Hackflex library preparation enables low-cost metagenomic profiling.

Shotgun metagenomic sequencing provides valuable insights into microbial communities, but the high cost of library preparation with standard kits and protocols is a barrier for many. New methods such as Hackflex use diluted commercially available reagents to greatly reduce library preparation costs. However, these methods have not been systematically validated for metagenomic sequencing. Here, we evaluate Hackflex performance by sequencing metagenomic libraries from known mock communities as well as mouse fecal samples prepared by Hackflex, Illumina DNA Prep, and Illumina TruSeq methods. Hackflex successfully recovered all members of the Zymo mock community, performing best for samples with DNA concentrations <1 ng/µL. Furthermore, Hackflex was able to delineate microbiota of individual inbred mice from the same breeding stock at the same mouse facility, and statistical modeling indicated that mouse ID explained a greater fraction of the variance in metagenomic composition than did library preparation method. These results show that Hackflex is suitable for generating inventories of bacterial communities through metagenomic sequencing.

Hackflex library preparation enables low-cost metagenomic profiling.

Shotgun metagenomic sequencing provides valuable insights into microbial communities, but the high cost of library preparation with standard kits and protocols is a barrier for many. New methods such as Hackflex use diluted commercially available reagents to greatly reduce library preparation costs. However, these methods have not been systematically validated for metagenomic sequencing. Here, we evaluate Hackflex performance by sequencing metagenomic libraries from known mock communities as well as mouse fecal samples prepared by Hackflex, Illumina DNA Prep, and Illumina TruSeq methods. Hackflex successfully recovered all members of the Zymo mock community, performing best for samples with DNA concentrations <1 ng/uL. Furthermore, Hackflex was able to delineate microbiota of individual inbred mice from the same breeding stock at the same mouse facility, and statistical modeling indicated that mouse ID explained a greater fraction of the variance in metagenomic composition than did library preparation method. These results show that Hackflex is suitable for generating inventories of bacterial communities through metagenomic sequencing.

Sex-specific competitive social feedback amplifies the role of early life contingency in male mice.

Contingency (or 'luck') in early life plays an important role in shaping individuals' development. When individuals live within larger societies, social experiences may cause the importance of early contingencies to be magnified or dampened. Here we test the hypothesis that competition magnifies the importance of early contingency in a sex-specific manner by comparing the developmental trajectories of genetically identical, free-living mice who either experienced high levels of territorial competition (males) or did not (females). We show that male territoriality results in a competitive feedback loop that magnifies the importance of early contingency and pushes individuals onto divergent, self-reinforcing life trajectories, while the same process appears absent in females. Our results indicate that the strength of sexual selection may be self-limiting, as within-sex competition increases the importance of early life contingency, thereby reducing the ability of selection to lead to evolution. They also demonstrate the potential for contingency to lead to dramatic differences in life outcomes, even in the absence of any underlying differences in ability ('merit').

Female behavior drives the formation of distinct social structures in C57BL/6J versus wild-derived outbred mice in field enclosures.

BACKGROUND: Social behavior and social organization have major influences on individual health and fitness. Yet, biomedical research focuses on studying a few genotypes under impoverished social conditions. Understanding how lab conditions have modified social organizations of model organisms, such as lab mice, relative to natural populations is a missing link between socioecology and biomedical science. RESULTS: Using a common garden design, we describe the formation of social structure in the well-studied laboratory mouse strain, C57BL/6J, in replicated mixed-sex populations over 10-day trials compared to control trials with wild-derived outbred house mice in outdoor field enclosures. We focus on three key features of mouse social systems: (i) territory establishment in males, (ii) female social relationships, and (iii) the social networks formed by the populations. Male territorial behaviors were similar but muted in C57 compared to wild-derived mice. Female C57 sharply differed from wild-derived females, showing little social bias toward cage mates and exploring substantially more of the enclosures compared to all other groups. Female behavior consistently generated denser social networks in C57 than in wild-derived mice. CONCLUSIONS: C57 and wild-derived mice individually vary in their social and spatial behaviors which scale to shape overall social organization. The repeatable societies formed under field conditions highlights opportunities to experimentally study the interplay between society and individual biology using model organisms.

