Characterizing the processes that drive microbial community assembly remains a key challenge in ecology. Several recent studies have argued that pairwise interactions may be insufficient to explain co-occurrence patterns in complex microbial communities, but most such studies have focused on synthetic communities not found in nature or microbes grown in contexts that differ widely from their natural environment. Moreover, most models of pairwise interactions rely on equilibrium assumptions that are not relevant to all natural communities, such as gut microbiomes or species in fluctuating environments. Inclusion of appropriate demographic factors into models of pairwise interactions could be a potential approach to better capture patterns of community assembly. In this study, we investigated whether multispecies co-occurrence patterns can be predicted from pairwise interactions for microbes isolated from sourdough starters. Interaction parameters inferred from pairwise growth trajectories were suggestive of widespread coexistence between pairs of microbes in our species pool. In communities of up to nine species, most species' presence and relative abundance could be reliably predicted based on a model of pairwise interactions. The inclusion of nonequilibrium demography in our model further improved the accuracy of our pairwise model. Our work contributes to the broader debate on the processes underlying community assembly by showing that pairwise interactions are predictive of community structure in a system of moderate species complexity.

Microbial interactions in cheese rinds influence community structure, food safety, and product quality. But the chemical mechanisms that mediate microbial interactions in cheeses and other fermented foods are generally not known. Here, we investigate how the spoilage mold Aspergillus westerdijkiae chemically inhibits beneficial cheese-rind bacteria using a combination of omics technologies. In cheese-rind community and co-culture experiments, A. westerdijkiae strongly inhibited most cheese-rind community members. In co-culture with Staphylococcus equorum, A. westerdijkiae strongly affected bacterial gene expression, including upregulation of a putative bceAB gene cluster that is associated with resistance to antimicrobial compounds in other bacteria. Mass spectrometry imaging revealed spatially localized production of secondary metabolites, including penicillic acid and ochratoxin B at the fungal-bacterial interface with Brachybacterium alimentarium. Integration of liquid chromatography-tandem mass spectrometry and genome annotations confirmed the presence of additional bioactive metabolites, such as notoamides and circumdatins. Fungal metabolic responses varied by bacterial partner, suggesting species-specific chemical strategies. Notably, penicillic acid levels increased 2.5-fold during interaction with B. alimentarium, and experiments with purified penicillic acid showed inhibition in a dose-dependent manner against this rind bacterium. These findings show that A. westerdijkiae deploys a context-dependent suite of mycotoxins and other metabolites, disrupting microbial community assembly in cheese rinds.IMPORTANCEThis study identifies the chemical mechanisms underlying the negative impacts of Aspergillus westerdijkiae on cheese-rind development, revealing how specialized metabolites like penicillic acid and ochratoxin B influence rind bacterial communities. By integrating biosynthetic gene cluster analyses with mass spectrometry, we demonstrate how chemical communication shapes microbial interactions, with possible implications for food safety and cheese quality. Understanding these interactions is essential for assessing the risks of fungal-driven spoilage and mycotoxin production in cheese-rind maturation. Beyond cheese, these findings contribute to broader microbiome ecology, emphasizing how secondary metabolites mediate microbial competition in natural and fermented food environments.

