggests frogs in captivity may support a reduced and diverged bacterial community in comparison to wild populations of the same species, which could have particular relevance for ex situ conservation projects.

Microbiome composition is linked to host functional traits including metabolism and immune function. Drivers of microbiome composition are increasingly well-characterised; however, evidence of group-level microbiome convergence is limited and may represent a multi-level trait (i.e. across individuals and groups), whereby heritable phenotypes are influenced by social interactions. Here, we investigate the influence of spatial structuring and social interactions on the gut microbiome composition of Welsh mountain ponies. We show that semi-feral ponies exhibit variation in microbiome composition according to band (group) membership, in addition to considerable within-individual variation. Spatial structuring was also identified within bands, suggesting that despite communal living, social behaviours still influence microbiome composition. Indeed, we show that specific interactions (i.e. mother-offspring and stallion-mare) lead to more similar microbiomes, further supporting the notion that individuals influence the microbiome composition of one another and ultimately the group. Foals exhibited different microbiome composition to sub-adults and adults, most likely related to differences in diet. We provide novel evidence that microbiome composition is structured at multiple levels within populations of social mammals and thus may form a unit on which selection can act. High levels of within-individual variation in microbiome composition, combined with the potential for social interactions to influence microbiome composition, suggest the direction of microbiome selection may be influenced by the individual members present in the group. Although the functional implications of this require further research, these results lend support to the idea that multi-level selection can act on microbiomes.

Marine sponges are hosts to large, diverse communities of microorganisms. These microbiomes are distinct among sponge species and from seawater bacterial communities, indicating a key role of host identity in shaping its resident microbial community. However, the factors governing intraspecific microbiome variability are underexplored and may shed light on the evolutionary and ecological relationships between host and microbiome. Here, we examined the influence of genetic variation and geographic location on the composition of the Ircinia campana microbiome. We developed new microsatellite markers to genotype I. campana from two locations in the Florida Keys, USA, and characterized their microbiomes using V4 16S rRNA amplicon sequencing. We show that microbial community composition and diversity is influenced by host genotype, with more genetically similar sponges hosting more similar microbial communities. We also found that although I. campana was not genetically differentiated between sites, microbiome composition differed by location. Our results demonstrate that both host genetics and geography influence the composition of the sponge microbiome. Host genotypic influence on microbiome composition may be due to stable vertical transmission of the microbial community from parent to offspring, making microbiomes more similar by descent. Alternatively, sponge genotypic variation may reflect variation in functional traits that influence the acquisition of environmental microbes. This study reveals drivers of microbiome variation within and among locations, and shows the importance of intraspecific variability in mediating eco-evolutionary dynamics of host-associated microbiomes.

Tree pathogens are a major threat to forest ecosystems. Conservation management strategies can exploit natural mechanisms of resistance, such as tree genotype and host‐associated microbial communities. However, fungal and bacterial communities are rarely looked at in the same framework, particularly in conjunction with host genotype. Here, we explore these relationships and their influence on ash dieback disease, caused by the pathogenHymenoscyphus fraxineus, in European common ash trees. We collected leaves from UK ash trees and used microsatellite markers to genotype trees, quantitative PCR to quantifyH. fraxineusinfection load and ITS and 16S rRNA amplicon sequencing to identify fungal and bacterial communities, respectively. There was a significant association betweenH. fraxineusinfection intensity and ash leaf fungal and bacterial community composition. Higher infection levels were positively correlated with fungal community alpha‐diversity, and a number of fungal and bacterial genera were significantly associated with infection presence and intensity. Under higher infection loads, leaf microbial networks were characterized by stronger associations between fewer members than those associated with lower infection levels. Together these results suggest thatH. fraxineusdisrupts stable endophyte communities after a particular infection threshold is reached, and may enable proliferation of opportunistic microbes. We identified three microbial genera associated with an absence of infection, potentially indicating an antagonistic relationship withH. fraxineusthat could be utilized in the development of anti‐pathogen treatments. Host genotype did not directly affect infection, but did significantly affect leaf fungal community composition. Thus, host genotype could have the potential to indirectly affect disease susceptibility through genotype × microbiome interactions, and should be considered when selectively breeding trees. Synthesis. We show that the diversity, composition and network structure of ash leaf microbial communities are associated with the severity of infection from ash dieback disease, with evidence of disease‐induced dysbiosis. We also show that host genotype influences leaf fungal community composition, but does not directly influence tree infection. These findings help to elucidate relationships between host genetics, the microbiome and a tree pathogen, highlighting potential resistance mechanisms and possible co‐infection concerns that could inform ash tree management.