Microbial transmission in the social microbiome and host health and disease.

Although social interactions are known to drive pathogen transmission, the contributions of socially transmissible host-associated mutualists and commensals to host health and disease remain poorly explored. We use the concept of the social microbiome-the microbial metacommunity of a social network of hosts-to analyze the implications of social microbial transmission for host health and disease. We investigate the contributions of socially transmissible microbes to both eco-evolutionary microbiome community processes (colonization resistance, the evolution of virulence, and reactions to ecological disturbance) and microbial transmission-based processes (transmission of microbes with metabolic and immune effects, inter-specific transmission, transmission of antibiotic-resistant microbes, and transmission of viruses). We consider the implications of social microbial transmission for communicable and non-communicable diseases and evaluate the importance of a socially transmissible component underlying canonically non-communicable diseases. The social transmission of mutualists and commensals may play a significant, under-appreciated role in the social determinants of health and may act as a hidden force in social evolution.

Major urinary protein (Mup) gene family deletion drives sex-specific alterations in the house-mouse gut microbiota.

The gut microbiota is shaped by host metabolism. In house mice (Mus musculus), major urinary protein (MUP) pheromone production represents a considerable energy investment, particularly in sexually mature males. Deletion of the Mup gene family shifts mouse metabolism toward an anabolic state, marked by lipogenesis, lipid accumulation, and body mass increases. Given the metabolic implications of MUPs, they may also influence the gut microbiota. Here, we investigated the effect of a deletion of the Mup gene family on the gut microbiota of sexually mature mice. Shotgun metagenomics revealed distinct taxonomic and functional profiles between wild-type and knockout males but not females. Deletion of the Mup gene cluster significantly reduced diversity in microbial families and functions in male mice. Additionally, a species of Ruminococcaceae and several microbial functions, such as transporters involved in vitamin B5 acquisition, were significantly depleted in the microbiota of Mup knockout males. Altogether, these results show that MUPs significantly affect the gut microbiota of house mouse in a sex-specific manner.IMPORTANCEThe community of microorganisms that inhabits the gastrointestinal tract can have profound effects on host phenotypes. The gut microbiota is in turn shaped by host genes, including those involved with host metabolism. In adult male house mice, expression of the major urinary protein (Mup) gene cluster represents a substantial energy investment, and deletion of the Mup gene family leads to fat accumulation and weight gain in males. We show that deleting Mup genes also alters the gut microbiota of male, but not female, mice in terms of both taxonomic and functional compositions. Male mice without Mup genes harbored fewer gut bacterial families and reduced abundance of a species of Ruminococcaceae, a family that has been previously shown to reduce obesity risk. Studying the impact of the Mup gene family on the gut microbiota has the potential to reveal the ways in which these genes affect host phenotypes.

Convergence of gut microbiota in myrmecophagous amphibians.

The gut microbiome composition of terrestrial vertebrates is known to converge in response to common specialized dietary strategies, like leaf-eating (folivory) or ant- and termite-eating (myrmecophagy). To date, such convergence has been studied in mammals and birds, but has been neglected in amphibians. Here, we analysed 15 anuran species (frogs and toads) representing five Neotropical families and demonstrated the compositional convergence of the gut microbiomes of distantly related myrmecophagous species. Specifically, we found that the gut microbial communities of bufonids and microhylids, which have independently evolved myrmecophagy, were significantly more similar than expected based on their hosts' evolutionary divergence. Conversely, we found that gut microbiome composition was significantly associated with host evolutionary history in some cases. For instance, the microbiome composition of Xenohyla truncata, one of the few known amphibians that eat fruits, was not different from those of closely related tree frogs with an arthropod generalist diet. Bacterial taxa overrepresented in myrmecophagous species relative to other host families include Paludibacter, Treponema, and Rikenellaceae, suggesting diet-mediated selection and prey-to-predator transmission likely driving the observed compositional convergence. This study provides a basis for examining the roles of the gut microbiome in host tolerance and sequestration of toxic alkaloids from ants and termites.