Filamentous fungi play important roles in the development of surface-ripened cheese microbial communities and contribute to the aesthetics and flavors of these products. Much is known about the diversity and ecology of desirable cheese fungi, but our understanding of the natural history of cheese spoilage molds is limited. The goal of this work was to characterize the genomic diversity of Aspergillus species contaminating artisan cheeses and to identify how the abiotic environment of cheese (the substrate itself and temperature) may constrain the growth of Aspergillus. Comparative genomics identified two main species of Aspergillus, A. westerdijkiae and A. ostianus, as the spoilage molds across three different facilities in the Northeastern United States that experienced contamination events. Multiple genomic types of A. westerdijkiae were found across the different cheese production facilities, indicating that these contamination events are not caused by a single clonal strain. All A. westerdijkiae isolates produced ochratoxin A, but concentrations varied greatly across strains. RNA-sequencing of A. westerdijkiae on nutrient-rich lab media (malt extract agar) versus cheese curd agar identified a suite of pathways enriched in expression on cheese, including degradation of amino and fatty acids. Experiments measuring growth over a range of temperatures identified that spoilage Aspergillus species have a higher optimal growth temperature compared to desirable fungal species in cheese rinds and are outcompeted by Penicillium species at temperatures lower than 15°C. Global fungal metabarcoding databases suggest that A. westerdijkiae is not normally found in natural habitats of the Northeastern United States, and it may be introduced to this region.IMPORTANCEOver the past decade, disruptive contamination events of Aspergillus spoilage molds have occurred at cheese production facilities in Massachusetts, Connecticut, and Vermont in the United States, causing aesthetic, flavor, and potential safety issues. Our work highlights independent introductions of different strains of A. westerdijkiae into multiple cheese facilities and suggests that temperature could be used to control the abundance of Aspergillus spoilage molds. Based on our analysis of the global distribution of A. westerdijkiae, it is not invading cheese facilities from local fungal populations and may be a contaminant in materials used for cheese production.

ABSTRACT While the domestication of plants and animals is widely recognized for its role in the rise of human civilization, humans have also cultivated microbes over millennia to produce food and beverages. One microbe in particular, Saccharomyces cerevisiae , is associated with a wide variety of human-fermentation environments, including wine, beer, and notably bread, such that it is often referred to as “baker’s yeast.” To better illuminate the domestication history of baking associated yeast, we isolated 38 Saccharomyces cerevisiae strains from sourdough starters donated by bakers throughout North America and compared them to thousands of S. cerevisiae isolates from a variety of wild and human-fermentation environments. We identified 6 major clades with two primary domestication hubs, Mediterranean liquid-state fermentation and Asian solid-state fermentation, diverging across Eurasia that gave rise to human-associated lineages. Population genomic analyses demonstrate that S. cerevisiae strains found in sourdough starters are genetically distinct from commercial baking strains and do not come from the surrounding wild environment. Our results show that sourdough yeast strains are closely related to each other and have shared ancestry with strains isolated from various Asian solid state grain fermentations such Japanese sake, Asian rice wines, Chinese distilled spirits (baijiu), and Chinese steamed bread (mantou). We found evidence of significant admixture throughout S. cerevisiae populations, including baking-associated lineages, likely facilitated by human activity. Pangenome gene content largely captures S. cerevisiae traditional genomic sequence-based population structure and reflects human cultural practices, with differences in gene content and copy number between baking associated strains and other groups. Overall, we show that many generalized hallmarks of domestication, such as genome contraction, loss of genetic diversity, and lack of niche expansion, are not universal features of S. cerevisiae domestication, and that baking-associated yeasts have a complex evolutionary history heavily shaped by human culture.

Shotgun metagenomes were used to identify microbes associated with a pink discoloration of an unpasteurized blue cheese made in the United States. Taxonomic assessments of individual reads and metagenome-assembled genomes revealed that the genus Virgibacillus was present in the pink paste, but not in unaffected paste.

Studies of microbial interactions often emphasize large, easily measurable growth differences and short-term ecological outcomes spanning just a few generations. However, more subtle interactions, such as those without obvious phenotypes, may play a significant role in shaping both the short-term ecological dynamics and the long-term evolutionary trajectories of microbial species. We used the cheese rind model microbiome to examine how two fungal species, Penicillium camemberti and Geotrichum candidum, impact global gene expression and genome evolution of the bacterium Pseudomonas carnis. Even though fungi had limited impacts on the growth of P. carnis, ~4-40% of its genome was differentially expressed, depending on the specific fungal partner. When we evolved this Pseudomonas strain alone or in co-culture with each of the fungi, we observed frequent mutations in global regulators of nitrogen regulation, secondary metabolite production, and motility, depending on the fungus. Strikingly, many strains with mutations in the nitrogen regulatory gene ntrB emerged when evolved alone or with G. candidum, but not with P. camemberti. Metabolomic and fitness experiments demonstrate that release of free amino acids by P. camemberti removes the fitness advantages conferred by ntrB mutations. Collectively, these results demonstrate that even in the absence of major short-term growth effects, fungi can have substantial impacts on the transcriptome and genomic evolution of bacterial species.