Assessing co-diversification in host-associated microbiomes.

When lineages of hosts and microbial symbionts engage in intimate interactions over evolutionary timescales, they can diversify in parallel (i.e., co-diversify), producing associations between the lineages' phylogenetic histories. Tests for co-diversification of individual microbial lineages and their hosts have been developed previously, and these have been applied to discover ancient symbioses in diverse branches of the tree of life. However, most host-microbe relationships are not binary but multipartite, in that a single host-associated microbiota can contain many microbial lineages, generating challenges for assessing co-diversification. Here, we review recent evidence for co-diversification in complex microbiota, highlight the limitations of prior studies, and outline a hypothesis testing approach designed to overcome some of these limitations. We advocate for the use of microbiota-wide scans for co-diversifying symbiont lineages and discuss tools developed for this purpose. Tests for co-diversification for simple host symbiont systems can be extended to entire phylogenies of microbial lineages (e.g., metagenome-assembled or isolate genomes, amplicon sequence variants) sampled from host clades, thereby providing a means for identifying co-diversifying symbionts present within complex microbiota. The relative ages of symbiont clades can corroborate co-diversification, and multi-level permutation tests can account for multiple comparisons and phylogenetic non-independence introduced by repeated sampling of host species. Discovering co-diversifying lineages will generate powerful opportunities for interrogating the molecular evolution and lineage turnover of ancestral, host-species specific symbionts within host-associated microbiota.

Major urinary protein (Mup) gene family deletion drives sex-specific alterations on the house mouse gut microbiota.

The gut microbiota is shaped by host metabolism. In house mice (Mus musculus), major urinary protein (MUP) pheromone production represents a considerable energy investment, particularly in sexually mature males. Deletion of the Mup gene family shifts mouse metabolism towards an anabolic state, marked by lipogenesis, lipid accumulation, and body mass increases. Given the metabolic implications of MUPs, they may also influence the gut microbiota. Here, we investigated the effect of deletion of the Mup gene family on the gut microbiota of sexually mature mice. Shotgun metagenomics revealed distinct taxonomic and functional profiles between wildtype and knockout males, but not females. Deletion of the Mup gene cluster significantly reduced diversity in microbial families and functions in male mice. Additionally, specific taxa of the Ruminococcaceae family, which is associated with gut health and reduced risk of developing metabolic syndrome, and several microbial functions, such as transporters involved in vitamin B5 acquisition, were significantly depleted in the microbiota of Mup-knockout males. Altogether these results show that major urinary proteins significantly affect the gut microbiota of house mouse in a sex-specific manner.

Widespread extinctions of co-diversified primate gut bacterial symbionts from humans.

Humans and other primates harbour complex gut bacterial communities that influence health and disease, but the evolutionary histories of these symbioses remain unclear. This is partly due to limited information about the microbiota of ancestral primates. Here, using phylogenetic analyses of metagenome-assembled genomes (MAGs), we show that hundreds of gut bacterial clades diversified in parallel (that is, co-diversified) with primate species over millions of years, but that humans have experienced widespread losses of these ancestral symbionts. Analyses of 9,460 human and non-human primate MAGs, including newly generated MAGs from chimpanzees and bonobos, revealed significant co-diversification within ten gut bacterial phyla, including Firmicutes, Actinobacteriota and Bacteroidota. Strikingly, ~44% of the co-diversifying clades detected in African apes were absent from available metagenomic data from humans and ~54% were absent from industrialized human populations. In contrast, only ~3% of non-co-diversifying clades detected in African apes were absent from humans. Co-diversifying clades present in both humans and chimpanzees displayed consistent genomic signatures of natural selection between the two host species but differed in functional content from co-diversifying clades lost from humans, consistent with selection against certain functions. This study discovers host-species-specific bacterial symbionts that predate hominid diversification, many of which have undergone accelerated extinctions from human populations.

Home-site advantage for host species-specific gut microbiota.

Mammalian species harbor compositionally distinct gut microbial communities, but the mechanisms that maintain specificity of symbionts to host species remain unclear. Here, we show that natural selection within house mice (Mus musculus domesticus) drives deterministic assembly of the house-mouse gut microbiota from mixtures of native and non-native microbiotas. Competing microbiotas from wild-derived lines of house mice and other mouse species (Mus and Peromyscus spp.) within germ-free wild-type (WT) and Rag1-knockout (Rag1-/-) house mice revealed widespread fitness advantages for native gut bacteria. Native bacterial lineages significantly outcompeted non-native lineages in both WT and Rag1-/- mice, indicating home-site advantage for native microbiota independent of host adaptive immunity. However, a minority of native Bacteriodetes and Firmicutes favored by selection in WT hosts were not favored or disfavored in Rag1-/- hosts, indicating that Rag1 mediates fitness advantages of these strains. This study demonstrates home-site advantage for native gut bacteria, consistent with local adaptation of gut microbiota to their mammalian species.

Metagenomic signatures of balancing selection in the human gut.

Bacterial lineages that populate the human gut microbiota contend with spatial and temporal fluctuations in numerous environmental variables, including bouts of extreme selective agents such as antibiotics. Oscillations in the adaptive landscape can impose balancing selection on populations, leaving characteristic signatures in the sequence variation of functionally significant genomic loci. Despite their potential importance for gut bacterial adaptation, the metagenomic targets of balancing selection have not been identified. Here, I present population genetic evidence that balancing selection maintains allelic diversity in multidrug efflux pumps of multiple predominant gut bacterial species. Metagenome-wide scans of 566,958 genes from 287 bacterial species represented by 118,617 metagenome-assembled genomes indicated that most genes have been conserved by purifying selection. However, dozens of core open reading frames (CORFs) displayed positive Tajima's D values that deviated significantly from their species' genomic backgrounds, indicating the action of balancing selection. Multidrug efflux pumps (MEPs) from a diversity of bacterial species were significantly enriched among the CORFs with Tajima's D values >3 in industrialized, but not nonindustrialized, human populations. The AcrB subunit of an MEP from Bacteroides dorei displayed the highest Tajima's D of any CORF. Divergent haplotypes of this CORF displayed evidence of positive selection and homology to an Escherichia coli AcrB subunit that binds tetracycline and macrolide antibiotics, suggesting functional significance and implicating medical antibiotics as an agent of selection acting on this locus. Other proteins identified as targets of balancing selection included peptidoglycan/LPS O-acetylases and ion transporters. Intriguingly, the degree of balancing selection acting on gut bacterial species was associated with species abundance in the gut based on metagenomic data, further suggesting fitness benefits of the allelic variation identified.

Convergent remodelling of the gut microbiome is associated with host energetic condition over long-distance migration.