Previous comparative and experimental evolution studies have suggested how fungi may rapidly adapt to new environments, but direct observation of in situ selection in fungal populations is rare due to challenges with tracking populations over human time scales. We monitored a population of Penicillium solitum over eight years in a cheese cave and documented a phenotypic shift from predominantly green to white strains. Diverse mutations in the alb1 gene, which encodes the first protein in the dihydroxynaphthalene (DHN)-melanin biosynthesis pathway, explained the green-to-white shift. A similar phenotypic shift was recapitulated with an alb1 knockout and experimental evolution in laboratory populations. The most common genetic disruption of the alb1 genomic region was caused by putative transposable element insertions upstream of the gene. White strains had substantial downregulation in global transcription, with genetically distinct white strains possessing divergent shifts in the expression of different biological processes. White strains outcompeted green strains in co-culture, but this competitive advantage was only observed in the absence of light. Our results illustrate how fermented food production by humans provides opportunities for relaxed selection of key fungal traits over short time scales. The local adaptation we observed may be part of a domestication process that could provide opportunities to generate new strains for innovation in fermented food production.

Previous comparative and experimental evolution studies have suggested how fungi may rapidly adapt to new environments, but direct observation of in situ selection in fungal populations is rare due to challenges with tracking populations over human time scales. We monitored a population of Penicillium solitum over eight years in a cheese cave and documented a phenotypic shift from predominantly green to white strains. Diverse mutations in the alb1 gene, which encodes the first protein in the DHN-melanin biosynthesis pathway, explained the green to white shift. A similar phenotypic shift was recapitulated with an alb1 knockout and experimental evolution in laboratory populations. The most common genetic disruption of the alb1 genomic region was caused by putative transposable element insertions upstream of the gene. White strains had substantial downregulation in global transcription, with genetically distinct white strains possessing divergent shifts in expression of different biological processes. White strains outcompeted green strains in co-culture, but this competitive advantage was only observed in the absence of light, suggesting that loss of melanin is only adaptive in dark conditions. Our results illustrate how fermented food production by humans provides opportunities for relaxed selection of key fungal traits over short time scales. Unintentional domestication of microbes by cheesemakers may provide opportunities to generate new strains for innovation in traditional cheese production. ### Competing Interest Statement M.K. is a producer of the cheese studied in this work. He helped coordinate the research on site and provided samples and observations. Although he has a clear financial stake in the success of the blue cheese studied, he did not influence the outcomes or findings of this work. The other authors declare no competing interests.

There is increasing interest in applying resilience concepts at different scales of biological organization to address major interdisciplinary challenges from cancer to climate change. It is unclear, however, whether resilience can be a unifying concept consistently applied across the breadth of the biological sciences, or whether there is limited capacity for integration. In this review, we draw on literature from molecular biology to community ecology to ascertain commonalities and shortcomings in how resilience is measured and interpreted. Resilience is studied at all levels of biological organization, although the term is often not used. There is a suite of resilience mechanisms conserved across biological scales, and there are tradeoffs that affect resilience. Resilience is conceptually useful to help diverse researchers think about how biological systems respond to perturbations, but we need a richer lexicon to describe the diversity of perturbations, and we lack widely applicable metrics of resilience.

While research on the sourdough microbiome has primarily focused on lactic acid bacteria (LAB) and yeast, recent studies have found that acetic acid bacteria (AAB) are also common members. However, the ecology, genomic diversity, and functional contributions of AAB in sourdough remain unknown. To address this gap, we sequenced 29 AAB genomes, including three that represent putatively novel species, from a collection of over 500 sourdough starters surveyed globally from community scientists. We found variations in metabolic traits related to carbohydrate utilization, nitrogen metabolism, and alcohol production, as well as in genes related to mobile elements and defense mechanisms. Sourdough AAB genomes did not cluster when compared to AAB isolated from other environments, although a subset of gene functions was enriched in sourdough isolates. The lack of a sourdough-specific genomic cluster may reflect the nomadic lifestyle of AAB. To assess the consequences of AAB on the emergent function of sourdough starter microbiomes, we constructed synthetic starter microbiomes, varying only the AAB strain included. All AAB strains increased the acidification of synthetic sourdough starters relative to yeast and LAB by 18.5% on average. Different strains of AAB had distinct effects on the profile of synthetic starter volatiles. Taken together, our results begin to define the ways in which AAB shape emergent properties of sourdough and suggest that differences in gene content resulting from intraspecies diversification can have community-wide consequences on emergent function. IMPORTANCEThis study is a comprehensive genomic and ecological survey of acetic acid bacteria (AAB) isolated from sourdough starters. By combining comparative genomics with manipulative experiments using synthetic microbiomes, we demonstrate that even strains with >97% average nucleotide identity can shift important microbiome functions, underscoring the importance of species and strain diversity in microbial systems. We also demonstrate the utility of sourdough starters as a model system to understand the consequences of genomic diversity at the strain and species level on multispecies communities. These results are also relevant to industrial and home-bakers as we uncover the importance of AAB in shaping properties of sourdough starters that have direct impacts on sensory notes and the quality of sourdough bread.

Although Penicillium molds can have significant impacts on agricultural, industrial, and biomedical systems, the ecological roles of Penicillium species in many microbiomes are not well characterized. Here we utilized a collection of 35 Penicillium strains isolated from cheese rinds to broadly investigate the genomic potential for secondary metabolism in cheese-associated Penicillium species, the impact of Penicillium on bacterial community assembly, and mechanisms of Penicillium-bacteria interactions. Using antiSMASH, we identified 1558 biosynthetic gene clusters, 406 of which were mapped to known pathways, including several mycotoxins and antimicrobial compounds. By measuring bacterial abundance and fungal mRNA expression when culturing representative Penicillium strains with a cheese rind bacterial community, we observed divergent impacts of different Penicillium strains, from strong inhibitors of bacterial growth to those with no impact on bacterial growth or community composition. Through differential mRNA expression analyses, Penicillium strains demonstrated limited differential gene expression in response to the bacterial community. We identified a few shared responses between the eight tested Penicillium strains, primarily upregulation of nutrient metabolic pathways, but we did not identify a conserved fungal response to growth in a multispecies community. These results in tandem suggest high variation among cheese-associated Penicillium species in their ability to shape bacterial community development and highlight important ecological diversity within this iconic genus.

Sourdough fermentation is an ancient leavening method that uses wild yeasts to produce carbon dioxide, contributing to bread rise, and bacteria which produce organic acids. Sourdough starter cultures are known to be diverse in terms of the microorganisms they comprise and while specific genera and species of microorganisms have been identified from starters and associated with specific attributes, overarching relationships between sourdough starter culture microbiomes and bread quality are not well understood. The objective of this study was to characterize differences in the physical and chemical properties of breads produced with sourdough starter cultures with unique microbiomes. Sourdough starter cultures (n = 20) of known microbial populations were used to produce wheat-based dough and bread, which were analyzed for chemical and physical properties then compared to their microbial populations in order to identify relationships between microbial profiles and dough/bread qualities. All samples were also compared to bread produced only with Saccharomyces cerevisiae (baker's yeast). Significant differences among pH, titratable acidity, loaf volume, crumb firmness, crust color, free amino acids, and organic acids were observed when comparing sourdough breads to the yeast-only control (p <= 0.05). Furthermore, bacterial diversity of sourdough starter cultures was correlated with lactic acid and free amino acid in the dough and loaf volume and crumb firmness of baked breads. No significant correlations were found between fungal diversity and measured outcomes. These data demonstrate the importance of considering sourdough starter microbiomes as an ingredient in baked goods and they contribute to quality and safety outcomes in bread production.