The gut microbiome can be thought of as a virtual organ given its immense metabolic capacity and profound effects on host physiology. Migratory birds are capable of adaptively modulating many aspects of their physiology to facilitate long-distance movements, raising the hypothesis that their microbiome may undergo a parallel remodeling process that helps to meet the energetic demands of migration.To test this hypothesis, we investigated changes in gut microbiome composition and function over the fall migration of the Blackpoll Warbler (Setophaga striata), which exhibits one of the longest known autumnal migratory routes of any songbird and rapidly undergoes extensive physiological remodeling during migration.Overall, our results showed that the Blackpoll Warbler microbiome differed significantly across phases of fall migration. This pattern was driven by a dramatic increase in the relative abundance of Proteobacteria, and more specifically a single 16S rRNA gene amplicon sequence variant belonging to the family Enterobacteriaceae. Further, Blackpoll Warblers exhibited a progressive reduction in microbiome diversity and within-group variance over migration, indicating convergence of microbiome composition among individuals during long-distance migration. Metagenomic analysis revealed that the gut microbiome of staging individuals was enriched in bacterial pathways involved in vitamin, amino acid, and fatty acid biosynthesis, as well as carbohydrate metabolism, and that these pathways were in turn positively associated with host body mass and subcutaneous fat deposits.Together, these results provide evidence that the gut microbiome of migratory birds may undergo adaptive remodeling to meet the physiological and energetic demands of long-distance migration.

A low-cost genomics workflow enables isolate screening and strain-level analyses within microbiomes.

Earth's environments harbor complex consortia of microbes that affect processes ranging from host health to biogeochemical cycles. Understanding their evolution and function is limited by an inability to isolate genomes in a high-throughput manner. Here, we present a workflow for bacterial whole-genome sequencing using open-source labware and the OpenTrons robotics platform, reducing costs to approximately $10 per genome. We assess genomic diversity within 45 gut bacterial species from wild-living chimpanzees and bonobos. We quantify intraspecific genomic diversity and reveal divergence of homologous plasmids between hosts. This enables population genetic analyses of bacterial strains not currently possible with metagenomic data alone.

Loyal gut microbes.

Bacterial strains in the gut microbiota diversified as humans spread across the globe.

Humanization of wildlife gut microbiota in urban environments.

Urbanization is rapidly altering Earth's environments, demanding investigation of the impacts on resident wildlife. Here, we show that urban populations of coyotes (Canis latrans), crested anole lizards (Anolis cristatellus), and white-crowned sparrows (Zonotrichia leucophrys) acquire gut microbiota constituents found in humans, including gut bacterial lineages associated with urbanization in humans. Comparisons of urban and rural wildlife and human populations revealed significant convergence of gut microbiota among urban populations relative to rural populations. All bacterial lineages overrepresented in urban wildlife relative to rural wildlife and differentially abundant between urban and rural humans were also overrepresented in urban humans relative to rural humans. Remarkably, the bacterial lineage most overrepresented in urban anoles was a Bacteroides sequence variant that was also the most significantly overrepresented in urban human populations. These results indicate parallel effects of urbanization on human and wildlife gut microbiota and suggest spillover of bacteria from humans into wildlife in cities.

Vertebrate species, such as reptiles, birds or mammals, harbour distinct communities of microbes in their digestive systems. These miniature ecosystems ­ also known as microbiomes ­ are unique to each owner and species, reflecting their diverse lifestyles and evolutionary history. Urbanisation can disrupt these delicate intestinal communities. Humans and other animals living in cities have different gut microbes to their counterparts living in rural areas. And captive species in homes and zoos often acquire human gut bacteria in their digestive systems, which can lead to health problems in these animals. So far, it has been unclear whether such a humanization of gut bacteria also affects wild animals living in and around cities. To investigate this further, Dillard et al. compared the gut microbes of wild reptiles, birds, and mammals living in close contact with humans in North America, such as coyotes, crested anole lizards and white-crowned sparrows. DNA sequencing showed that in urban environments, the composition of gut bacteria living in all three wildlife species resembled the ones in humans. The types of bacteria overrepresented in the guts of urban humans were also overrepresented in urban wildlife. This suggests that urbanization can affect the composition of gut bacteria in wildlife species by disrupting or replacing portions of their microbiome. The reason for this pattern is unclear. It is possible that humans might be sharing their gut microbes directly with city animals, or that a human-like diet is causing the change. Given the role that gut microbes play in health and disease, it is important to find out whether these changes cause the animals any harm.

Robustness of Mammalian Gut Microbiota to Humanization in Captivity.