Evolutionary processes may have substantial impacts on community assembly, but evidence for phylogenetic relatedness as a determinant of interspecific interaction strength remains mixed. In this perspective, we consider a possible role for discordance between gene trees and species trees in the interpretation of phylogenetic signal in studies of community ecology. Modern genomic data show that the evolutionary histories of many taxa are better described by a patchwork of histories that vary along the genome rather than a single species tree. If a subset of genomic loci harbor trait-related genetic variation, then the phylogeny at these loci may be more informative of interspecific trait differences than the genome background. We develop a simple method to detect loci harboring phylogenetic signal and demonstrate its application through a proof of principle analysis of Penicillium genomes and pairwise interaction strength. Our results show that phylogenetic signal that may be masked genome-wide could be detectable using phylogenomic techniques and may provide a window into the genetic basis for interspecific interactions. Data & code accessibility Data and code for this project are freely available in the repository linked below and will be permanently archived upon publication. Statement of authorship NL, BEW, and LHU designed the research; NL performed experiments; NL and LHU performed computational analyses; NL, BEW, and LHU wrote the manuscript. Code repository https://github.com/uricchio/ILSComp

Fermented foods and beverages have been produced around the world for millennia, providing humans with a range of gastronomic, cultural, health, and scientific benefits. Building on these traditional forms, a convergence of factors, including culinary innovation, globalization, shifts in consumer preferences, and advances in microbiome sciences, has led to the emergence of so-called 'novel fermentations'. In this review, we define novel fermentation as the confluence of traditional food practices and rational microbiome design. Using principles of microbial ecology and evolution, we develop a microbiological framework that outlines several strategies for producing and characterizing novel fermentations, including switching substrates, engrafting target species, assembling whole-community chimeras, and generating novel phenotypes. A subsequent analysis of existing traditional ferments points to gaps in 'fermentation space' where novel ferments could potentially be produced using new combinations of microbes and food substrates. We highlight some important safety and sociocultural issues presented by the repurposing and modification of microbes from traditional ferments that fermented-food producers and microbiologists need to address.

Bacterial-fungal interactions (BFIs) can shape the structure of microbial communities, but the small molecules mediating these BFIs are often understudied. We explored various optimization steps for our microbial culture and chemical extraction protocols for bacterial-fungal co-cultures, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that metabolomic profiles are mainly comprised of fungi derived features, indicating that fungi are the key contributors to small molecules in BFIs. LC-inductively coupled plasma MS (LC-ICP-MS) and MS/MS based dereplication using database searching revealed the presence of several known fungal specialized metabolites and structurally related analogues in these extracts, including siderophores such as desferrichrome, desferricoprogen, and palmitoylcoprogen. Among these analogues, a novel putative coprogen analogue possessing a terminal carboxylic acid motif was identified from Scopulariopsis sp. JB370, a common cheese rind fungus, and its structure was elucidated via MS/MS fragmentation. Based on these findings, filamentous fungal species appear to be capable of producing multiple siderophores with potentially different biological roles (i.e. various affinities for different forms of iron). These findings highlight that fungal species are important contributors to microbiomes via their production of abundant specialized metabolites and that elucidating their role in complex communities should continue to be a priority.

Summary Global change is reshaping Earth's biodiversity, but the changing distributions of nonpathogenic fungi remain largely undocumented, as do mechanisms enabling invasions. The ectomycorrhizal Amanita phalloides is native to Europe and invasive in North America. Using population genetics and genomics, we sought to describe the life history traits of this successfully invading symbiotic fungus. To test whether death caps spread underground using hyphae, or aboveground using sexual spores, we mapped and genotyped mushrooms from European and US sites. Larger genetic individuals (genets) would suggest spread mediated by vegetative growth, while many small genets would suggest dispersal mediated by spores. To test whether genets are ephemeral or persistent, we also sampled from populations over time. At nearly every site and across all time points, mushrooms resolve into small genets. Individuals frequently establish from sexual spores. But at one Californian site, a single individual measuring nearly 10 m across dominated. At two Californian sites, the same genetic individuals were discovered in 2004, 2014, and 2015, suggesting single individuals (both large and small) can reproduce repeatedly over relatively long timescales. A flexible life history strategy combining both mycelial growth and spore dispersal appears to underpin the invasion of this deadly perennial ectomycorrhizal fungus.

Experimental studies of microbial evolution have largely focused on monocultures of model organisms, but most microbes live in communities where interactions with other species may impact rates and modes of evolution. Using the cheese rind model microbial community, we determined how species interactions shape the evolution of the widespread food- and animal-associated bacterium Staphylococcus xylosus. We evolved S. xylosus for 450 generations alone or in co-culture with one of three microbes: the yeast Debaryomyces hansenii, the bacterium Brevibacterium aurantiacum, and the mold Penicillium solitum. We used the frequency of colony morphology mutants (pigment and colony texture phenotypes) and whole-genome sequencing of isolates to quantify phenotypic and genomic evolution. The yeast D. hansenii strongly promoted diversification of S. xylosus. By the end of the experiment, all populations co-cultured with the yeast were dominated by pigment and colony morphology mutant phenotypes. Populations of S. xylosus grown alone, with B. aurantiacum, or with P. solitum did not evolve novel phenotypic diversity. Whole-genome sequencing of individual mutant isolates across all four treatments identified numerous unique mutations in the operons for the SigB, Agr, and WalRK global regulators, but only in the D. hansenii treatment. Phenotyping and RNA-seq experiments highlighted altered pigment and biofilm production, spreading, stress tolerance, and metabolism of S. xylosus mutants. Fitness experiments revealed antagonistic pleiotropy, where beneficial mutations that evolved in the presence of the yeast had strong negative fitness effects in other biotic environments. This work demonstrates that bacterial-fungal interactions can have long-term evolutionary consequences within multispecies microbiomes by facilitating the evolution of strain diversity.

For thousands of years, humans have enjoyed the novel flavors, increased shelf-life, and nutritional benefits that microbes provide in fermented foods and beverages. Recent sequencing surveys of ferments have mapped patterns of microbial diversity across space, time, and production practices. But a mechanistic understanding of how fermented food microbiomes assemble has only recently begun to emerge. Using three foods as case studies (surface-ripened cheese, sourdough starters, and fermented vegetables), we use an ecological and evolutionary framework to identify how microbial communities assemble in ferments. By combining in situ sequencing surveys with in vitro models, we are beginning to understand how dispersal, selection, diversification, and drift generate the diversity of fermented food communities. Most food producers are unaware of the ecological processes occurring in their production environments, but the theory and models of ecology and evolution can provide new approaches for managing fermented food microbiomes, from farm to ferment.

The observation that Penicillium molds can inhibit the growth of Staphylococcus was a catalyst for the antibiotic revolution. Considerable attention has been paid to purified Penicillium metabolites that inhibit bacteria, but little is known about how Penicillium species impact the ecology and evolution of bacteria in multispecies microbial communities. Here, we investigated how four different species of Penicillium can impact global transcription and evolution of a widespread Staphylococcus species (S. equorum) using the cheese rind model microbiome. Through RNA sequencing, we identified a core transcriptional response of S. equorum against all five tested Penicillium strains, including upregulation of thiamine biosynthesis, fatty acid degradation, and amino acid metabolism as well as downregulation of genes involved in the transport of siderophores. In a 12-week evolution experiment where we co-cultured S. equorum with the same Penicillium strains, we observed surprisingly few non-synonymous mutations across S. equorum populations evolved with the Penicillium species. A mutation in a putative DHH family phosphoesterase gene only occurred in populations evolved without Penicillium and decreased the fitness of S. equorum when co-cultured with an antagonistic Penicillium strain. Our results highlight the potential for conserved mechanisms of Staphylococcus-Penicillium interactions and demonstrate how fungal biotic environments may constrain the evolution of bacterial species.IMPORTANCEFungi and bacteria are commonly found co-occurring both in natural and synthetic microbiomes, but our understanding of fungal-bacterial interactions is limited to a handful of species. Conserved mechanisms of interactions and evolutionary consequences of fungal-bacterial interactions are largely unknown. Our RNA sequencing and experimental evolution data with Penicillium species and the bacterium S. equorum demonstrate that divergent fungal species can elicit conserved transcriptional and genomic responses in co-occurring bacteria. Penicillium molds are integral to the discovery of novel antibiotics and production of certain foods. By understanding how Penicillium species affect bacteria, our work can further efforts to design and manage Penicillium-dominated microbial communities in industry and food production.

Cheese rinds are economically important microbial communities where fungi can impact food quality and aesthetics. The specific mechanisms by which fungi can regulate bacterial community assembly in cheeses, other fermented foods, and microbiomes in general are largely unknown.