In mammals, the composition of the gut microbiota is associated with host phylogenetic history, and host-lineage specific microbiota have been shown, in some cases, to contribute to fitness-related traits of their hosts. However, in primates, captivity can disrupt the native microbiota through a process of humanization in which captive hosts acquire gut microbiota constituents found in humans. Despite the potential importance of this process for the health of captive hosts, the degree to which captivity humanizes the gut microbiota of other mammalian taxa has not been explored. Here, we analyzed hundreds of published gut microbiota profiles generated from wild and captive hosts spanning seven mammalian families to investigate the extent of humanization of the gut microbiota in captivity across the mammalian phylogeny. Comparisons of these hosts revealed compositional convergence between captive mammal and human gut microbiota in the majority of mammalian families examined. This convergence was driven by a diversity of microbial lineages, including members of the Archaea, Clostridium, and Bacteroides. However, the gut microbiota of two families-Giraffidae and Bovidae-were remarkably robust to humanization in captivity, showing no evidence of gut microbiota acquisition from humans relative to their wild confamiliars. These results demonstrate that humanization of the gut microbiota is widespread in captive mammals, but that certain mammalian lineages are resistant to colonization by human-associated gut bacteria.

Culture-enriched community profiling improves resolution of the vertebrate gut microbiota.

Vertebrates harbour gut microbial communities containing hundreds of bacterial species, most of which have never been cultivated or isolated in the laboratory. The lack of cultured representatives from vertebrate gut microbiotas limits the description and experimental interrogation of these communities. Here, we show that representatives from >50% of the bacterial genera detected by culture-independent sequencing in the gut microbiotas of fence lizards, house mice, chimpanzees, and humans were recovered in mixed cultures from frozen faecal samples plated on a panel of nine media under a single growth condition. In addition, culturing captured >100 rare bacterial genera overlooked by culture-independent sequencing, more than doubling the total number of bacterial sequence variants detected. Our approach recovered representatives from 23 previously uncultured candidate bacterial genera, 12 of which were not detected by culture-independent sequencing. Results identified strategies for both indiscriminate and selective culturing of the gut microbiota that were reproducible across vertebrate species. Isolation followed by whole-genome sequencing of 161 bacterial colonies from wild chimpanzees enabled the discovery of candidate novel species closely related to the opportunistic pathogens of humans Clostridium difficile and Hungatella hathewayi. This study establishes culturing methods that improve inventories and facilitate isolation of gut microbiota constituents from a wide diversity of vertebrate species.

The microbiome: A heritable contributor to bone morphology?

Bone provides structure to the vertebrate body that allows for movement and mechanical stimuli that enable and the proper development of neighboring organs. Bone morphology and density is also highly heritable. In humans, heritability of bone mineral density has been estimated to be 50-80%. However, genome wide association studies have so far explained only 25% of the variation in bone mineral density, suggesting that a substantial portion of the heritability of bone mineral density may be due to environmental factors. Here we explore the idea that the gut microbiome is a heritable environmental factor that contributes to bone morphology and density. The vertebrae skeleton has evolved over the past ~500 million years in the presence of commensal microbial communities. The composition of the commensal microbial communities has co-evolved with the hosts resulting in species-specific microbial populations associated with vertebrate phylogeny. Furthermore, a substantial portion of the gut microbiome is acquired through familial transfer. Recent studies suggest that the gut microbiome also influences postnatal development. Here we review studies from the past decade in mice that have shown that the presence of the gut microbiome can influence postnatal bone growth regulating bone morphology and density. These studies indicate that the presence of the gut microbiome may increase longitudinal bone growth and appositional bone growth, resulting differences cortical bone morphology in long bones. More surprising, however are recent studies showing that transfer of the gut microbiota among inbred mouse strains with distinct bone phenotypes can alter postnatal development and adult bone morphology. Together these studies support the concept that the gut microbiome is a contributor to skeletal phenotype.